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Role of ferroxidases in iron absorption and metabolism

Permanent Link: http://ufdc.ufl.edu/UFE0044968/00001

Material Information

Title: Role of ferroxidases in iron absorption and metabolism
Physical Description: 1 online resource (167 p.)
Language: english
Creator: Lu, Yan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: copper -- ferroxidase -- intestine -- iron
Nutritional Sciences -- Dissertations, Academic -- UF
Genre: Nutritional Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Iron is an essential trace mineral, acting as a cofactor for many enzymes and functioning in oxygen transport. Abnormal iron accumulation results in oxidative stress and associated cellular damage. Due to a lack of active excretory mechanisms, iron levels are tightly regulated by modulation of intestinal absorption. Previous studies demonstrated that serum and hepatic copper levels increase during iron deficiency, implicating copper in the control of iron metabolism. The most obvious intersection between iron and copper is the multi-copper ferroxidases(FOX), ceruloplasmin (Cp) and hephaestin (Heph). The liver-derived, circulating Cp protein facilitates iron release from certain tissues (including perhapsintestine), while the membrane-bound FOX Heph works with ferroportin 1 (Fpn1)to mediate iron export from intestinal epithelial cells (IECs). Furthermore, during iron deficiency, we noted elevated copper levels and increased expression of an intestinal copper exporter, the Menkes copper transporting ATPase(Atp7a), in rodent IECs. These observations led us to hypothesize that Cp and Heph expression and activity are enhanced during iron deficiency to maximize intestinal iron absorption. Studies in iron-deficient rats demonstrated that Cp protein expression and serum FOX activity were elevated; Cp was previouslysuggested to enhance iron export from IECs during states of low iron stress, but this remains controversial. Further investigations noted that, unexpectedly, Heph protein and ferroxidase activity are present in thecytosolic/soluble fraction of rodent enterocytes. Importantly, cytosolic FOX activity increased ~30% upon iron deprivation, while membrane FOX activity (likely representing membrane-bound Heph) was unchanged. Moreover, studies in a number of mutant and knockout mouse models demonstrated that cytosolic FOX activity could not be fully explained by Heph; thus rodent IECs contain an unknown FOX (termed cytoFOX). It was further noted that cyto FOX activity was not contributed by Cp, amyloid precursor protein (App; a ferroxidase discoveredin brain), or ferritin (an intracellular iron storage protein with ferroxidase activity). We thus conclude that: 1) Cp expression and activity are enhanced during hepatic copper loading associated with iron deficiency, and 2) a soluble ferroxidase exists in rodent enterocytes, perhaps complementing membrane Heph activity and playing an important role in iron export fromthe mammalian intestine.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yan Lu.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Collins, James Forrest.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044968:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044968/00001

Material Information

Title: Role of ferroxidases in iron absorption and metabolism
Physical Description: 1 online resource (167 p.)
Language: english
Creator: Lu, Yan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: copper -- ferroxidase -- intestine -- iron
Nutritional Sciences -- Dissertations, Academic -- UF
Genre: Nutritional Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Iron is an essential trace mineral, acting as a cofactor for many enzymes and functioning in oxygen transport. Abnormal iron accumulation results in oxidative stress and associated cellular damage. Due to a lack of active excretory mechanisms, iron levels are tightly regulated by modulation of intestinal absorption. Previous studies demonstrated that serum and hepatic copper levels increase during iron deficiency, implicating copper in the control of iron metabolism. The most obvious intersection between iron and copper is the multi-copper ferroxidases(FOX), ceruloplasmin (Cp) and hephaestin (Heph). The liver-derived, circulating Cp protein facilitates iron release from certain tissues (including perhapsintestine), while the membrane-bound FOX Heph works with ferroportin 1 (Fpn1)to mediate iron export from intestinal epithelial cells (IECs). Furthermore, during iron deficiency, we noted elevated copper levels and increased expression of an intestinal copper exporter, the Menkes copper transporting ATPase(Atp7a), in rodent IECs. These observations led us to hypothesize that Cp and Heph expression and activity are enhanced during iron deficiency to maximize intestinal iron absorption. Studies in iron-deficient rats demonstrated that Cp protein expression and serum FOX activity were elevated; Cp was previouslysuggested to enhance iron export from IECs during states of low iron stress, but this remains controversial. Further investigations noted that, unexpectedly, Heph protein and ferroxidase activity are present in thecytosolic/soluble fraction of rodent enterocytes. Importantly, cytosolic FOX activity increased ~30% upon iron deprivation, while membrane FOX activity (likely representing membrane-bound Heph) was unchanged. Moreover, studies in a number of mutant and knockout mouse models demonstrated that cytosolic FOX activity could not be fully explained by Heph; thus rodent IECs contain an unknown FOX (termed cytoFOX). It was further noted that cyto FOX activity was not contributed by Cp, amyloid precursor protein (App; a ferroxidase discoveredin brain), or ferritin (an intracellular iron storage protein with ferroxidase activity). We thus conclude that: 1) Cp expression and activity are enhanced during hepatic copper loading associated with iron deficiency, and 2) a soluble ferroxidase exists in rodent enterocytes, perhaps complementing membrane Heph activity and playing an important role in iron export fromthe mammalian intestine.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yan Lu.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Collins, James Forrest.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044968:00001


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1 ROLE OF FERROXIDASES IN IRON ABSORPTION A ND METABOLISM By YAN LU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSO PHY UNIVERSITY OF FLORIDA 2012

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2 2012 Yan Lu

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3 To those who gave their love to me

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4 ACKNOWLEDGMENTS I would like to give my gratitude to many people, who helped me in different ways on this journey. Witho ut them, this work would not be possible My deepest gratitude goes to my major professor Dr. James F. Collins. expres s how fortunate I feel to have h im always be there to listen and give advi c e. His kindness, encouragement and patience guide d my way throughout the process support ed me to ove rcome crisis moments, provide d me incredible opportunities and helped me finish this dissertation strong Dr. P erungavur N. Ranganathan is the one I want to give my special thanks to. I am deeply grateful for having him in the lab His experience is a valuable resource for me and he contributed to building the fundamental observations for this dissertation. Throughout all those long discussion s, I learned not only the knowledge but most importantly, how to thi nk differently and cri tically. These are two of many indispensable characteristic s for a scientist and necessary for making novel findings. I am grateful to Dr. Greg ory J. Anderson, who generously provided me the resources I needed for my research during my stay in Queensland Institute of Medical Research ( QIMR ) I am also thankful to him for always be ing there to listen patiently and give advice. His insightful discussions and comments greatly enhanced my understanding s and enriched my work I would lik e to acknowledge my committee members, Dr. Robert J. Cousins, Dr. Mitchell D. Knuts on and Dr. Thomas Clanton for the ir support Their helpful discussions, reviewing and commenting on my research and this dissertation made it a better product.

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5 Dr. Chris Vu lpe and his Ph.D. student Brie K. Fuqua from University of California, Berkeley (UCB) are the ones I want to express my gratitude to. Thank them for kindly providing me the knockout m ouse strains they originally generated Especially Brie, she assisted me greatly with my experiments in both UCB and QIMR. All those long discussions we had sparkle new ideas I am also thankful to my current and former lab members fellow graduate students and IT staff from Food Science and Human Nutrition D epartment for provi ding various support during my graduate studies Liwei Xie, Lingli Jiang, Rebecca Creasy and many others. Many of them became my friends, who stayed with me through hard ti mes; their support, care and encouragement helped me overcome setbacks and kept me going. I deeply appreciate their belief in me and greatly value their friendship. Lastly, I want to give my greatest gratitude to my p arents and family whose love, understanding and support enable d me to follow my dreams throughout the se years

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6 TABLE O F CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Iron and Copper Metabolism ................................ ................................ ................... 16 Iron Copper Interactions ................................ ................................ ......................... 19 Cerul oplasmin ................................ ................................ ................................ ......... 21 Hephaestin ................................ ................................ ................................ .............. 22 The Novel Cytosolic/soluble Ferroxidase ................................ ................................ 24 The Essential Role of Ferroxidase in Iron Metabolism ................................ ............ 24 Other Ferroxidases as Candidates for Cytofox ................................ ....................... 25 Zyklopen (Zp) ................................ ................................ ................................ ... 25 Cp ................................ ................................ ................................ ..................... 26 H Ferritin ................................ ................................ ................................ .......... 26 Beta amyloid Precursor Protein (App) ................................ .............................. 27 Overall Hypotheses ................................ ................................ ................................ 28 Specific Aims ................................ ................................ ................................ .......... 28 2 MATERIALS AND METHODS ................................ ................................ ................ 29 Chemicals and Reagents ................................ ................................ ........................ 29 Animals and Diets ................................ ................................ ................................ ... 29 Elemental Analyses, H emoglobin (Hb) and Hematocrit (Hct) Measurements ......... 32 Enterocyte Isolation and Subcellular Fractionation ................................ ................. 33 Subcellular Fractionat ion ................................ ................................ .................. 33 Method I (grinding) ................................ ................................ ..................... 33 Method II (hypotonic lysis) ................................ ................................ ......... 34 Met hod III (freeze/thaw) ................................ ................................ ............. 34 Western Blot Analysis ................................ ................................ ............................. 34 Real Time PCR ................................ ................................ ................................ ....... 36 Ferroxidase Enzyme Activity Assays ................................ ................................ ...... 37 In gel Assays ................................ ................................ ................................ .......... 37 Spectrophotometric Assays ................................ ................................ .................... 38 para Phenylenediamine ( p PD) Assay ................................ .............................. 38

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7 Ferrozine (Fz) Assay ................................ ................................ ........................ 38 p PD and Fz Assays in Australia ................................ ................................ ....... 39 pPD assays ................................ ................................ ................................ 39 Fz assays ................................ ................................ ................................ ... 39 Transferrin Coupled FOX Initial Velocity Assay ................................ ................ 40 Thin Layer Chromatography ................................ ................................ ................... 40 Cell Culture, Immunocytochemistry (ICC) and Immunohistochemistry (IHC) Analyses ................................ ................................ ................................ ............ 40 Statistical Analyses ................................ ................................ ................................ 41 Figures and Tables ................................ ................................ ................................ 43 3 ROLE OF CERULOPLASMIN IN IR ON METABOLISM ................................ .......... 47 Serum Ceruloplasmin Protein Expression and Activity in Rats ............................... 47 Summary ................................ ................................ ................................ .......... 47 Introduction ................................ ................................ ................................ ....... 48 Results ................................ ................................ ................................ ............. 49 Dietary Feeding Strategy ................................ ................................ ........... 49 Iron and Copper Levels in Rat Sera and Liver ................................ ........... 50 Hematological Status as a Function of Diet ................................ ............... 50 Expression of Cu homeostasis related genes in liver. ............................... 51 Quantification of Immunoreactive Ceruloplasmin Protein Levels ............... 51 In Gel Serum Ferroxidas e and Amine Oxidase Activity Assays ................. 52 Spectrophotometric Serum Ferroxidase and Amine Oxidase Activity Assays ................................ ................................ ................................ .... 52 Discussion ................................ ................................ ................................ ........ 53 Serum Ceruloplasmin Protein Expression and Activity in Mice ............................... 59 Materials and Methods ................................ ................................ ..................... 59 Results ................................ ................................ ................................ ............. 59 Figures and Tables ................................ ................................ ................................ 61 4 A NOVEL CYTOSOLIC/SOLUBLE FERROXIDASE IN RODENT ENTEROCYTES ................................ ................................ ................................ ..... 73 Discovery of the Novel Cytosolic/Soluble Ferroxidase ................................ ............ 73 Summary ................................ ................................ ................................ .......... 73 Intr oduction ................................ ................................ ................................ ....... 74 Results ................................ ................................ ................................ ............. 75 Analysis of experimental animals ................................ ............................... 75 FOX a ctivity in rat enterocyte fractions ................................ ...................... 76 Purity of rat enterocyte subcellular fractions ................................ .............. 76 FOX activity in cytosolic fractions pre pared by additional methods ............ 77 Inhibition studies of rat enterocyte cyto FOX ................................ ............. 77 Cyto FOX activity in copper deficient rats ................................ .................. 77 Analysis of cyto FOX activity in mutant mouse models .............................. 78 Discussion ................................ ................................ ................................ ........ 78 Further Investigation of the Cytosolic/Soluble Ferroxidase ................................ ..... 81

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8 Cytosolic/soluble Heph ................................ ................................ ..................... 82 H Ferritin (H Ft) ................................ ................................ ................................ 82 Ceruloplasmin (Cp) ................................ ................................ .......................... 83 Amyloid Precursor Protein (App) ................................ ................................ ...... 84 Atp7b ................................ ................................ ................................ ................ 86 Hephaestin (Heph) ................................ ................................ ........................... 86 Heph /y group 1 ................................ ................................ ........................... 87 Heph /y group 2 ................................ ................................ ........................... 88 Heph int/int ................................ ................................ ................................ ..... 89 Heph /y / Cp / mice ................................ ................................ .............................. 89 Conclusions ................................ ................................ ................................ ...... 90 Figures and Tables ................................ ................................ ................................ 92 5 CYTOSOLIC HEPHAESTIN ................................ ................................ ................. 124 Summary ................................ ................................ ................................ .............. 124 Introduction ................................ ................................ ................................ ........... 125 Results and discussion ................................ ................................ ......................... 126 Heph Expression in Rat Intestinal Cytosol and Fractio n Purity ....................... 126 Detection of Heph by Immunoflourescence ................................ .................... 127 Heph Expression in Response to Iron and Copper Deficiency ....................... 128 Ferroxidase (FOX) Activity in Rat Enterocyte Cytosol ................................ .... 129 Could Intestinal Ceruloplasmin (Cp) Explain Cytosolic FOX Activity? ............ 130 Conclusions ................................ ................................ ................................ .......... 131 Figures ................................ ................................ ................................ .................. 132 6 CONCLUSIONS ................................ ................................ ................................ ... 136 Ceruloplamin Protein Expression and Activity ................................ ...................... 136 Hephaestin Protein Expression and Activity ................................ ......................... 136 Possible Identities for CytoFOX ................................ ................................ ............ 137 APPENDIX MULTIPLE MENKES COPPER ATPASE (ATP7A) TRANSCRIPT AND PROTEIN VARIANTS ARE INDUCED BY IRON DEFICIENCY IN RAT DOUDENAL ENTEROCYTES ................................ ................................ ................................ ... 139 Summary ................................ ................................ ................................ .............. 139 Introduction ................................ ................................ ................................ ........... 140 Methods ................................ ................................ ................................ ................ 141 Results and Discussion ................................ ................................ ......................... 144 Conclusions ................................ ................................ ................................ .......... 148 Figures ................................ ................................ ................................ .................. 149 LIST OF REFERENCES ................................ ................................ ............................. 155 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 167

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9 LIST OF TABLES Table page 2 1 Ingredient list for QIMR home made diets. ................................ ......................... 44 2 2 Composition for mineral/vitamin mix used in the QIMR home made diet (7.8 g/ kg diet) ................................ ................................ ................................ ............ 45 2 3 Primer sequences ................................ ................................ ............................... 46 3 1 Iron and copper levels in experimental diets ................................ ...................... 69 3 2 Hb and Hct levels of ex perimental groups ................................ .......................... 70 3 3 Correlation analysis of different biochemical and physiological parameters with Cp activity ................................ ................................ ................................ .... 71 3 4 Hema tological parameters of mice on different diets. ................................ ......... 72 4 1 Hematological parameters and mineral levels of experimental rodents ............ 115 4 2 Hematological parameters of wt and Cp / mice. ................................ ............... 116 4 3 Hematological parameters of wt and App / mice fed FeD diet. ......................... 117 4 4 Hematological parameters of wt and App / mice fed standard rodent diet ...... 118 4 5 Hematological parameters of wt and Atp7b / mice. ................................ .......... 119 4 6 Hematological parameters of wt and Heph /y mice in group 1. .......................... 120 4 7 Hematological parameters of wt and Heph /y mice in group 2. .......................... 121 4 8 Hematological parameters of Heph Flox and Heph int/int mice. .............................. 122 4 9 Hematological parameters for wt and Heph /y / Cp / mice ................................ .. 123

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10 LIST OF FIGUR ES Figure page 2 1 A letter from QIMR Animal Ethics Committee. ................................ .................... 43 3 1 Iron and copper levels in rat liver and serum. ................................ ..................... 61 3 2 qRT PCR analysis of hepatic gene expression. ................................ ................. 62 3 3 Serum Cp protein expression in rat serum. ................................ ........................ 63 3 4 In gel Cp enzyme activity assays. ................................ ................................ ...... 64 3 5 Spectrophotometric Cp enzyme activity assays. ................................ ................ 64 3 6 Relative Cp activity as a function of liver and serum copper levels. ................... 65 3 7 Ferritin (Ft) expression in enterocyte cytosol. ................................ ..................... 65 3 8 Serum ceruloplasmin (Cp) expression in mouse serum from dietary treatment groups. ................................ ................................ ................................ ............... 66 3 9 Serum p PD assay from mice fed diets containing different iron levels. .............. 67 3 10 Serum ferrozine (Fz) assay from mice fed diets containing different iron levels. ................................ ................................ ................................ ................. 68 4 1 Ferroxidase activity in rat enterocyte fractions. ................................ ................... 92 4 2 Enterocyte purity and alternative methods. ................................ ........................ 93 4 3 Chemical properties of cytosolic FOX. ................................ ................................ 94 4 4 Effect of copper deficiency on cytosolic FOX activity. ................................ ......... 95 4 5 FOX activity in mutant mouse models. ................................ ............................... 96 4 6 Heph protein expression and FOX activity in enterocyte cytosol from Heph KO mice. ................................ ................................ ................................ ............. 97 4 7 Hephaestin protein expression in enterocyte cytosol and membrane fractions from mice on d ifferent diets. ................................ ................................ ............... 98 4 8 Cytosol and membrane ferroxidase (FOX) activity by Tf assay. ......................... 99 4 9 Cytosol ferroxidase activity after incubation at room temperature or after heat treatment. ................................ ................................ ................................ ......... 100

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11 4 10 Serum Cp protein expression in Cp / and wt mice. ................................ ........... 100 4 11 Seru m p PD assay for wt and Cp / mice. ................................ ........................... 101 4 12 Serum Fz assay for wt and Cp / mice. ................................ .............................. 101 4 13 NaN 3 inhibition of serum p PD oxidase activity from wt and Cp / mice at 2 mM or 10 mM. ................................ ................................ ................................ ......... 102 4 14 DP inhibition of serum p PD oxidase activity from wt and Cp / mice. ................. 102 4 15 Cytosol and membrane ferroxidase activity by Tf assay in wt and Cp / mice. .. 103 4 16 Serum p PD and Fz assays for wt and App / mice fed an FeD diet. .................. 103 4 17 Serum p PD assay with DP inhibition in wt and App / mice fed an FeD diet. .... 104 4 18 Serum Fz assay with DP inhibition in wt and App / mice fed an FeD diet ....... 104 4 19 Serum p PD and Fz assays for wt and App / mice fed a standard rodent diet 105 4 20 Serum p PD assay with DP inhibiti on in wt and App / mice fed a standard rodent diet ................................ ................................ ................................ ....... 105 4 21 Serum Fz assay with DP inhibition in wt and App / mice fed a standard rodent diet ................................ ................................ ................................ ....... 106 4 22 Enterocyte cytosolic and membrane FOX activity in wt and App / mice fed an FeD diet. ................................ ................................ ................................ ........... 106 4 23 Cytosolic FOX activity in wt and App / mice fed a standard rodent d iet. ........... 107 4 24 Hephaestin protein expression in enterocyte cytosol and membrane fractions of wt and Atp7b / mouse enterocytes. ................................ .............................. 107 4 25 Enterocyte cytosol and membrane FOX activity from wt and Atp7b / mice by Tf assay. ................................ ................................ ................................ ........... 108 4 26 Western blot analysis of Heph protein expression in enterocyte cytosol and membrane fractio ns from wt and Heph /y mice. ................................ ................ 108 4 27 Cytosol and membrane enterocyte FOX activity in wt and Heph /y mice. .......... 109 4 28 Membrane Tf assay of native or heated samples. ................................ ............ 109 4 30 Enterocyte cytosol and membrane FOX activities by Tf assay. ........................ 110 4 31 Ferritin protei n expression in the enterocyte cytosol from Heph /y and wt mice. ................................ ................................ ................................ ................. 111

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12 4 32 Heph protein expression in enterocyte cytosol and membrane fractions of Heph Flox and Heph int/int mice. ................................ ................................ ............. 111 4 33 Enterocyte cytosol and membrane FOX activities from Heph Flox and Heph int/int mice by Tf assay. ................................ ................................ ............. 112 4 34 Ferritin protein expression in enterocyte cytosol from Heph Flox and Heph int/int mi ce. ................................ ................................ ................................ ................. 112 4 35 Western blot analysis of serum Cp protein expression in wt and Heph /y /Cp / mice. ................................ ................................ ................................ ................. 113 4 36 Western blot analysis of Heph protein expression in enterocyte cytosol and membrane fractions of wt and Heph /y / Cp / mice. ................................ ............. 113 4 37 Enterocyte cytosol and membr ane FOX activities from wt and Heph /y / Cp / mice by Tf assay. ................................ ................................ .............................. 114 4 38 Ferritin protein expression in enterocyte cytosol from wt and Heph /y / Cp / mice. ................................ ................................ ................................ ................. 114 A 1 Northern blot analysis using full length cDNA probes with RNA extracted from duodenal enterocytes of Ctrl and FeD rats and IEC 6 cells. ..................... 149 A 2 Full length At splice variants. ................................ ................................ ................................ .. 150 A 3 IP of IEC 6 cell extract and membrane preps using three different anti Atp7a antibodies. ................................ ................................ ................................ ........ 151 A 4 Immunoreactive protein expression in Atp7a specific shRNA expressing 6 cells. ........................ 152 A 5 Atp7a protein expression in cytosol and membrane fractions of isolated enterocytes from control and iron deficient (FeD) rats. ................................ ..... 153 A 6 Immunocytochemical analysis of IEC 6 cells by Lon g Ab.. ............................... 154

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13 LIST OF ABBREVIATION S APP A m y loid precursor protein C P Ceruloplasmin C TRL Control DKO Double knockout F E D Iron deficien t F E E Iron excess GPI C P G lycosyl phosphatidylinositol anchored Cp H EPH Hephaestin FOX Ferro xidase FT Ferritin H B Hemoglobin H CT Hematocrit KO Knockout MCH Mean cell hemoglobin MCHC M ean cell hemoglobin concentration MCV Mean cell volume RBC Red blood cell S LA Sex linked anemia

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14 Abstract of Dissertation Presented to the Graduate School of the U niversity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF FERROXIDASES IN IRON ABSORPTION A ND METABOLISM By Yan Lu December 2012 Chair: James F. Collins Major: Nutritional Sciences I ron is an essential trace mineral, acting as a cofactor for many enzymes and functioning in oxygen transport. Abnormal iron accumulation results in oxidative stress and associated cellular damage. Due to a lack of active excretory mechanisms, iron levels a re tightly regulated by modulation of intestinal absorption. Previous studies demonstrated that serum and hepatic copper levels increase during iron deficiency, implicating copper in the control of iron metabolism. The most obvious intersection between iro n and copper is the multi copper ferroxidases (FOX), ceruloplasmin (Cp) and hephaestin (Heph). The liver derived, circulating Cp protein facilitates iron release from certain tissues (including perhaps intestine), while the membrane bound FOX Heph works wi th ferroportin 1 (Fpn1) to mediate iron export from intestinal epithelial cells (IECs). Furthermore, during iron deficiency, we noted elevated copper levels and increased expression of an intestinal cop per exporter, the Menkes copper transporting ATPase (A tp7a), in rodent IECs. These observations led us to hypothesize that Cp and Heph expression and activity are enhanced during iron deficiency to maximize intestinal iron absorption. Studies in iron deficient rats demonstrated that Cp protein expression and serum FOX activity were elevated; Cp was previously suggested to enhance iron

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15 export from IECs during states of low iron stress, but this remains controversial. Further investigation noted that, unexpectedly, Heph protein and ferroxidase activity are prese nt in the cytosolic/soluble fraction of rodent enterocytes. Importantly, cytosolic FOX activity increased ~30% upon iron deprivation, while membrane FOX activity (likely representing membrane bound Heph) was unchanged. Moreover, studies in a number of muta nt and knockout mouse models demonstrated that cytosolic FOX activity could not be fully explained by Heph; thus rodent IECs contain an unknown FOX (termed cytoFOX). It was further noted that cytoFOX activity was not contributed by Cp, amyloid precursor pr otein (App; a ferroxidase discovered in brain), or ferritin (an intracellular iron storage protein with ferroxidase activity). We thus conclude that: 1) Cp expression and activity are enhanced during hepatic copper loading associated with iron deficiency, and 2) a soluble ferroxidase exists in rodent enterocytes, perhaps complementing membrane Heph activity and playing an important role in iron export from the mammalian intestine.

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16 CHAPTER 1 INTRODUCTION Iron and Copper Metabolism Iron is the fourth mos t abundant element i n earth and it plays important roles in the body. It is essential in cellular functions and is involved in many metabolic processes. Among the functions it carries out iron is vital for hemoglobin synthesis ( to transport oxyge n) and mitochondria l function (cytochromes of the electron transport chain) (147) Excess free iron can be toxic and cause s oxidative stress, and there is no active mechanism for iron excretion; thus, it is tightly regulated by control of intestinal absorption (143) Various perturbations in iron metabolism lead to iron overload and deficiency diseases like hemochromatosis and anemia, respectively (94) Conditions such as inflammation, infection (72) and cancer (141) interfere with normal intestinal iron absorption, resulting in lower body iron storage and anemia. Even though, with the advances in molecular bio logy and genetics in the past decades, we have obtained a vast amount of information regarding how iron is metabolized and regulated in our body, to date, the precise mechanisms of the tightly controlled intestinal iron transport are not fully understood. Most iron is absorbed in the proximal part of the intestine, namely, duodenum and proximal jejunum (147) Dietary iron, w hich is usually present in the ferric state (Fe 3+ ), is reduced by a reductase (e.g. duodenal cytochrome b) (89) on the apical membrane of enterocytes before being taken up by divalent metal transporter 1 (Dmt1) (48) If body iron storage is adequate, the reduce d iron (Fe 2+ ) inside enterocytes will be obtained by ferritin in the cytosol and lost during normal cell turnover with feces. If the body needs more iron, the imported ferrous iron is

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17 exported by ferroportin 1 (FPN1) on the basolateral membrane (140) In order for Fe 3+ to bind to circulating apo transferrin ( apo Tf), F e 2+ needs to be oxidized. The current evidence suggests that most likely, hephaestin (Heph) mediates the oxidation as a ferroxidase and it may physically interact with Fpn1 (152) The circulating multi copper ferrox idase ceruloplasmin (Cp) may also contribute to this process. Beside the well studied pathway as described in the previous paragraph, iron movement in enterocytes was also proposed to be handled via a transcytosis pathway to avoid free flowing reactive fer rous iron within the cytosol due to lack of known iron chaperones. This was suggested by earlier in vitro and in vivo studies. Iron loading in Caco 2 cells growing in bicameral chambers induced the colocalization of both DMT1 and apo Isolated endosomes from those cells were found to have both proteins (83) Furthermore, iron gavage of rats demonstrated that, in intestinal epithelial cells, Dmt1 and Fpn1 were relocated from t he basolateral membrane to intracellular compartments upon this treatment (97) However, an iron oxidation step would be needed to convert Fe 2+ to Fe 3+ inside the vesicles before release into the circulation. Intes tinal iron absorption is mainly regulated by body iron stores and erythropoiesis. When storage is low and/or the demand for making erythrocytes is increased, iron absorption is enhanced. Decreased iron storage can be caused by chronic malnutrition, chronic blood loss (for instance, digestive tract ulcers), and increased iron demand during certain physiological conditions such as puberty, pregnancy and lactation, etc. During some pathophysiological conditions, for

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18 example thalassemia, the demand for making new erythrocytes is high, which also promotes iron absorption (60) Hepcidin is a 25 amino acid peptide that is synthesized in liver and circulates in the plasma. It is considered to be the key regulator in iron absorption and metaboli sm (9 5) Hepcidin expression is enhanced during iron overload and decreased during iron deficiency. Besides iron status, infection and inflammation can also induce hepcidin expression (96) In iron deficient rats, hepci din level was found to be inversely correlated with the levels of Dcytb, Dmt1 and Fpn1 (45) Enhanced erythropoiesis induced by hemolysis suppressed hepcidin expression (43) Later, it was found that hepcidin regulates plasma and extracellular iron levels through its action on Fpn1. Nemeth et al. demonstrated that hepcidin can bind to Fpn1 and internalize it to reduce cellular iron efflux (96) This effect has been shown in a few cell types in mice, including enterocytes, hepatocytes and macrophages (116) Recently, hypoxia inducible factors (HIFs) were also shown to regulate intestinal iron absorption. Normally, the intestinal epithelium is mildly hypoxia, and thus, induction of HIFs can be protective d uring inflammation and helpful for intestinal barrier maintenance (reviewed in (128) ). Recently, HIF regulate iron absorption related proteins. Shah et al. and Mastrogiannaki et al. demonstrated that during iron deprivation, Dmt1 and Dcytb are direct targets of HIF (87, 121) transcriptionally activate FPN1 and modulate intestinal iron absorption (129) This helps expla in the opposite FPN1 expression patterns during iron deficiency in liver

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19 and intestine, where in the former case, FPN1 decreased, but in the later case, it increased (1) Copper is also an essential trace mineral t hat serves as a co factor for cuproenzymes. Th e se redox enzymes are necessary for many physiological functions such as connective tissue formation, iron metabolism, central nervous system function and development, cardiac function and cholesterol metabolis m (135) In the intestine, similar to iron, dietary copper needs to be reduced first in order to be absorbed into the enterocyte by Copper Transporter 1 (Ctr1) (122) Unlike iron, copper is actively exported by two ATPases Atp7a in enterocytes and A tp7b copper ATPase) normally traffics in enterocytes betw een the trans Golgi apparatus, where it facilitates copper incorporation into newly synthesized cuproenzymes, and the basolateral membrane, where it exports copper when intracellular copper level is elevated (98) V arious mutations in ATP7A systemic copper deficiency due to an inability to export absorbed dietary copper (32) tocytes behaves similarly to ATP7A (118) It delivers copper to apo ceruloplasmin (Cp) (126) which is then re leased into the blood from hepatocytes, and functions in peripheral tissue iron release via its ferroxidase activity. Most importantly, Atp7b eliminates excess copper into bile when the hepatic copper level is elevated. cause copper accumulation induced organ damage in brain, liver, kidneys and eye (78) Iron Copper Interactions The interaction between iron and copper has been recognized fo r many decades. As reviewed by Fox, P. in (42) physicians reported that providing copper and iron to

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20 chlorosis (a form of anemia that was common among young women at that time) patients helped them to recover from this common disease. Later, Wintrobe and colleagues m ade the initial observations that copper is required for iron release from liver and peripheral tissues; moreover, copper is necessary for hemoglobin synthesis (145) Later, studies in copper deficient swine indicated that during copper deprivation, intestinal iron absorption and iron release from tissues were impaired, and the utilization of iron for erythropoiesis was adversely affected (56, 80) The connection was strengthened by the discovery of two multi copper ferroxidases (FOX), ceruloplasmin (Cp) (64) and hephaestin (Heph) (138) which may facilitate essential steps during iron absorption and utilization Both of these FOXs will be described in detail in the following sections. In the past few years, some observations from our lab strengthened the concept that copper dependent processes are also involved in iron homeostasis. In iron deficient rats microarray analysis of duodenal epithelia cells showed that not only iron absorption related genes were upregulated (e.g. Dmt1 and Dcytb), but also a few genes related to copper homeostasis were also strongly induced (e.g. Atp7a and Metallothionein ) (25) Later, a follow up study in iron deficient rats confirmed that Atp7a mRNA and protein were indeed strongly induced Elevated c opper transport and accumulated copper in intestine and liver were also seen in those rats (111) Thus, the observations regarding Atp7a and increased copper levels led to the hypothesi s that multi coppe r ferroxidases which are the most obvious link between iron and copper, might function to maximize intestinal iron absorption during iron deprivation

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21 Ceruloplasmin Cp was first purified by Holmberg and Laurell in 1948 (64) but it was recognized earlier as the blue protein in serum (42) Soluble ceruloplasmin is synthesized in liv er and released from hepatocytes. It circulates in the serum and functions as a ferroxidase. As a major copper carrier, it can incorporate 6 copper atoms and this process is shown to occur in the later stages of the secretory pathway, likely in the trans G olgi network. A study in copper deficient rats showed that copper administration increased Cp oxidase activity, but none of the injected copper was bound to circulating apo Cp (66) Another study in co ntrol and copper deficient rats indicated that copper appeared to be essential for its ferroxidase activity, but had no effect on mRNA expression or rate for biosynthesis (51) Copper incorporation in apo Cp greatly extended its half life, from 5 hrs to 5 days (49, 88) As modeled in Long Evans Cinnamon (LEC) rats with mutations in Atp7b, due to their spontaneous hepatitis, copper failed to incorporate into Cp, leading to hepatic copper acc umulation and decreased serum Cp activity (130) Hellman et al. studied a missense mutant Cp that was found in an aceruloplasminemia patient and their results showed that the endoplasmic reticul um (ER) was the cellular compartment where the mutant Cp was retained. This suggested that the ER, as an early step of the secretory pathway, was not involved in copper incorporation (62) Cp not only transports copper in the blood as a major copper carrier, but also acts as a multi copper ferroxida se, which facilitates iron release from certain tissues and allows optimal iron metabolism. Early studies in swine demonstrated that injection of Cp into Cu deficient, but iron storage adequate swine resulted in a rapid

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22 increase of plasma iron concentratio n, suggesting Cp promoted iron release from cells to plasma (105, 119) This is further exemplified by disruption of Cp in mice, which results in no disturbance in copper homeostasis but mild anemia and hepatic iron overload (59) These observations are similar to humans with aceruloplasminemia (50) Moreover, a glyco syl phosphatidylinositol (GPI) linked Cp was found to exist in brain (103) immune cells, hepatocytes and other organs and tissues (85, 92) Although the precise function of GPI Cp is unknown, current evidence suggests that it has protective effects in those tissues against oxidative stress caused by iron overload. Given the role of Cp in iron metabolism, during iron deficiency, i t could be induced to maximize intestinal iron absorption and utilization. However, previous studies produced conflicting results. Wollenberg, et al. suggested that in iron deficient rats, Cp injection did not increase iron absorption; however, Cp was much more efficient in accelerating iron release from portal blood (146) Another study using control and Cu deficient rats also failed to provide evidence to connect Cp ferroxidase activity and intestinal iron absorption (11) In contrast, when mice were su bjected to erythropoie tic stress, immunoreactive Cp was noted in the lamina propria of the intestine (21) The authors proposed a model in which the presence of Cp in intestine during iron deficiency may enhance iro n absorption. Hephaestin Heph was discovered as the mutant gene that is responsible for the phenotype of the sex linked anemic ( sla ) mouse (138) Sla mice have a 194 a mino a cid deletion in the Heph protein, causing decreased expression and mis localiz ation in enterocytes

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23 (77) Sla mice generally exhibit a mild iron deficient phenotype (16, 75) In addition to anemia, sla mice also had increased intestinal iron and decreased hepatic iron levels (20) indicating normal apical import but impaired iron export from enterocytes Heph protein was never purified from natural sources to provide direct information about its structural properties; instead, the amino acid (a.a.) sequence was deduced from the cDNA sequence (138) After comparing the sequence with Cp, Heph was found to share ~ 50% homology with this circulating multi copper ferroxidase. In addition to the conserved copper binding sites, Heph was predicted to have a C terminal transmembrane domain presumably anchoring it to the basolateral membrane of enterocytes (138) Heph is expressed in many tissues and organs, but the hi ghest expression was seen in duodenum (44, 138) Heph was found to have ferroxidase activity (6) and it is likely to function in tandem with the ferrous iron exporter FPN1 for iron release f rom the enterocytes. The interaction between these two proteins was found to present both in vivo and in vitro (57, 151) As a multi copper ferroxidase, copper status would affect Heph protein expression and enzyme activity. This was demonstrated by dramatically decreased Heph protein expression and activity in rodents fed a copper deficient diet, when compared to those fed a control diet (18, 115) Though Heph is predicted to be present on the basolateral membrane of (44, 76, 124) Findings in rat and mouse from my dissertation research sugge sted that a soluble version of Heph exists in enterocytes and it contributes to the cytosolic ferroxidase activity (109) This may support the proposed transcytosis pathway of intestinal iron absorption. Moreover, the fact that sla mice with mutant Heph can still

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24 absorb iron and have only minor iron deficiency, suggests that other oxidases may exist. In support of this conce pt, Griffiths et al. used a recombinant Heph and showed that, Heph as a catalyst, has very slow enzymatic activity (54) Overall, these facts suggested that Heph may not be adequate for intestinal iron transport to provide sufficient iron for va rious biological processes, including erythropoiesis The Novel Cytosolic/soluble Ferroxidase If sHeph is the only ferroxidase that contributes to the cytosolic FOX activity, presumably, the activity would be absent in enterocytes that do not have any Heph However, studies in Heph /y mice showed that despite the lack of Heph protein, the cytosol still have ~65% of the ferroxidase activity as compared to wt mice. This suggested the existence of a novel ferroxidase (named cytoFOX) and it may compensate for t he loss of Heph to provide sufficient ferroxidase activity for intestinal iron absorption. The possible requirement for a FOX in intestinal iron absorption is considered in the next section. The Essential Role of Ferroxidase in Iron Metabolism Current evid ence suggests that oxidation of iron is necessary for optimal intestinal iron absorption and normal body iron homeostasis. As proposed by Osaki in 1966, spontaneous iron oxidation could not provide enough iron to support erythropoiesis, while the enzymatic route could (101) In this regard, one might wonder why iron would require an oxidase, while copper is thought to spontaneously oxidize in the interstitial fluids of the lamina propria after transit through enterocytes. This can in part be explained by comparing the basic chemical properties of the two ions. At pH 7.0, the standard reduction potential for Fe 2+ is 0.77 V while that for Cu 1+ is 0.15 V, indicating a much greater propensity for spontaneous oxidation of cuprous copper at

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25 physiologic oxygen tensions. Consi deration of iron oxidation is an important issue, as only ferric iron can bind to apo transferrin(Tf) and be transported to body cells and tissues (and iron within the intestinal epithelium is ferrous iron). The importance of Tf for iron homeostasis is cle arly demonstrated in mice that lack functional transfer due to a point mutation in the gene (the hypotransferrinemic mice [ Tfr hpx/hpx ]). These mice die of severe iron deficiency anemia soon after birth if left untreated (134) Injection of the mutant mice in the neonatal period with Tf can rescue them from death, but they su bsequently develop severe iron loading in some tissues and redevelop anemia due to an inability to deliver adequate iron to the bone marrow for red blood cell production. These observations reveal key concepts in iron biology, including that iron oxidation in the intestinal epithelium is required to maintain iron delivery to the bone marrow for hemoglobin production, and that transferrin is also required. A thorough understanding of the molecular mechanisms which mediate iron oxidation at the level of the e nterocyte is thus critical, particularly given the fact that this process can occur efficiently in the absence of the only known intestinal ferroxidase, Heph. Other Ferroxidases as Candidates for Cytofox There are several proteins that were identified in e arlier studies which were shown to have ferroxidase activity and if they are present in the enterocytes, they could be the novel cytoFOX. Thus, each of them is taken into consideration here, and studies to investigate their potential roles in intestinal ir on oxidation were undertaken. Zyklopen (Zp) Zyklopen (Zp) is a newly identified member of the multi copper ferroxidase work, this protein is mainly expressed in placenta and mammary gland. No Zp protein

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26 expression was found in enterocytes however (17) so it was not considered as a potential intestinal FOX. Cp Soluble Cp is the circulating multicopper ferroxidase in serum. As disc ussed in the previous section, o ne study using anti Cp antibody showed that Cp is expressed in wt mouse duodenum after p hlebotomy (21) Thus, Cp might play a role in intestinal iron absorption and experiments were p erformed as part of my dissertation research in Cp / mice to consider this possibility. H Ferritin Ferritin is an intracellular iron storage protein. It takes up excess iron in cells to prevent toxicity. Studies have shown that one Ft molecule can uptake ~4 5 00 ferric iron atoms (84) One molecule of ferritin consists of 12 pairs of heavy (H) and light (L) chain subunits (86) Depending on which cells the ferritin molecules are loc ated in, they have distinct properties. The iron atoms contained in ferritin can be trapped in enterocytes and lost during normal cell turnover; they can be used as a reservoir of iron in n macrophages or be use immediately by erythrocytes (details reviewed in (131) ). As ferric iron i s reduced by DcytB (or other proteins) to ferrous iron and imported into enterocytes by Dmt1 on the apical surface, intracelluar iron oxidation is necessary for ferritin iron uptake and stor age The Ft H subunit was indeed shown to have ferroxidase activity by Tf assay or measurement of oxygen consumption (79, 149) Deletion of the H ferritin gene led to early embryonic lethality in mice (40) which demonstrated its importance.

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27 To determine whether H ferritin contributed to cytosolic FOX activity, one known aspect of F t was taken advantage of namely heat stability. Ferri tin is a very stable protein, as mentioned in previous studies by Tosha et al. and Kim et al. (74, 133) Heating the protein homogenate at 65 C for 15 min can denature and precipitate soluble non ferritin proteins An earlier study showed that 90% of functional, recombinant F t can be recovered after incubation at 75 C for 10 min (82) Thus, the author hypothesized that if the cytosol samples are heated moderately, ferritin will be present in the supernatant and rem ain s active while most other soluble proteins should be denatured and inactive. Beta amyloid Precursor Protein ( App ) amyloid precursor protein (App) is considered to play an important role in the plaques that are seen in the brains of AD patients (99) As reviewed in (34) biometals, such as copper, zinc and iron, are closely related to the pathological process of AD. Under normal conditions, can be removed from the synaptic cleft by degradation or by traveling to peripheral tissues. But in AD patients abnormal metal interaction increases and this prevents the removal of Zinc and iron were found to leads to oxidative stress and damage of important biomolecule s such as proteins and DNA. Recently, Duce et al. found that recombinant, soluble App has robust FOX activity and App / mouse brains had significantly decreased FOX activity (35) Furthermore, Puig et al. detected immunoreactive App protein in mouse ileum segment (104) Thus, App could be a candidate for th e novel cytoFOX and studies were performed in App / mice to consider its role in intestinal iron oxidation.

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28 Overall Hypotheses As discussed in the previous sections, iron oxidation in enterocyte s is critical for efficient iron delivery to bone marrow for erythropoiesis and Heph is not adequate for this process During iron deficiency, copper loading was seen in both liver and intestine, and they are key organs and tissues for iron metabolism. This suggested that copper and iron are intimately connected wit h each other, and that copper influences iron homeostasis. Thus, it is proposed that the two known multi copper ferroxidases, i.e. Cp derived from liver and Heph in enterocytes, would enhance iron absorption during iron deficiency. These observations led t o the hypotheses of my dissertation as listed here: 1. Copper loading during iron deficiency enhances dietary iron absorption by promoting multi copper ferroxidase (Cp and Heph) protein expression and enzyme activity; 2. Iron oxidation is required for normal ir on metabolism and Heph alone is not adequate to provide enough iron for body utilization, thus other unidentified FOXs may exist; 3. Cytolic FOX activity cannot be explained by the known FOXs, Cp, H Ft, Heph or App. Specific Aims Aim 1: To investigate how iro n deficiency and hepatic copper loading influence serum Cp protein expression and enzymatic activity. Aim 2: To investigate how iron or copper deficiency influence enterocyte Heph protein expression and ferroxidase activity. Aim 3: To investigate whether t he novel cytosolic/soluble ferroxidase (cytoFOX) could be Cp, H Ft or App.

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29 CHAPTER 2 MATERIALS AND M ETHODS Chemicals and R eagents Chemicals were obtained from Sigma Aldrich and Fisher Scientific, and were of analytical grade or high purity. Animals and D iets All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Florida and the Queensland Institute of Medical Research (QIMR) Animal Ethics Committee An approval letter from QIMR Animal Ethics Committee is attached (Figure 2 1) and the author was included in that project during her stay in QIMR. Weanling, male Sprague Dawley rats were purchased from Harlan (Indianapolis, IN) and were raised in the departmental animal quarters in overhanging, wire mesh bott omed cages under 12 hr dark/light cycle and killed between 9 11 a.m. to minimize diurnal variation. Twenty four animals were utilized in the first experiment (4 on each dietary treatment) and then a separate 24 animals were studied in a subsequent experime nt. Note that for some analyses reported herein, the n is < or >8. When it is lower, insufficient sample quantity was obtained to measure the desired parameter. When n is >8, additional samples were included that were derived from rats under the exact same dietary protocol and at the exact same ages, otherwise used in an independent investigation that was parallel for the parameter in question. All analyses were done on individual animals. The animals had free access to food and iron and copper free water. The experimental design co n sisted of six different AIN 93G based diets (Dyets, Inc., Bethlehem, PA). The diets were identical except for having variable

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30 Fe and Cu levels. The diets and the corresponding groups of rats consuming them were designated as fol lows: Control (Ctrl), Fe Deficient (FeD), Copper Deficient (CuD), Fe and Cu Deficient (FeDCuD), Copper Extra (CuE) and Fe Deficient, Copper Extra (FeDCuE). All diets with normal iron levels (Ctrl, CuD, CuE) were modeled after standard rodent chows to conta in ~200 mg/kg ( ppm ) iron (added as ferric citrate). Low Fe diets (FeD, FeDCuD, FeDCuE) contained <9 ppm iron. All normal copper containing diets (Ctrl, FeD) contained ~6 ppm Cu, low Cu diets (CuD, FeDCuD) contained ~1 ppm Cu and the extra copper diets (CuE FeDCuE) contained ~28 ppm Cu. Animals were placed on each diet for 32 35 days. At the end of the feeding regime, rats were anesthetized by CO 2 exposure and killed by cervical dislocation. Blood was collected by cardiac puncture and transferred to pre ch illed polypropylene tubes. After one hour on ice to allow for clotting, the tubes were centrifuged at 1,500 x g for 15 min at 4 C. The supernatants (sera) were separated and stored at 4 C, and used for Cp enzyme activity assays within 3 days (note that t he vast majority of samples were assayed with 48 hours). Portions of livers were snap frozen in liquid N 2 then stored at 80 C for RNA isolation, or stored at 20 C for mineral analysis The knockout mice mentioned in the following chapters were stud ied at Queensland Institute of Medical Research (QIMR) (Herston, Queensland, Australia) unless otherwise noted. They were maintained on a standard rodent diet with natural ingredients containing ~160 ppm iron (Norco Stockfeeds, South Lismore, NSW, This was originally published in 110. Ranganathan PN, Lu Y, Jiang L, Kim C, Collins JF. 2011. Serum ceruloplasmin protein expression and activity increases in iron deficient rats and is further enhanced by higher dietary copper intake. Blood 118: 3146 53

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31 Australi a). Cp / Heph / y Heph int/int Heph / y / Cp / and A pp / mice were on the C57BL/6J background. The Cp / strain was originally created by Dr. Leah Harris at Vanderbilt University ( Nashville, Tennessee, USA ) (59) The strain in QIMR was obtained from Dr. Joshua Dunaief at the University of Pennsylvania and is also available from The Jackson Laboratory (Strain name Cp tm1Hr s /J, stock # 003582. Bar Harbor, Maine, USA.). Heph / y m ouse of California, Berkeley (Berkeley, California, USA) using the Cre Lox system targeting Exon 4 of Heph gene (the 2nd codi ng exon). For Heph int/int strain, a Villin Cre expression system was used. App / mice were originally obtained from t he Jackson Laboratory (Strain name B6.129S7 App tm1Dbo /J, stock #004133) and the mouse strain available at the QIMR was derived from the co lony maintained in Dr. Ashley I. University of Melbourne (Melbourne, Victoria, Australia). Heph / / Cp / mice were generated by crossing the H eph / mouse strain with the Cp / mouse strain for several generations. Atp7b / mice were orig inally obta ined from Dr. Svetlana Lutsenko at Johns Hopkins University (Baltimore, MD, USA) and the details of the knockout mouse model were described in Buiakova et al. (12) Genotypes for all experimental m ice were determined by gene specific PCR reactions using tail tip DNA. Subsequently, absence of proteins in knockout mice was confirmed by specific antibodies using Western Blotting. WT mice used to investigate serum FOX activity and ferritin FOX activity (Chapter s 3 and 4) were fed home made purified diets containing different iron levels Weanling wt mice were given control, iron deficient or iron loaded diet s for 6 weeks before experiments. Four iron deficient mice were housed in one cage with a meshed

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32 bottom, while four control mice and five iron overloaded mice were kept in separate conventional cages. All groups had free access to food and water. The three diets were made in house and the iron levels were described previously (44) Brief ly, the composition of the iron deficient diet was originally based on the work of Valberg et al (136) A detailed ingredient list is shown in Tables 2 1 (Ingredients for iron deficient diet) and 2 2 ( Composition of m ineral and v itamin m ix) To make the control diet, ferrous ammonium sulfate was ad ded at 0.42 g/kg diet, which is equivalent to 82 mg iron/kg diet (82 ppm). For iron overload diet, 10 g carbonyl iron was added to 1 kg base diet to achieve 1% wt/wt ratio being equivalent to 2.86 g iron/kg diet (2860 ppm). The copper content o f these thr ee diets is 33.6 mg copper/kg diet (33.6 ppm). Diets were stored at 20 C before use Elemental A nalyses Hemoglobin ( Hb ) and Hematocrit ( Hct ) Measurements Rat livers and serum were submitted to the Diagnostic Center for Population and Animal Health (D CPAH) at Michigan State University for mineral analysis using standard methods. Briefly, liver samples were dried and weighed followed by wet digestion with nitric acid at 95 C overnight. Then, the digested tissues/ diluted serum samples were subjected to Inductively Coupled Plasma Mass Spectrometry (ICP MS) for analysis. Serum transferrin bound iron was measured with the Olympus Iron Reagent on an Olympus Chemistry Immuno Analyzer (Olympus America Inc., Melville, ctions. Experiments that were done in the US measured Hemoglobin (Hb) and Hematocrit (Hct) using a HemoCue hemoglobin

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33 analyzer (Hemocue AB, Sweden), and a Readacrit hematocrit system (Clay Adams, NJ) respectively At the QIMR, mice were anesthetized by C O 2 exposure and blood was drawn through the inferior vena cava Part of the fresh blood sample was immediately transferred to a clean Eppendorf tube and allowed to clot on ice to obtain serum as described earlier for rats; the remainder (~200 L) was colle cted in Terumo CAPIJECT tubes, which contain EDTA K2 to prevent clotting. Hematological parameters of the mice studied at the QIMR were measured by a COULTER AcT diffTM Analyzer (Beckman Coulter, Brea, CA, USA) using the unclotted blood Enterocyte Isol ation and S ubcellular Fractionation Enterocytes were isolated from duodenum and proximal jejunum of rats as described (71) Th is section was published in (108) Subc ellular F ractionation Method I ( g rinding) Cytosolic and solubilized, particulate membrane fraction preparations were as described (27) All steps were performed at 4 C. Briefly, enterocytes were homogenized by a tissue grind plus protease inhibitor cocktail) and centrifuged at 16,000 g for 15 min. Cytosolic fractions were obtained by re centrifuging the supernatants at 110,000 g for 1 h. The pellets were resuspended in bu ffer 2 (buffer 1 with 0.25% [v/v] Tween 20), sonicated 2 x This paragraph was originally published in 110. Ranganathan PN, Lu Y, Jiang L, Kim C, Collins JF. 2011. Serum ce ruloplasmin protein expression and activity increases in iron deficient rats and is further enhanced by higher dietary copper intake. Blood 118: 3146 53

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34 5 sec at 5 watts in an ice:water slurry with 15 sec chilling in between and re centrifuged at 16,000 g for 30 min. These supernatants were termed solubilized membrane fraction. Method II ( h ypotoni c l ysis) Enterocytes were incubated in buffer 1 on ice for 30 min with frequent mixing with 1 ml pipette tips and centrifuged at 16,000 g for 15 min. Subsequent steps were identical to method I Method III ( f reeze/ t haw) Enterocytes were suspended in buf fer 1 and snap frozen in liquid nitrogen first, then quickly thawed in a 37 o C water bath. This temperature change cycle was repeated 5 times and lysis of cells was confirmed visually under a microscope. Subsequent steps were identical to method I Wester n Blot Analysis Protein concentrations were determined by Pierce 660 nm Assay (Pierce 660 nm was measured using a microplate reader. Thirty g of serum cytosolic or memb rane protein was resolved by electrophoresis through SDS 7.5% p olyacrylamide gels, followed by transfer to PVDF membranes (Millipore, MA) as described previously (28) Serum b lots were subsequently reacted with chic ken anti ceruloplasmin antibody at a 1:5000 fold dilution followed by peroxidase coupled secondary antibody at a 1:10,000 fold dilution (Cat. # GW20074F and A9046, respectively; kind gifts from Sigma Aldrich, St. Louis, MO). Cytosolic and membrane protein blots were processed following a standard protocol (107) The anti (111) (called 54 10), and one anti Heph antibody (14) (called D4 a kind gift from Dr. Chris Vulpe, UC Berkeley, USA ) are well validated. The other anti Heph antibody was

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35 from GeneTex (Cat # GTX115300, Irvine, CA). The D4 antibody recognizes an epitope in the N terminal portion of the Heph protein whi le the GeneTex antibody recognizes the N terminus. Anti Cp Ab was from Sigma (Cat # GW20074F, St. Louis, MO ) Anti Ferroportin Ab was from Santa Cruz Biotechnology (Cat # sc 49668, Santa Cruz, CA). KNRK (normal rat kidney [NRK] cell line transformed by Ki rsten murine sarcoma virus) lysate a kind gift from Santa Cruz Biotech (Cat # sc 2214, Santa Cruz CA) was used as a positive control for immunoreactive FPN 1 protein Rabbit anti Ferritin Ab was from MP Biochemicals (Cat # 650771). Goat anti rabbit HRP co njugated secondary antibody was from Bethyl Laboratories, Montgomery, TX (Cat # A120 101P) and Invitrogen (Cat # 65 6120) Donkey anti goat HRP conjugated secondary Ab was also a kind gift from Santa Cruz Biotechnology (Cat # sc 2020, Santa Cruz, CA). Rabb it anti chicken IgY HRP Ab was a kind gift from Sigma Aldrich (Cat # A9046, St. Louis, MO USA ). Immunoreactive bands were visualized by enhanced chemiluminescence (Reagent A: 0.4 mM coumaric acid and 2.5 mM luminol in 0.1 M Tris HCl, pH 8.5; Reagent B: 0. 018% H 2 O 2 in 0.1 M Tris HCl, pH 8.5) and autoradiography. In QIMR, a few blots were visualized by ImageQuant LAS500 system (product code 29 0050 63, GE Healthcare). Blots were stained with Ponceau S solution to confirm equal sample loading and efficient tr ansfer. This approach avoids potential confounding variation in any single The optical density of immunoreactive bands on film and proteins on stained blots was quantified using the digitizing software UN SCAN IT (Silk Scientific, O rem, UT) and the average pixel numbers were used for normalization and comparison. The intensity of immunoreactive bands on film was normalized to the intensity of proteins on stained blots.

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36 Real Time PCR Total RNA was purified from rat liver and enterocy tes by TRIzol reagent (Invitrogen, Carlsbad, CA) and qRT PCR was performed as previously described (26) Briefly, one g of RNA was converted to cDNA with the Bio Rad iScript cDNA synthesis kit, in a 20 l reactio n. One l of the cDNA sample was subjected to PCR amplification using 10 l of SYBR Green master mix (Bio Rad) and 0.75 l (0.25 pM) of each forward and reverse, gene specific primer (sequences listed in Table 2 3 ), in a 20 l reaction. Primers were desi gned to span large introns to eliminate amplification from genomic DNA. Reactions were run in 96 well plates on a Bio Rad iCycler C1000 with the following cycling parameters: 95 C for 3 min, then 39 cycles with 95 C for 10 s and 58 C for 30 sec. A mel t curve was routinely performed after amplification by increasing the temperature from 65 95 C with 0.5 C increments for 5 sec at each temperature; single amplicons were detected in all cases. Preliminary experiments established the validity of each pri mer pair in that each set was able to linearly amplify each transcript across a range of template concentrations. Each RT reaction was analyzed in duplicate for 18S rRNA ankyrin repeat domain containing protein 37 ( Ankrd37 ), Menkes copper ATPase ( Atp7a ), Atp7b ), Cp, copper transporter 1 ( Ctr1 ), hepcidin ( Hamp ), metallothionein 1 ( Mt1a ), and vascular endothelial growth factor ( Vegf ) in each of the individual animals. Then, the average of 18S was subtracted from the experimental gen This part was originally published in 110. Ranganathan PN Lu Y, Jiang L, Kim C, Collins JF. 2011. Serum ceruloplasmin protein expression and activity increases in iron deficient rats and is further enhanced by higher dietary copper intake. Blood 118: 3146 53

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37 were calculated for experimental genes, for all experimental groups vs. the control (Ctrl) group. Mean fold change = 2 One comparison tests was used to determine significance between means. Ferroxidase Enzyme Activity Assays Principles for p PD and Fz assays we re described in detail earlier (110) Briefly, para Phenylenediamine ( p PD) is converted to a color ed compound upon oxidation, which has an absorption maxima at ~530 nm and it can be quantified in a spectrophotometer by reading at A 530 this reaction is considered an indirect way of measuring Cp FOX activit y. The substrate disappearing Fz assay is a more direct method to measure FOX activity. Ferrozine and Fe 2+ form a pink colored complex. This has an A max at ~570n m and it can be quantified by reading at A 570 in a spectrophotometer. In gel Assay s One mg o f serum protein was electrophoresed through a native 7.5% polyacrylamide gel at a constant 80 V in native running buffer (0.12 M Tris and 0.04 M glycine) at 4 C, until the t racking dye just left the gel. For the p PD assay, the gel was briefly rinsed in wa ter, incubated in 30 ml 0.1% p PD in 0.1 M CH 3 COONa CH 3 COOH bu f fer, pH 5.0 for 2 3 hours in the dark with gentle shaking, rinsed in Milli Q water and air dried overnight in a gel frame. For the ferrozine assay, the gel was incubated in 200 M Fe(NH 4 ) 2 (SO 4 ) 2 (H 2 O) 6 in 0.1 M CH 3 COONa CH 3 COOH buffer, pH 5.0 for 2 3 hours in the dark with gentle orbital sh aking. After rinsing with Milli Q water, the gel was reacted with 15 mM ferrozine solution and color development was allowed to proceed. The results were record ed by scanning the gels in a scanner.

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38 Spectrophotometric Assays para Phenylenediamine ( p PD) A ssay Serum p PD and Ferrozine assays related to the work of Ranganathan et al (110) were performed in US by Dr. P.N. Ran ganthan following the protocols described in th is and the subsequent sections. The reaction mix contained equal quantities of serum protein (either 200 or 500 g) from each animal, with 0.1 % p PD in 0.1 M CH 3 COONa CH 3 COOH buffer, pH 5.0, with/without inhib itors, in 1 ml final volume, and was incubated at 37 C. The volume of serum was kept at 15 20 l (providing up to 500 g total protein), in order to minimize serum induced variations and higher concentrations of endogenous inhibitors such as Cl citrate, PO 4 3 etc. After considering the relative apparent K ms of the substrates used, potential backward reaction, the low quantity of sera (hence the enzyme) used and after extensive preliminary trials, a 1 hour reaction time, end point assay was chosen. Earlier reports have used longer times such as 90 minutes and even up to 3 hours to measure serum Cp activity (119, 127) The reaction was stopped by addition of NaN 3 to a final conc entration of 10 mM, mixed, and absorbanc e was read at 530 nm in a Beckman DU 640 spectrophotometer. Blank (complete reaction buffer devoid of serum, i.e., enzyme source) readings were su btracted from sample readings. Ferrozine ( Fz ) Assay The reaction conditions were as mentioned above except the reaction was stopped by addition of Fz to a final concentration of 3 mM, mixed, and absorbance was read at 570 nm in a Beckman DU 640 spectrophotometer. Blank readings were subtracted from sample readings. For both methods, sera were also assayed in th e

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39 presence of 10 mM NaN 3 a well identity (i.e. Cp). p PD and Fz A ssays in Australia Serum data generated in QIMR was obtained using a 96 well plate based format and absorbance was measured in a BioTek Spect rophotometer (BioTek Instruments, Inc., Australia ) This greatly improved the efficiency in both experimental practice and data management. pPD a ssays Briefly, a total volume of 450 L reaction mix consisted of the following: 100 L 0.5M CH 3 COONa CH 3 COOH b uffer, pH 5.0, 250 g serum pr otein; Milli Q water was added to fill the volume to 450 L. Assay b lank contained buffer and water. For inhibition studies, inhibitors were added and the tubes were mix gently for ~1 hour before the initiation of the reaction s. One hundred and eighty L of the reaction mix was added into each well in duplicate in a 96 well plate Prior to reading in a pre heated (37 C) plate reader, 20 L of fresh made 1% p PD solution was added to each well (final reaction volume in each well wa s 200 L) and mixed by shaking Absorbance at 530nm was recorded every 15 min for 3 hrs. Fz a ssays Twenty L of 30 mM ferrozine solution was added to 96 well plates in the dark before the initiation of the reaction s Briefly, a 2000 L reaction consist ed of 400 L 0.5M CH 3 COONa CH 3 COOH buffer, pH 5.0, 250 or 500 g serum protein and Milli Q water was added up to 1800 L. Initiation of the reaction was achieved by adding 200 L of 500 M Fe(NH 4 ) 2 (SO 4 ) 2 (H 2 O) 6 solution into the reaction and after a quick m ixing,

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40 200 L aliquots of the mixture w ere added to the wells containing ferrozine solution at different time points (every 15 min for 2 hrs) and read at A 570 in a plate reader. Transferrin Coupled FOX Initial Velocity Assay One hundred g cytosol or 60 g membrane proteins from isolated enterocytes of each animal, in 0.125 M CH 3 COONa CH 3 COOH buffer, pH 5.0, were mixed with 50 M bovine apo transferrin (Sigma Aldrich, St. Louis, MO). Reaction was initiated by adding (NH 4 ) 2 Fe(SO 4 ) 2 to a 50 M final concent ration, in 200 l total volume. Absorbance at 460 nm was recorded every 5 seconds from time 0 to 120 s in a Thermo Biomate 3 spectrophotometer (Thermo Scientific, Rochester, NY, USA) at room temperature. Blanks (contain ing sample buffer instead of samples) were run for each experiment. The i nitial velocities ( dA 460 /dt) at every 15 s w ere calculated and plotted on graphs. Thin Layer Chromatography Following the general method of Bligh and Dyer (9) membrane and cy tosolic fractions were extracted with 3:2 CHCl 3 :CH 3 OH, and developed on a silica gel plate, using 2:1 C 6 H 14 :(CH 3 ) 2 CHOH as the mobile phase, along with purified phospho lipids that were run as a positive control. Cell Culture, Immunocytochemistry ( ICC ) and I mmunohistoc hemistry ( IHC ) Analyses IEC 6 and Caco 2 cells, obtained from ATCC, were cultured according to recommendations (www.atcc.org). Cells were seeded onto poly D lysine (Cat # P6407, Sigma Aldrich, St. Louis, MO) coated cover slips in 6 well plates. Upon reaching ~80% confluence, me dium was removed, cells were washed twice with ice cold PBS and fixed with freshly made 4% formaldehyde in PBS at room temperature for 20 min. After three

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41 washes with PBS, the cells were incubated with IHC blocking buffer (Bethyl Laboratories) for 30 min, followed by anti Heph antibody (GeneTex [1:500] or D4 [1:1000]) or PBS, for 2 hrs at room temperature with gentle rocking. After three brief washes with PBS, the cells were incubated with a fluorescent tagged secondary antibody (Alexa Fluor 647 goat anti r abbit IgG [1:2000]; Invitrogen Molecular Probes) in PBS for 30 min at room temperature. After 3 washes with PBS, the cover slips were drained, mounted on glass slides with fluorescent mounting medium and dried in the dark. The fluorescent signals were visu alized with a Leica SP5 Confocal Microscope at the Interdisciplinary Center for Biotechnology (ICBR) of the University of Florida. Confocal settings were kept identical across different samples in the same experiment, enabling direct comparison of fluoresc ence intensity. Transverse s egments from the proximal rat duodenum were fixed in formalin and embedded in paraffin. 10 m sections were cut and affixed onto positively charged microscope s lides. After de paraffinization and a few other steps, tissue secti ons were reacted with IHC blocking buffer for 30 min followed by with anti Heph antibody (D4) overnight at 4C in a humidified chamber. The remaining steps were as stated above for ICC analysis. Statistical Analyses For rat studies, e ach group had a minim um of eight rats, although in some cases data are reported for different numbers for reasons explained above. M ouse studies had various n umbers of animals in each group; detailed information is listed in the text or in the figure legends All r esults are e xpressed as mean SD. Outliers in each group were determined by GraphPad outlier calculator ( http://www.graphpad.com/quickcalcs /Grubbs1.cfm ) and eliminated from subsequent analyses. The data were analyzed by

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42 descriptive statistics, one and Pearson correlation and regression analyses with SPSS version 19.0 and GraphPad Prism (San Diego, CA) software. P < 0.05 was considered statistically significant. Liver copper was normalized by log transformation prior to Pearson correlation and nonlinear regression analyses to correct for skewed distribution. In all bar graphs, data with different letters are statistically different from one another a nd those that share a letter are not statistically different from each other. In m ouse studies, the number of animals in each group is provided in the results section or the figure legends. For enzyme activity assays, w hen two groups of values at the same time point were compared, due to small sample size, a two tailed, two sample test was utilized with Microsoft Excel For comparison of more than two groups, one was condu cted with GraphPad Prism. P <0.05 was considered statistically significant

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43 Figures and Tables Figure 2 1 A letter from QIMR Animal Ethics Committee.

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44 Table 2 1 Ingredient list for QIMR home made diets Ingredients Per 1 Kg Cane sugar 300 g Skim milk powder 600 g Mineral/ Vitamin mix (prepared as in Table 2 2 ) 7.8 g D,L a tocopherol 0.86 mL Vitamin K 0.12 mL Polyunsaturated vegetable oil 37.8 mL Lard 49.8 mL

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45 Table 2 2 C omposition for mineral/vitamin mix used in the QIMR home made diet (7.8 g/ kg diet) Vitamin/ Mineral Mix g/ 7.8 g Sodium chloride 6.625 Choline Cl 2.5875 Manganous sulfate 0.22125 m Inositol 0.21875 Cupric sulfate 0.08375 Calcium pantoth enate 0.013125 Retinol palmitate 0.0125 Nicotinic acid 0.010938 Thiamine HCl 0.010938 p Aminobenzoic acid 0.010938 Pyridoxine 0.005469 Riboflavin 0.005469 Ergocalciferol 0.001563 Folic acid 0.001094 Sodium iodate 0.000688 Biotin 0.000219 Vitamin B12 0.000188

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46 Table 2 3 Primer sequences Name F/R Sequence 18s Forward 18s Reverse Ctr1 Forward Ctr1 Reverse Atp7a common Forward Atp7a common Reverse Hepcidin Forward Hepcidin Reverse Cp Forward Cp Reverse Atp7b Forward Atp7b Reverse Mt1a Forward Mt1a Reverse Vegf Forward Vegf Reverse Ankrd37 Forward Ankrd37 Reverse

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47 CHAPTER 3 ROLE OF CERULOPLASMI N IN IRON METABOLISM Serum Ceruloplasmin Protein Expression and Activity in Rat s The work in this section has been published and permission for reprint ing it has been obtained § Summary Increases in serum and liver copper content are noted during iron deficiency in mammals, suggesting that copper dependent processes participate durin g iron deprivation. One point of intersection between the two metals is the liver derived, multi copper ferroxidase, ceruloplasmin (Cp), which is important for iron release from body stores. The current studies sought to explore Cp expression and activity during physiological states in which hepatic copper loading occurs (e.g. iron deficiency). Weanling rats were fed control or low Fe diets containing low, normal or high copper for ~5 weeks, and parameters of iron homeostasis were measured Liver copper inc reased in control and Fe deficient rats fed extra Cu. Hepatic Cp mRNA levels did not change, however, serum Cp protein was higher during iron deprivation and with higher copper consumption In gel and spectrophotometric ferroxidase and amine oxidase assay s demonstrated that Cp activity was enhanced when hepatic copper loading occurred Interestingly, liver copper levels strongly correlated with Cp protein expression and activity. These observations support the possibility that liver copper loading increases metallation of the Cp protein leading to increased production of the holo enzyme. § This research was originally published in Blood Ranganathan PN, Lu Y, Jiang L, Kim C, Collins JF. Serum ceruloplasmin protein expression and activity increases in iron deficient rats and is further enhanced by higher dietary copper intake. Blood 2011 Sep 15;118(11):3146 53. the American Society of He matology

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48 Moreover, this phenomenon may play a n important role in the compensatory response to maintain iron homeostasis during iron deficiency Introduction Iron deficiency enhances absorption of dietary iron in several mammalian species (106, 117) via a host of genetic and morphological adaptations intended to maximize extraction of iron from the diet. Several key genes encoding iron transpor t related proteins are strongly induced during iron deprivation (24, 25, 36) some by post transcriptional stabilization of mRNA transcripts (120) and others via transcriptional induction mediated at least in part by a hypoxia responsi ve trans (87, 121) In addition, genes related to intestinal copper homeostasis are induced during iron deprivation in rats (25, 111) suggesting that co pper is important in the physiological response of the intestinal epithelium to iron deficiency. Copper levels are known to increase in the liver and serum of iron deficient mammals (38, 123, 153) further hinting a t an important physiological role for copper during iron deprivation. The best known links between iron and copper are the multi copper ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp is a liver derived, circulating protein that is important for iron release from certain tissues, as evidenced by the iron overload phenotype in humans that lack ceruloplasmin (50) Although Cp contains the predominance of serum copper in many species, the lack of Cp does not lead to perturbations in copper homeostasis, rather exerting preferential effects on iron metabolism (90) Heph has a highly similar functional (ferroxidase) domain, but also has a singl e transmembrane segment that presumably anchors it to the plasma membrane of certain cells. Heph is expressed in several tissues, with high expression levels in the basolateral membranes of enterocytes of the proximal small intestine (68) Mice

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49 expressing a mutant Heph protein (sex linked anemia [ sla ] mice) have reduced intestinal iron absorption and develop moderate iron deficiency, especially during early life (138) Moreove r, Cp and Heph activities decrease during dietary copper deprivation, as recent studies in laboratory rodents have demonstrated (18, 113, 114) The current investigation was undertaken to further explore the potent ial influence of copper on homeostatic processes related to dietary iron absorption and test hypotheses on the mechanism for such an influence. As previous studies suggested that Heph is not strongly regulated during iron deficiency (4) Cp is the focus of the current study. Since Cp is a copper dependent enzyme, the design was to determine if Cp expression or activity increased during conditions in which liver copper loading occurs, such as during iron deficiency or when increased dietary copper is consumed. Weanling rats were thus placed on control or low Fe die ts containing variable copper levels (high, normal, low). After ~5 weeks on the diets, animals were killed and various parameters of iron and copper homeostasis were measured, including Cp mRNA and protein expression and serum ferroxidase activity. Results showed notable effects of iron deprivation and high copper intake on Cp protein expression and serum ferroxidase activity in the absence of alterations in Cp mRNA levels, implicating a post transcriptional molecular mechanism. Results Dietary Feeding Stra tegy The dietary feeding regime used in this study was developed over several years a time period when numerous dietary iron and copper deprivation studies were performed in Sprague Dawley rats (24 26) These studi es showed that diets containing <10 ppm Fe lead to sever e iron deficiency anemia and that diets containing ~1 ppm

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50 copper resulted in notable symptoms of copper deficiency. Moreover, we found that feeding weanling rats (~21 days of age) these deficient diet s for a minimum of 30 days led to maximal effects on various iron and copper related parameters. The dietary feeding regime used for this investigation was successful at meeting the following goals: 1) rats consuming Fe deficient diets were severely iron d eficient, 2) rats consuming low Cu diets were severely copper deficient and 3) rats consuming extra copper containing diets showed increase d body copper content. The data supporting these statements are delineated throughout the Results section below Iro n and Copper Levels in Rat Sera and Liver Hepatic and serum iron levels were reduced 80 90% in the three groups consuming iron deficient chows ( p <0.05 as compared to control values) (Figure 3 1). Hepatic iron increased in the CuD group (53%; p <0.001 ), wh ile it was not different from controls in the CuE group. Serum iron decreased in the CuD group (51%; p <0.001 ) but no change was noted in the CuE group. Serum copper levels were significantly decreased in rats consuming the low Cu diets (~95%; p <0.001 ), whi le although hepatic copper levels were decreased by ~70% in the same dietary groups, the difference did not reach statistical significance (i.e. p > 0.05 ). Furthermore, a trend towards increased liver copper was apparent in the FeD and CuE groups, but again, statistical significance was not achieved due to large variation between individual rats. Serum copper was notably higher in the rats consuming higher copper diets (CuE and FeDCuE groups; 65 70%, p <0.001 ) and in FeD group (~40%, p <0.05) all as compared to controls. Hematological Status as a Function of Diet The effects of varying the iron and copper content of the diets were even more apparent when we considered hematological parameters ( Table 3 2 ). Hb in the FeD

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51 and FeDCuD group s was decreased ~85% ( p < 0.001 ), while Hb was also significantly decreased in the FeDCuE group (~50%; p <0.001 ), all as compared to controls. Hb in the CuD group was reduced ~60% ( p <0.001 ) while the CuE diet had no effect on Hb levels (both compared to controls). Hct levels followe d a similar trend with significant decreases seen in the FeD and FeDCuD groups (~70%, p <0.001 ), with a less severe decrease noted in the FeDCuE group (32%, p <0.001 ). Hct was reduced less in the CuD group as compared to the reduction seen in the FeD group ( 51% p <0.001 ) and no change was noted in the CuE group (as compared to controls). Expression of Cu homeostasis related genes in liver As hepatic iron and copper levels are modulated by various transporters and metal binding proteins, we considered that c hanges in expression of the genes encoding these proteins could reveal mechanistic insight into the inverse relationship between iron and copper during states of deficiency. qRT PCR analysis of hepatic genes related to copper (and Fe) homeostasis was thus done in all rats from the 6 dietary groups. No differences were noted among the dietary groups for the following genes: Atp7a Atp7b, Ctr1, Mt1a and Vegf (data not shown), or Cp (Figure 3 2 ). Hamp and Ankrd37 did however show significant differences betwee n groups (Figure 3 2 ). Hamp (hepcidin) mRNA expression was significantly reduced in the FeD, CuD, FeDCuD and FeDCuE groups compared to controls while Ankrd37 was increased in the FeD and FeDCuD groups Quantification of Immunoreactive Ceruloplasmin Protei n Levels Given the striking changes in serum Cu (Fig. 3 1) and the high proportion of serum Cu that Cp represents, we asked if Cp reflected serum Cu changes. Serum Cp levels were thus quantified from individual rats in the different dietary treatment grou ps.

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52 Results from a representative experiment are shown in Fig ure 3 3 along with quantitative data from all animals studied Cp was detected in all 6 experimental groups, but both groups consuming low copper (CuD and Fe D CuD) showed a significant decrease compared to control s ( ~70% reduction; p <0.001) Dietary iron deprivation with and without supplemental Cu (FeD and FeDCuE group s ) led to an in crease in Cp expression (50 60%; p <0.001). Moreover, Cp expression in rats consuming the CuE diet was moderately increased (~27%), but this increase did not reach statistical significance. In Gel Serum Ferroxidase and Amine Oxidase Activity Assays Figure 3 4 panel A depicts the product of the reaction catalyzed by serum protein using p PD as a substrate Activity in the FeD CuE and FeDCuE groups was noticeably higher than that of control s, while no activity was noted in samples derived from animals consuming the low Cu diets ( CuD and Fe D CuD ). Results in Figure 3 4 panel B show Fe 2+ to Fe 3+ oxidation catalyzed by serum ferroxidase. In the image, translucent whitish band s represent the absence of the Fe 2+ ferrozine complex at the site of the immobilized ferroxidase since the latter has oxidized from Fe 2+ to Fe 3+ Results were almost identical to p PD assay results above with higher activity detected in the FeD CuE and FeDCuE groups, and no activity being detected in the CuD and Fe D CuD groups. Spectrophotometric Serum Ferroxidase and Amine Oxidase Activity Assays As a complementary approach to in gel assays s pect rophotometric Cp assays were developed, because quantification of data is more precise and different reaction times/conditions can be used. Extensive preliminary experiments were performed to optimize reaction conditions and to determine the stability of t he enzyme in rat serum.

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53 Pilot studies determined that Cp enzyme activity was relatively stable for up to 3 days when serum was processed on ice and stored at 4 o C. As such, all assays were performed by the end of the third day, with the vast majority of as says being done within 48 hours. Results from serum ferroxidase assays utilizing ferrozine (Fz) are depicted in Figure 3 5 panel A. Ferroxidase activity was significantly increased in the FeD, CuE and FeDCuE groups as compared to controls (~90%, 80% and 120%, respectively; p <0.001). Activity was essentially absent in the rats consuming the low Cu diets (CuD and FeDCuD groups; p <0.001), while activity was completely abolished in all samples tested using NaN 3 as an inhibitor (data not shown). Note that NaN 3 is a known inhibitor of FOX I (i.e. Cp). To confirm the increase in Cp activity noted in the FeD group and using the CuD and FeDCuD groups as controls, experiments were also performed spectorphotometrically using p PD as the substrate ; serum amine oxidase activity, likely mediated by Cp, was thus measured. Figure 3 5 panel B depicts results obtained. As is evident, activity was increased in the FeD group (~3.6 fold; p <0.05) and activity was again absent with dietary copper deprivation (values <0 in the Cu D and FeDCuD groups). Enzyme activity in the Ctrl and FeD groups was once again abolished by NaN 3 (data not shown). Discussion Results described herein provide evidence of increased serum ferroxidase activity during iron deficiency anemia. This observati on concurs with scientific findings that were documented several decades ago (42) A seminal observation was made over 80 years ago when it was demonstrated that anemia induced in bi rds by bloodletting resulted in increased protein bound copper in plasma (139) Additional investigations in the 1930s and 40s documented increased serum copper in humans suffering from

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54 anemia associated with dietary iron deficiency, massive hemorrhage (42) pregnancy (39) and infection or inflammation (13) Once it was recognized that most copper in the blood is bound to Cp (55) noted increases in serum copper were shown to be due to elevated Cp in most of these and other pathological conditions (e.g. sickle cell disease and renal failure). The induction of Cp ac tivity during infection and inflammation, and pregnancy is probably the result of cytokine and hormonal stimulation of Cp expression. However, increased Cp activity during dietary iron deficiency, an observation that has been singularly reported in the lit erature (137) likely reflects a distinct regulatory pathway and exemplifies the relationship between Cp activity and body iron levels. The current investigat ion provides evidence that dietary iron deficiency in rats leads to notable increases in serum copper and Cp activity. Extensive molecular analysis of tissue and serum samples allowed comparison of functional changes in circulating Cp to the Cp gene expres sion pathway, permitting inference to be drawn regarding the molecular mechanism of Cp induction. Moreover, tissue mineral analyses reported herein allowed consideration of potential relationships between the different physiological parameters analyzed. Th e comprehensive nature of the current investigation has thus revealed novel aspects of this phenomenon that were not appreciated previously, including the observation that Cp expression and activity are enhanced in response to liver copper loading and that the underlying molecular mechanism is likely related to increased metallation of the Cp protein leading to higher circulating levels of the holo (i.e. functional) form of the enzyme. The biological purpose for enhanced Cp activity probably relates to the role of Cp in iron homeostasis. During dietary iron deficiency, Cp induction would increase iron

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55 mobilization from storage depots, for example from the liver, thereby maximizing iron delivery to the bone marrow for erythropoiesis (22) It is also possible that Cp plays a role in enhancing iron extraction from the diet during states when body iron stores are depleted. Several investigations have examined this possibility, with mixed results. Some studies do not support a role for Cp in intestinal iron absorption (11, 30) Another invest igation however provides strong evidence of a positive effect of Cp in this process (146) Of particular note is the observation that bleeding of mice combined with low dietary iron intake resulted in a dramatic shift of Cp into the lamina propria of the intestin al villus (21) A model was proposed in which Cp was present in enterocytes and then released into the lamina propria in response to iron deficiency, where it presumably enhances iron release from enterocytes. Furth ermore, although Cp knockout mice did not exhibit notable defects in intestinal iron transport (58) the role of Cp during states of low iron related stress was not determined. Also important to consider is the role of hephaestin in intestinal iron transport. While Heph plays an undisputed role in iron absorption, the phenotype of sex linked anemia ( sla ) mice (which lack functional Heph), suggests that another, redundant ferroxidase exists, as the anemia seen in these mutant mice resolves as they age (138) Additional studies are thus clearly warranted to further consider the physiological role of Cp in iron absorption, particularly during states of low iron stress. A previous investigation demonstrated that the induction of Cp in HepG2 cells (a human, hepatoma cell line) by iron chelation or hypoxia was transcriptionally mediated via activation of the Cp (93) The current observations however are not consistent with a hypoxia responsive transcription

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56 factor mediating the induction of Cp, as no change in Cp mRNA expres sion was noted. The livers of the experimental animals in the FeD groups were likely hypoxic as hemoglobin levels were significantly decreased. Moreover, there was a significant induction of hepatic Ankrd37 mRNA. Ankrd37 is a sensitive hypoxia responsive g ene and a proven HIF (7) ; in fact, i t was previously shown to be the most strongly upregulated gene (~15 fold) in intestinal epithelial cells treated with an iron chelator that mimics hypoxia (67) The current investigation thus demonstrates that the induction of Cp during dietary iron deficiency in this in vivo rodent model is mediated by a post transcriptional mechanism; findings that lead to speculation that increased production of the active, Cu containing form of the enzyme occurs in the copper lo aded livers of these experimental animals. Cp is synthesized in the liver in the apo (devoid of copper) and holo (replete with copper) forms in more or less equal quantities (61) The holo form, which is functional Cp enzyme, has a half life of ~5 days (125) while that of the apo form is estimated to be <6 hours (88) The predominance of Cp in the circulation is thus holo Cp. Serum Cp activity is drastica lly reduced during copper deprivation (51) likely due to the fact that most Cp enzyme synthesized during low copper conditions is apo Cp, which is rapidly catabolized. Given this role for copper in the synthesis of the functional Cp enzyme, it was important to also qu antify Cp expression and activity when liver copper levels increase (e.g. during iron deficiency). This is particularly relevant given the fact that scant experimental evidence documenting increases in serum Cp activity due to dietary iron deficiency exis ts (137) and no plausible insight as to a potential mechanism was proposed. Interestingly, this report, which describes increased serum amine oxidase

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57 (i.e. Cp question as to what initiates and maintains the increase in circulating ceruloplasmin in (137) This investigation documents increased Cp protein levels and serum ferroxidase activity in iron deficient rats and in those consuming higher copper levels. Statistical comparisons of the biological parameters analyzed here reveal strong correlations between Cp activity and serum and liver copper (Table 3 2 and Figure 3 6 ) allowing a reasonable conclusion to be drawn. It is hypothesized that increases in live r copper result in enhanced metallation of the Cp protein leading to a higher proportion of the holo form of the enzyme. This supposition is supported by the observation that Cp protein was increased and serum ferroxidase and amine oxidase activities were also significantly higher in rats with higher liver copper levels. Moreover, since Cu is a cofactor that is inserted co translationally into the Cp protein in an energy independent fashion, it is reasonable to predict Cu (content) to be rate limiting. A re cent in vitro study in fact provides support for this hypothesis, as it was demonstrated that Cp protein expression increased in copper loaded HepG2 cells with no corresponding changes in mRNA expression (41) Wheth er such a phenomenon occurs in vivo in other mammalian species is unknown, but the authors note a published report that documented increased Cp activity in a human patient suffering from acute copper toxicosis (65) Perhaps the most common clinical condition in which patients present (31) in which the ATP7B copper exporter is dysfunctional. These individuals are unable to synthesis functional Cp enzyme though, as the ability of ATP7B to deliver copper to the secretory pathway is abolished.

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58 consider how this could relate to the current observations. Dietary cupric copper must first be reduced by a brush border reductase (perhaps Cybrd1) followed by transport into enterocytes via copper transporter 1 (Ctr1) (29) Once in enterocytes, copper is bound to various chaperones that deliver i t to different cellular locations. Atox1 delivers copper to the trans Golgi where Cu + is taken up by the Menkes copper ATPase (Atp7a), allowing synthesis of cuproenzymes as part of the secretory pathway. Under conditions of copper excess, Atp7a trafficks to the basolateral membrane of cells where it mediates copper export (23) Interestingly, under low iron conditions in rats, Atp7a expression is strongly induced (24, 25) and the protein is present on the basolateral membrane of enterocytes, suggesting enhanced copper uptake (111) Copper then binds to albumin and other serum proteins for delivery to and transport into hepatocytes by Ct r1. It is tempting to speculate that the induction of Atp7a (and presumed increased copper export from enterocytes) increases copper delivery to hepatocytes, secondarily leading to increased holo Cp production. Whether this is the case or if decreased copp er excretion into the bile is responsible for liver copper loading during iron deficiency is currently not known. In summary, data presented in this manuscript support the possibility that Cp protein expression and activity are determined by liver copper l evels. It was previously noted that this relationship existed in the negative direction whereby decreased hepatic copper led to the production of non functional Cp enzyme, but the current investigation now suggests that such a relationship also exists in t he positive direction. Moreover, observations described in this manuscript provide evidence of a post transcriptional

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59 mechanism underlying the induction of Cp protein and activity levels during iron deficiency anemia. A clear focus for future studies relat es to recapitulating these observations in vitro so as to be able to precisely define the molecular mechanism underlying the induction of Cp protein and activity during hepatic copper loading. Serum Ceruloplasmin Protein Expression and Activity in Mice ** Th e p revious section noted that when rats were iron deficient Cp protein expression and activity increased These observations are in agreement with what has been observed in iron deficient humans (137) Currently, mice are widely used as genetic models due to the availability of knockouts The authors and other investigators in the iron field have noticed that mice and rat s respond differently upon various iron treatment s. Thus, it would be informative to conduct a dietary experiment to study how Cp changes during iron deprivation and overload in mice. Materials and Method s A d etailed dietary regime was described in Chapter 2, Animals and Diets section. Ctrl and FeD group s ha d 4 male mice per group while the FeE group ha d 5 male mice. Serum was collected for analysis of Cp protein expression and FOX activity. Enterocytes were isolated and fractionated for Western blot and FOX activity assays (results are show n in Chapter 4) Results Iron status of mice at the end of the experiment was determined by hematological analysis (Table 3 4) and cytosol Ft W estern blot (Figure 3 7 ). Complete blood analysis indicated that mice from the FeD group had significantly lower Hb ( FeD: ** Studies described in this section were performed at QIMR by the author of this dissertation with minimal technical assistance from Brie Fuqua.

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60 89.63 14.82 g/L vs. Ctrl: 160.33 10.27 g/L p <0.001 ) and Hct ( FeD: 0.30 0.047 L/L vs. Ctrl: 0.52 0.039 L/L p <0.001), while the FeE group had similar levels to the Ctrl group (Table 3 4). I t thus becomes apparent that mice are more difficult t o make iron deficient by feeding FeD diet only T he extent of anemia in the FeD mice is not as severe as rats that were fed the FeD diet (Table 4 1). Enterocyte cytosol Ft protein expression reflected the dietary treatment. The FeD group had virtually no F t present in the cytosol and the FeE group had doubled Ft expression (Ctrl: 1.00 0.58, FeD: 0.12 0.05, FeE: 2.15 0.23 in relative densitometric units ; statistical analysis show n in the figure ). Serum Cp protein expression was not different between g roups (Ctrl: 1.00 0.20; FeD: 0.83 0.04; FeE: 1.03 0.16 in relative densitometric units ; non significant from each other ) (Figure 3 8 ). Serum p PD assay showed that FeD mice had lower p PD oxidase activity, but the differences did not reach statistical significance. Mice from the Ctrl and FeE groups had similar p PD oxidase activity (Figure 3 9 ). Serum Fz assay exhibited similar results (Figure 3 10 ).

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61 Figures and Tables Figure 3 1 Iron and copper levels in rat liver and serum Values from individual rats are shown. Liver samples were dried and wet ashed before ICP MS analysis; data are presented as ppm (g/g dry weight). Serum samples were diluted and analyzed similarly; data are presented as ppm (g/ml) for Cu and g/dL for Fe. Plots with d ifferent letters on top are statistically different from one another ( p <0.05). N = 7 9 per group.

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62 Figure 3 2 qRT PCR analysis of hepatic gene expression. The expression of several genes was measu red in individual rats from the 6 dietary treatment groups. Shown are the 2 genes that showed significant changes in expression among the groups (A,C) and the data for Cp (which showed no significant differences; B). In all panels, bars with different lett ers atop or beneath error bars are statistically different from one other ( P < .05). Hamp, hepcidin; Ankrd37, ankyrin repeat domain protein 37. n = 7 for CuD group and n = 8 for all other groups. Data are expressed as means SD except for data in panel C, which are expressed as means SEM (because a log scale is used).

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63 Figure 3 3 Serum Cp protein expression in rat serum A) A representative western blot is shown above, representing 2 rats per dietary treatment group. B) qu antitative data from all rats (n = 8 12 rats/group). Bars with different letters atop error bars are statistically different ( p <0.05) from each other. Results are expressed as mean SD. The image is of one blot from a single x ray film; the vertical black line indicates where a blank lane with non chemiluminescent molecular weight markers was removed from the image.

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64 Figure 3 4 In gel Cp enzyme activit y assays. Serum samples were separated by gel electrophoresis and subsequ ently reacted with different substrates A) p PD or B) ferrozine to estimate enzyme activity levels. In both panels, each treatment group is represented by 2 samples derived from 2 individual rats (except for 3 samples for FeDCuD). Figure 3 5 Spectrophotometric Cp enzyme activity assays. A) ferrozine assays. Ctrl, n=12; FeD and FeDCuE, n=11; FeDCuD and CuE, n=8; CuD, n=7. B) p PD assays Ctrl and FeD, n=7; CuD, n=2; FeDCuD, n=4. Bars with different letters atop error bars ar e statistically different (p<0.05) from each other. Results are expressed as mean SD Note that for ferrozine assay, we included additional samples derived from rats that were from a different study in which the dietary protocol and animal ages were iden tical. (The raw data from this figure courtesy of Dr. Ranganathan)

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65 Figure 3 6 Relative Cp activity as a function of liver and serum copper levels. Plots depict the relationship between serum (panel A) and liver (panel B) c opper and Cp activity. Lines fitting the data were derived by linear regression for Cp activity vs. serum and by nonlinear regression analysis for Cp activity vs. liver copper. Data for liver copper were log transformed and fit to a sigmoidal dose response curve; actual values for liver copper (in g/g dry weight) are shown below the y axis In panel A, p <0.0001. r, Pearson Correlation Coefficient. Figure 3 7 Ferritin (Ft) expression in enterocyte cytosol. A) Western blot of 4 enterocyte cytosol samples from three groups reacted with anti Ft antibody; stained blot i n the lower panel to show comparable protein loading and transfer. B) Densitometry analysis of the film and blot in panel A. Different letters indicate statistic al differences between groups (1 way ANOVA followed by Tukey multiple comparison test; p <0 .05). Results are expressed as mean SD.

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66 Figure 3 8 Serum ceruloplasmin (Cp) expression in m ouse serum from dietary treatment group s. A) Western blot analysis of serum samples reacted with anti Cp antibody; stained blot on the bottom to show comparable protein loading and transfer. B) Densitometry analysis of the film and blot in panel A. Results are expressed as mean SD.

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67 Figure 3 9 Serum p PD assay from mice fed diets containing different iron levels. A) Overall comparison among three groups. B) Comparison for Ctrl and FeD groups. C) Comparison for Ctrl and FeE groups. Results are expressed as mean SD. N=4 for C trl, 4 for FeD, and 5 for FeE.

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68 Figure 3 10 Serum ferrozine (Fz) assay from mice fed diets containing different iron levels. Results are expressed as mean SD. N=4 for C trl, 4 for FeD, and 5 for FeE

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69 Table 3 1 Iron and copper levels in experimental diets Mineral Content (ppm) Ctrl FeD CuD FeDCuD CuE FeDCuE Iron 200 <9 200 <9 200 <9 Copper 6 6 <1 <1 28 28

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70 Table 3 2 Hb and Hct levels of experime ntal groups Group Ctrl FeD CuD FeDCuD CuE FeDCuE Hb (g/dL) 13.03 0.88 3.33 0.67 b 5.31 1.40 c 2.47 0.77 b 13.43 0.69 a 6.57 1.22 d Hct (%) 45.63 3.69 a 14.94 4.70 b 22.17 7.41 c 13.01 4.50 b 49.16 3.96 a 31.03 4.78 d Different letters indicate statistical di fferences between groups within each parameter, p<0.05 Results are expressed as mean SD One multiple comparison tests.

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71 Table 3 3 Correlation analysis of different biochemical and physiological parameters with C p activity Different letters indicate statistical differences between groups (one way ANOVA followed comparison tests; p <0.05). Values are means for each parameter. *Pearson Correlation Coefficient (r). ** g/g dry tissue weight. # Strong positive correlation Cp Activity Cp mRNA Cp Protein Liver Fe (g/g)** Serum Fe (g/dL) Liver Cu (g/g) Serum Cu (g/ml)* Hb (g/dL) Hct (%) Ctrl 1 1 1 a 376.6 a 331.8 a 10.7 a,c,d 0.46 a 13.1 a 46 a CuE 1.80 b 0.89 1.26 a,b 379.6 a 312.8 a 17.1 d 0.76 b 13.4 a 49 a FeD 1.89 b,c 1.41 1.52 b 63.3 b 33.6 b 15.3 a,d 0.65 b 3.3 b 15 b FeDCuE 2.20 c 1.72 1.53 b 76.7 b 64.4 b 30.6 b 0.79 b 6.6 c 31 c CuD 0.04 d 2.30 0.15 c 578 c 165 c 3.56 c 0.03 c 4.8 d 22 d FeDCuD 0.04 d 1.31 0.16 c 85.5 b 33.13 b 3.03 a,c 0.02 c 2.5 b 13 b Correlation with Cp Activity* N/A 0.02 0.74 # 0.39 0.05 0.88 # 0.95 # 0.29 0.33 Sig (2 tailed) 0.901 <0.0001 0.008 0.736 <0.0001 <0.0001 0.05 0. 025

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72 Table 3 4 Hematological parameters of mice on different diets. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) Ctrl 9.88 0.84 a 160.33 10.27 a 0.52 0.039 a 52.85 2.37 a 16.27 0.74 a 307.58 3.20 FeD 8.13 1.23 b 89.63 14.82 b 0.30 0.047 b 36.73 1 .17 b 11.01 0.36 b 302.63 17.38 FeE 9.19 0.72 a,b 151.60 6.96 a 0.48 0.026 a 52.93 1.87 a 16.5 0.59 a 312.9 5.56 Different letters indicate statistical differences between groups within each parameter (1 way ANOVA followed by Tukey multiple co mparison test; p<0.05). Results are expressed as mean SD. Ctrl, control; FeD, iron deficient; FeE, iron overload. N=6 for Ctrl, 4 for FeD, and 5 for FeE. RBC: red blood cell count, Hb: hemoglobin, Hct: hematocrit, MCV: mean cell volume, MCH: mean cell he moglobin, MCHC: mean cell hemoglobin concentration.

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73 CHAPTER 4 A NOVEL CYTOSOLIC /SOLUBLE FERROXIDASE IN RODENT ENTEROCYTE S Discovery of the Novel Cytosolic/Soluble Ferroxidase The work in this section has been published in PNAS and permission for rep rint ing it has been obtained Summary Hephaestin (Heph), a membrane bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex linked anemia ( sla ) mice harboring a 194 amino acid deletion i n the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity. The current studies were undertaken to se arch for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin coupl ed assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method induced artifac t. Cytosolic FOX activity was abolished by SDS and heat (78 C), suggesting protein mediated iron oxidation, and was also sensitive to Triton X 100. Furthermore, cytosolic FOX activity increased 30% in iron de fi cient rats (compared with controls) but This research was originally published in Proc Natl Acad Sci U S A Ranganathan PN, Lu Y ** Fuqua BK, Collins JF. Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes. Proc Natl Acad Sci U S A 2012 Feb 28;109(9):3564 9. the National Academy of Sciences ** co first author

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74 was unchanged in copper de fi cient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper de fi ciency). Additional studies done in sla Heph knockout, and ceruloplasmin knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membranebound Heph. Introduction Absorption of die tary iron occurs in the duodenal epithelium via the activity of trans membrane iron transport proteins coupled to reduction and oxidation reactions at the apical and basolateral surfaces of enterocytes, respectively. Iron export from enterocytes, presumed to be the rate limiting step in body iron acquisition (19) is mediated by ferroportin 1 (Fpn1) but also requires an oxidase to convert ferrous (Fe 2+ ) iron to ferric (Fe 3+ ) iron, the transferrin (Tf) binding form. T his oxidation is mediated by a multicopper ferroxidase (FOX), hephaestin (Heph), which contains a single membrane spanning domain and is associated with the basolateral membrane of enterocytes (69, 77) Mice harbori ng a major deletion in the Heph gene (sex linked anemia; sla ) express lower levels of a truncated Heph protein that is also mislocalized in intestinal epithelial cells (77) Despite this anomaly, adult sla mice gene rally display a mild iron deficient (FeD) phenotype (16) (Table 3 2 ), suggesting that another FOX can compensate for the lack of Heph. One possibility is that a circulating multicopper FOX, ceruloplasmin (Cp), could play this compensatory role. Cp was noted in the lamina propria of mouse villi under conditions of low iron stress and was hypothesized to facilitate iron absorption (21) Previous investigations of enterocyte Heph expression and activity were performed in whole cell lysates prepared in the presence of 1.5% (wt/vol) Triton X 100 (16, 19)

PAGE 75

75 The rationale for using this high detergent concentration, which is >70 times the critic al micelle concentration for Triton X 100, was not explained. Therefore the possibility that the activity of additional FOXs could have been masked was considered. Enterocyte lysates were prepared and separated into cytosolic/soluble and membrane/particula te fractions, and a spectrophotometric Tf coupled FOX assay was performed. Soluble/cytosolic fractions of rat enterocytes contained notable FOX activity. This activity could be a method induced artifact or caused by contamination of the cytosolic fraction with membrane proteins (e.g., Heph). Experiments designed to test these two possibilities eliminated any doubts. Studies were undertaken subsequently to determine the biochemical and functional properties of this FOX. Extensive additional experiments in Fe D and copper deficient (CuD) rats and in mice with mutations or deletions of known FOXs indicated that this cytosolic FOX (cyto FOX) activity is protein mediated and that it could not be fully explained by Heph or Cp. This FOX could complement membrane Hep h and may explain, in part, the lack of a severe FeD phenotype in sla mice. Results Analysis of e xperimental a nimals FeD and CuD rats were significantly anemic compared to controls ( Table 4 1 ), validating the dietary regimen. Enterocyte iron content of Fe D rats was reduced >90% compared with controls; mean copper levels, although notably higher in the FeD group, did not show a statistically significant increase ( P = 0.11). Quantitative RT PCR (qRT PCR) analysis of isolated rat enterocytes showed increases in Menkes copper ATPase (Atp7a; 9.3 fold; P < 0.05), copper transporter 1 ( 2.3 fold; P < 0.01), Heph ( 2.4 fold; P < 0.01), and metallothionein 1A ( 10.2 fold; P < 0.01) mRNA expression in FeD rats compared with controls ( n = 8 control and 8 FeD rats, each assayed separately).

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76 These ob servations provide further evidence of iron deficiency and are in agreement with previously published observations (24, 25) Furthermore, sla mice used for these studies were not anemic, as indicated by lack of diff erences in hemoglobin (Hb) and hematocrit (Hct) compared with WT mice, whereas Cp mice were mildly anemic with notable reductions in Hb and Hct ( Table 4 1 ). FOX a ctivity in r at e nterocyte f ractions To assess FOX activity in enterocytes, which previously had been measured only in total lysates prepared in the presence of 1.5% Triton X 100 (16, 19) cell lysates were fractionated to obtain cytosolic/soluble and membrane portions. FOX activity was then tested in these fractions from control and FeD rats using a coupled apo Tf assay. Surprisingly both fractions exhibited FOX activity, with the enzymatic rate (dA/dt) decreasing as a function of time, reflecting substrate depletion ( Fig. 4 1 ). Also notable was the increase in cyto FOX activity in FeD rats ( 30%), with no significant difference in membrane FOX activity noted between groups ( Fig. 4 1 A ). These data provide preliminary enzymatic evidence of a cytosolic FOX; subsequent experiments were designed to determine if this res ult was an artifact or caused by contamination with membrane bound Heph. Purity of r at e nterocyte s ubcellular f ractions First, the relative purity of the fractions was assessed. The cytosolic/soluble fraction contained robust lactate dehydrogenase (LDH) a ctivity (dA 340 /dt), representing an established marker enzyme for cytosol (Fig. 4 2A). LDH activity was significantly higher in cytosol than in whole cell lysate. Additional experiments were done by immunoblot analysis using antibodies against two transme mbrane proteins [zinc transporter 1 (ZnT1 ) (Fig. 4 2B) and Atp7a (Fig. 4 4B)]. Neither protein was detected in

PAGE 77

77 enterocyte cytosolic fractions (even upon overexposure of the blots), but strong immunoreactive bands were detected in membrane samples, as exp ected. FOX a ctivity in c ytosolic f ractions p repared by a dditional m ethods Next, to eliminate potential shear induced mechanical damage to the isolated proteins (and possible contamination of the cytosolic fraction with membrane proteins), cytosolic/soluble fractions were prepared by two complementary methods that achieved cell lysis by exposure to hypotonic solution or freeze thaw cycles. Two individual samples from different rats were tested by each of the three lysis methods (grinding, hypotonic, and free ze thaw). Progress curves from Tf coupled FOX assays demonstrated that FOX activity showed similar profiles in all methods tested (Fig. 4 2 C and D). The congruence of data from multiple methods enhances the validity of the observation that a soluble FOX exists in enterocyte cytosol. Inhibition s tudies of r at e nterocyte c yto FOX Whether cyto FOX activity was protein mediated and whether it was sensitive to a detergent used previously to study enterocyte Heph activity was determined next. Cyto FOX activity was abolished by treatment with 1% SDS and by heating samples to 78 C (Fig. 4 3A). When 1.5% Triton X 100 was added to samples just before FOX assay, a significant reduction in enzyme activity was observed, as indicated by the divergent slopes of the pro gress curves (Fig. 4 3B). Cyto FOX a ctivity in c opper d eficient r ats The expression and activity of two multicopper FOXs (Heph and Cp) are reduced significantly during copper deficiency in rodent and cell culture models (14, 18, 114) A logical question was whether cyto FOX activity was similarly responsive to dietary copper levels. A previously developed feeding regimen was used in which weanling rats

PAGE 78

78 were fed a low copper diet for 38 d (110) As expected, rats had significant reductions in Hb and Hct levels indicative of severe copper deficiency (Table 4 1 ). Moreover, serum Cp levels were reduced dramatically (Fig. 4 4A), and enterocyte At p7a expression was increased significantly (Fig. 4 4B), consistent with previous observations made in CuD mice (73) More importantly, cyto FOX activity was not different in the normal controls compared with the an emic CuD rats (Fig. 4 4C). Analysis of c yto FOX a ctivity in m utant m ouse m odels To further consider possible contributions of Heph or Cp to cyto FOX activity, studies were performed in sla Heph /y (i.e., Heph KO), and Cp KO mice. Immunoblot analysis sho wed that Heph protein was not detectable in cytosolic fractions of enterocytes isolated from sla mice (Fig. 4 5A) and that Cp protein was not detected in cytosolic fractions from enterocytes of any mice studied but was present in mouse serum (Fig. 4 5B). Moreover, Heph KO mice contained no immunoreactive Heph protein using two antibodies (one, D4, provided by Dr. Chris Vulpe, University of California, Berkeley, CA and one obtained from GeneTex), whereas WT controls had a strong immunoreactive Heph band. L ast, cyto FOX assays demonstrated very similar enzyme activity in sla and mice compared with WT mice (Fig. 4 5 C and D). Although cyto FOX activity was modestly reduced in Heph KO mice, the difference did not reach statistical significance (Fig. 4 6A). This observation was confirmed when FOX activity by ferrozine assay also showed no significant differences between WT controls and Heph KO mice (Fig. 4 6B). Discussion This study considered the possibility that additional, unidentified FOXs exist in mammalian enterocytes. In this regard, two important questions are worth considering:

PAGE 79

79 (i) Is a FOX absolutely required for intestinal iron absorption, and, if so, (ii) is Heph adequate? As to the first question, the predominance of evidence suggests that e nzymatic oxidation of iron is required to provide physiologic levels of iron for normal homeostasis. The chemical oxidation of Fe 2+ to Fe 3+ by O 2 is a complex process, influenced by pH, ionic strength, and temperature, involving many hard to characterize/ predict, metastable intermediates, and finally yielding not one but a variety of iron oxides (91) Even at pH 7.35 (in contrast to pH 5.0 that was used here), under air saturation, the enzymatic rate is four times as fast as the nonenzymatic rate (100) Most important, comparing the estimated rates of Fe 3+ Tf formation in human sera at pH 7.35 7 10% of th e former) routes, Osaki et al. (101) concluded that the chemical route is inadequate to supply sufficient iron for synthesis of biomolecules such as Hb. Furthe rmore, informative comparisons can be made with another redox active metal, copper, which traverses enterocytes as Cu+ but does not require an oxidation step for release from the intestinal epithelium and transport as Cu 2+ in the serum. Extrapolating the a pparent adequacy of chemical oxidation for Cu + to Cu 2+ to iron reveals distinct differences, because the standard reduction potential at pH 7.0 for Fe 3+ + e to Fe 2+ is 0.77 V, whereas that for Cu 2+ + e to Cu + is 0.15 V (19% of the former), indicating a greater propensity for copper to be oxidized spontaneously. Enzymatic oxidation of dietary iron thus appears to be critical for maintaining normal iron homeo stasis. As to whether Heph alone is adequate to mediate iron absorption, the mutation and mislocalization of Heph in sla mice does not result in a null phenotype for iron absorption, because only a mild/moderate iron deficiency is observed (53) (Table 3 1 ).

PAGE 80

80 Further, as a catalyst, the sole function of an enzyme is to help the reaction attain equilibrium much faster than via the uncatalyzed route, provided that Gibbs free energy cat of 2.0 estimated for Heph (54) is tantamount to an uncatalyz ed reaction, whereas many other enzymes have values three to seven orders of magnitude higher. For example, catalase, belonging to the same enzyme group, has a K cat of 100,000 (81) These facts raise questions about the adequacy of Heph as the sole enterocyte FOX to mobilize needed amounts of iron, in contrast to the proposal by Hellman and Gitlin (61) Two fortuitous alterations to previously used experimental approaches aided us in identifying an undiscovered cytosolic FOX activity in rat enterocytes: (i) Enterocyte lysates were prepared initially in the absence of detergent, and relatively pure cytosolic/soluble and membrane/particulate fractions ultimately were obtained; and (ii) a spectrophotometric, Tf coupled FOX assay was used, allowing more precise and sensitive measurement of enzyme activity. Initially it wa s considered that this cyto FOX activity could be the result of contamination of cytosolic fractions with membrane Heph or a method induced artifact. Several notable facts argue against both these possibilities: (i) Cytosolic fractions have robust activity of a commonly used marker enzyme; (ii) immunoblotting for two membrane proteins revealed little if any immunoreactive protein in cytosolic fractions; (iii) cytosolic fractions prepared using three distinct methods, two of which eliminate the grinding step all showed similar cyto FOX activity; (iv) cyto FOX activity is present in severely CuD rats, a physiologic state in which Heph expression and activity have been shown to decrease dramatically; and (v) cyto FOX activity is not different when comparing He ph mutant ( sla ) or Heph KO mice

PAGE 81

81 with WT controls. Thus it is unlikely that cyto FOX activity can be fully explained by Heph, although a possible contribution by a soluble/alternative form of Heph cannot be ruled out completely. Another possibility was that cyto FOX could be Cp, because Cp was detected in mouse enterocytes and in the lamina propria of mouse villi (21) To consider this alternative, a well established Cp antibody was used to probe Western blots of cyto solic proteins purified from isolated mouse enterocytes. Immunoreactive Cp was never detected in cytosolic fractions, but it was detected consistently in mouse serum. In summary, enzymatic oxidation of iron appears essential, and Heph alone may not be adeq uate. The current investigation provides multiple lines of evidence supporting the existence of a unique, soluble FOX in rodent enterocytes. Of significance is the fact that this cyto FOX activity increased during iron deficiency, whereas membrane FOX acti vity did not. Furthermore, the lack of a significant reduction in cyto FOX activity in CuD rats and the presence of cyto FOX activity in sla Heph /y and Cp KO mice, suggest that a previously undescribed or Heph unrelated protein mediates this activity. T his newly discovered FOX may have an important physiologic role in intestinal iron absorption, perhaps complementing the function of membrane bound Heph. Further I nvestigation of the Cytosolic/Soluble Ferroxidase As described in the previous section and C hapter 5, preliminary investigations in rats and knockout mice indicated that in enterocyte cytosol, the soluble version of Heph Studies d escribed in this section were performed at QIMR by the author of this dissertation with minimal technical assistance from Brie Fuqua.

PAGE 82

82 exist s F urthermore, strong evidence suggested that there is another ferroxidase, i.e. cytoFOX. To date there are a number of soluble proteins that are shown to have ferroxidase activity: cytosolic/soluble hephaestin, H ferritin soluble Cp, beta am y loid precursor protein (App), Zyklopen (Zp) and the novel cytoFOX. At QIMR, with the availability of various knockout mouse models, many of these candidates were studied. Cytosolic/soluble Heph Further studies on cytosolic/soluble Heph are beyond the scope of my current dissertation H Ferritin (H Ft) Whether H ferritin contributes to cytosolic FOX activity was investigated in wt mice fed control or iron overload diets (1% carbonyl iron). Dietary and experimental details are described in C hapter 2. Western blot analysis of cytosol F t confirmed the effectiveness of the dietary treatments (Figure 3 6 ). In the present experiment, Heph pro tein expression in enterocytes decrease d in cytosol and membrane fractions from FeE mice ( c ytosol: Ctrl 1.00 0.09 vs. FeE 0.19 0.15; membrane Ctrl 1.00 0.15 vs. FeE 0.22 0.05 in relative densitometric units p <0.05 ) (Figure 4 7, panel A and C ). Tf assay revealed that FeE mice had lower cytosolic FOX activity (Figure 4 8, panel B) ; however, enterocyte membrane fractions from both FeD and FeE groups had significantly lower activity than mice from the Ctrl group (Figure 4 8, panel C and D ). To determi ne whether H ferritin contributed to FOX activity, one known aspect of F t was taken advantage of namely heat stability. Ferritin is a very stable protein, as mentioned in previous studies by Tosha et al. and Kim et al. (74, 133) Heating the protein homogenate at 65 C for 15 min can denature and precipitate soluble non ferritin

PAGE 83

83 proteins. An earlier study showed that 90% of recombinant F t can be recovered after incubation at 75 C for 10 min (82) Thus, the author h ypothesized that if the cytosol samples are heated moderately, ferritin will be present in the supernatant and remain active while most other soluble proteins should be denatured and inactive. First, 65 C heat treatment was carried out for 15 min in a sha king heat block. Then, the heat ed samples were spun at 16,000 g for 10 min to remove precipitates (denatured proteins ) Equal volumes of supernatants from heated cytosol and untreated cytosol samples were then subjected to Tf assay. Results showed that all heated samples lost their FOX activity in Tf assay (dA 460 /dt=0 at all time points ; data not shown). Then, a lower temperature (i.e. 55 C) was utilized. Again, n one of the heated samples showed any FOX activity in Tf assays (Figure 4 9 ). Thus, the author conclude d from these experiments that H FT is not likely contributing to cytosolic FOX activity. Ceruloplasmin ( Cp ) Soluble Cp is the circulating multicopper ferroxidase in serum. One study using anti Cp antibody showed that Cp is expressed in wt mice duo denum after p hlebotomy (21) However, the well characterized Sigma anti Cp Ab could not detect any sign al in rat or mouse enterocyte cytosol ( Figure 4 5, panel B ). At QIMR, more Cp / mice were studied and the resul ts agreed with previous experiments in that cytosolic Tf activity did not differ between wt and Cp / mice. Hematological analysis indicated that the Cp knockout mice had mild microcytic anemia, as evidenced by lower Hb, Hct and MCV ( Table 4 2). These data are in accordance with a previous publication (22) Cp knockout was c onfirmed by serum Western Blot using the Sigma anti Cp anti b ody (Figure 4 10 ). S erum p PD assay detected no activity in Cp / mice, demonstrating that p PD oxidase activity is reflecting

PAGE 84

84 Cp activity (Figure 4 11 ). However, Fz assay showed no difference betwe en wt and Cp / mice (Figure 4 12). Given the fact that Fz assay measures FOX activity, this indicated that there should be at least one more FOX exist ing in serum. Studies by Gray et al. in wt and Cp / mice showed similar results by using p PD assay and E the substrate is Ferene S a colorimetric compound that is similar to Ferrozine ; binds to Fe 2+ ) (52) In that study, they also utilized two other FOX activity assays (Minotti/Ikeda Saito assay and Tf as say), and in these three assays plasma FOX activities in Cp / mice were not eliminated. This brings up the long time debate of the existence of FOX II in the serum. Back in 1970, Topham and Frieden had identified and purified FOX II patients (132) Other studies suggested that FOX II could be a peroxidized lipoprotein (46, 47) However, the true identity of FOX II is beyond the scope of the current dissertation. In inhibition studies, t wo con centrations of NaN 3 lowered p PD oxidase activity in wt mice to a similar extent, but no effect was noted in Cp / mice (Figure 4 1 3 ). The copper chelator, D penicillamine (DP), also decreased Cp activity in wt and Cp / mice in p PD assys (Figure 4 1 4 ). The results from both p PD and Fz assay suggested that p PD assay would be a better choice for measuring Cp activity. Cytosol and membrane Tf assay showed that there were no differences between wt and Cp / mice (Figure 4 1 5 ). This data is consistent with our previous ly published observations (108) Thus, the possibility that Cp could be the novel cyt osolic/soluble FOX is ruled out, but the issue regarding another serum FOX is not resolved Amyloid Precursor Protein ( Ap p ) In a personal communication with Dr. Ashley I. Bush from the Melbourne Brain Institute (Melbourne, Victoria, Australia), I learned that results from his lab strongly

PAGE 85

85 suggested that App is the other F OX that exists in serum ( i.e., FOX II ), contributing ~ 40% of total activity. Obviously, results the author obtained at QIMR from Cp / mice by p PD assay (described in the previous section Figure 4 11 ) do not support this idea, given the fact that App is also an amine oxidase (33) A peptide that contained the App putative iron binding motif was synthesized by Ebrahimi et al. and was then used in Tf assay to study its FOX activi ty. Surprisingly, this synthetic peptide did not exhibit FOX activity (37) as in a previous study (35) Thus, the statement that App has FOX ac tivity is open to question. However, in order to determine whether App could be the novel cytoFOX App / and wt mice were studied Two groups of adult, male App / mice were used; their hematological parameters were similar to wt mice regardless of which diet they were fed ( standard rodent diet or iron deficient diet) ( Table s 4 3 and 4 4 ). Serum p PD and Fz assay showed no differences between wt and App / mice ( FeD diet: Figures 4 1 6; s tandard rodent diet: Figure 4 19 ). Pre incubation with DP abolished the activities from all mice in both assays ( FeD diet: Figures 4 17 and 4 1 8; s tandard rodent diet: Figure s 4 20 and 4 21) This is consistent with the fact that Cp is the major ferroxidase in the serum. Thus, our data do not support the concept that App is F OX II and contributes to serum ferroxidase activity. As to its potential role in contributing to enterocyte FOX activity, W estern blot showed no difference in Heph protein expression between wt and App / mice (data not shown). Cytosolic Tf assay similarl y showed that no differences were detected between wt and App / mice, regardless of which diet they were fed (Figures 4 2 2 panel A and 4

PAGE 86

86 2 3 ). App / mice Thus, it is very unlikely that App w ould be the cytoFOX. Atp7b As a multi copper ferroxidase, Hephaestin needs to obtain copper by some mechanism. Previously, Weiss et al. (142) reported that the copper transporting ATPase, A tp7b was expressed in int estine, and it was found to be located on the Golgi apparatus. Thus, if ATP7B is the protein loading copper onto Heph, knockout of this protein will likely to have an impact on Heph stability, expression and/or activity and possibly iron absorption Two g roups of Atp7b / were studied. In total, the wt group had 6 male and 4 females, and the Atp7b / group had 5 males and 6 females. Their ages were from 11.2 to 18.5 weeks. All of the Atp7b / mice exhibited enlarged spleens (length in mm: wt 15.60 0.65, Atp7b / 25.33 2.42, n= 5 and 6, respectively, p< 0.0001) and yellowish serum. These phenotypes have not been reported before. The hematological parameters of the mice indicated that they have macrocytic anemia as indicated by deceased Hb Hct and eleva ted MCV ( Table 4 5 ). In both groups of wt and Atp7b / mice, Heph protein expression did not change in cytosol or membrane fractions of knockout mice (Figure 4 24 ). All Atp7b / mice also had similar cytosolic and membrane FOX activities as compared with w t mice (Figure 4 2 5 ). Thus, it is unlikely that ATP7B i s delivering copper to Heph in enterocytes. Heph aestin (Heph) In our p revious publication (108) only four Heph /y mice were used and the differences between wt and Heph /y mice were minimized by inter animal variation. Thus, the same experiment was conducted at QIMR using a larger number of Heph

PAGE 87

87 global knockout mice ( Heph /y ) and Heph intestinal specific knockout ( Hep h int/int ) mice. Previous investigations by Ch en et al. indicated that mice at different ages have different responses (15) Thus, two age groups ( group 1, older 10.4 to 14.9 weeks vs. group 2, younger, 5.5 to 7 weeks ) of Heph /y mice were studied. Heph /y g ro up 1 For the first group 7 wt males and 7 Heph /y males were used being between 10.4 to 14.9 weeks of age. Their hematological parameters showed that they have normal Hb and Hct, increased RBC number, decreased MCV, and decreased MCH (Table 4 6). Smaller RBC, normal Hb and Hct indicated that their erythropoiesis function was back to normal to produce more newly synthesized RBC which demonstrated the recover y process from anemia The knockout of the Heph gene was confirmed by the absence of Heph protein in enterocyte cytosol and membrane fractions by Western Blot using Heph D4 antibody (Figure 4 26 ). As indicated in Figure 4 27 in Heph /y mice, cytosolic FOX activity was significantly lower than in wt mice, suggesting that the difference was due to the sol uble, cytosolic form of Hephaestin (details described in Chapter 5). Surprisingly, there were no differences in FOX activity in membrane fractions when comparing wt and Heph /y mice. As far as we know, Heph should be the only FOX on the enterocyte membrane This unexpected result raised questions about the validity of the membrane Tf assay. There might be something else in the membrane fraction that exhibit s oxidase activity. One possibility is lipid peroxide molecules from the membrane fraction. Lipids are prone to oxidative degradation, which might oxidize Fe 2+ from the Tf assay reaction. Thus, heat reading for Tf assay.

PAGE 88

88 Portions of membrane fractions from wt mouse enterocytes were heated at 80 C for 15 min, and then the sample was spun at 16,000 g for 30 min to remove precipitate s. The supernatant was subjected to Tf assay according to the usual procedure Figure 4 28 illustrated the reaction rates for both samples. This indicated that the activit y show n in membrane fractions is mediated by proteins, not lipids. Another possibility is the inhibi tory effect of detergent on the FOX protein activity. In order to solublize membrane, detergent is required for micelle formation. As show n previously, Trit on X 100 (Tx 100) inhibits cytosolic FOX activity (Figure 4 3). The experiment was repeated on enterocyte total lysate ; two different concentrations of Tx 100 we re added into the lysate and FOX activity w as measured. Results indicated that Tx 100 indeed in hibits the activity in a dose dependent fashion (Figure 4 29 ). Heph /y g roup 2 Eight WT males and 10 Heph /y males were studied and their ages varied from 5.5 to 7 weeks. In these mice, the anemia was more pronounced than in the older group. Hb, Hct, MCV and MCH were significantly lower than in wt mice, indicating that they have mild microcytic anemia ( Table 4 7 ). Considering the role of Heph in iron absorption, this is most likely due to an insufficient supply of iron for erythropoiesis. In these groups cytosolic FOX activity was decreased in Heph /y mice, but the differences did not reach significance (Figure 4 30 panel A) Enterocyte membrane FOX activity did not change (Figure 4 30 panel B ). The hematological status of both groups of mice are consist ent with unpublished observations by Brie Fuqua in a greater number of mice at similar ages (>100; personal communication). In both older and younger mice, W estern blot analysis indicated that enterocyte FT expression was increased in the cytosol of Heph / y mice, which agrees with the hypothesis that Heph plays a role in iron export

PAGE 89

89 from enterocytes (wt 1.00 0.41 vs. Heph /y 3.96 2.09 in relative densitometric units p <0.001 ) (Figure 4 3 1 ). This observation is in agreement with earlier findings by Brie Fuqua that in the duodenal segments of Heph /y mice, more blue staining was seen in unpublished observation via personal communication). Heph int/int Six male wt and 6 male Heph int/int mice were used at the age of 10.1 to 15 weeks. Preliminary studies by Brie Fuqua showed that occasionally the Heph int/int knockout in enterocytes may not be 100% and residual activity may contribute to recovery of hematological parameters (unpublished obse rvation via personal communication). Results from this group of mice indicated that even though the western blot clearly showed that the intestinal specific knockout of the He ph gene was effective (Figure 4 3 2 ), they have differences in iron status, as ind icated by the large variation of hematological parameters ( Table 4 8 ). Cytosolic Tf assay showed that Heph int/int mice had lower activity, but the differences did not reach statistical significance at most time points (Figure 4 3 3 panel A) Heph int/int mi ce also had significantly lower membrane FOX activity (Figure 4 3 3 panel B ). Surprisingly cytosolic FT western blot showed that the Heph int/int mice did not load iron in enterocytes ( Heph Flox : 1.00 0.61 vs. Heph int/int : 1.25 0.33 in relative densi tometric units ) (Figure 4 3 4 ). Heph /y / Cp / mice Four male adult Heph /y / Cp / mice aged from 21 to 28 weeks were utilized. One obvious phenotype for them is that they were extremely pale. Their hematological data showed extremely low parameters and thes e indicated that they were suffering from severe microcytic anemia ( Table 4 9 ). The Cp knockout was confirmed by serum

PAGE 90

90 W estern blot and Heph knockout was confirmed by W estern blot for cytosolic and memb rane fractions (Figure s 4 3 5 and 4 3 6 ). As compared wi th Heph /y and Cp / mice, the Heph /y / Cp / mice had a more severe anemia phenotype. However, cytosolic Tf assay indicated that there were no differences between wt and Heph /y / Cp / while membrane FOX activity showed a non significant decrease (Figure 4 3 7 ). Cytosolic FT protein expression was greatly increased in the Heph /y / Cp / mice ( wt 1.00 0.25 vs. Heph /y / Cp / 3.68 0.86) (Figure 4 3 8 ). In a personal communication with Brie Fuqua on the absorption and distribution of iron in young mice fed wit h iron deficient diet, her results suggested that, despite the lack of Heph and Cp, these mice could still absorb iron (unpublished observations). The percentage of total 59 Fe dose retained in their body after 5 days of dosing is about twice the dose of wt The doses retained in GI tract and liver are significantly higher than wt mice. These data suggested that, knockout of Heph and Cp severe impaired iron utilization but the absorption step was unaffected and even up regulated. W hen mice are severely iron deficient, cytoFOX could compensate for the la ck of cytosol and membrane Heph and circulating Cp, perhaps allowing the mice to live Conclusions In summary, studies accomplished at QIMR further demonstrated that cytosolic FOX activity cannot be fully exp lained by Hephaestin and the results validated the conclusion from a previous publication (108) in that a novel cytosolic/soluble ferroxidase (cytoFOX) exists in rodent enterocytes. Dietary treatment with iron overl oad diet excludes the involvement of H ferritin; mutant mouse models ruled out possible contributions by Ceruloplasmin and amyloid precursor protein. Current evidence

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91 suggested that this cytoFOX could com pensate for circulating Cp, and both cytosol and membrane Heph, thus allow ing the mice to survive.

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92 Figures and Tables Figure 4 1 Ferroxid ase activity in rat enterocyte fractions Relative catalytic rate (dA 460 /dt) is shown at 1, 2 and 3 minute time points. A) and B) Mean SD of data from 12 individual rats in three separate experiments is shown. ** p <0.01, p <0.05. Ctrl control rats; FeD iron deficient rats. (The raw data from this figure courtesy of Dr. Ranganathan)

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93 Figure 4 2 Enterocyte purity and alternative methods. A ) Lactate dehydrogenase (LDH) activity assays. Catalytic rate at 75 sec is shown re flecting disappearance of the substrate pyruvate. Lysate and Cytosol are samples purified from rat enterocytes (n=3). 0.05 U, 0.1 U and 0.2 U indicate units of purified rabbit muscle LDH used as a positive control. **p<0.01 as compared to Lysate. B ) Wester n blot of 4 rat cytosol and 4 membrane samples reacted with anti ZnT1 antibody. Bottom row: Ponceau S stained blot showing comparable loading and transfer of proteins. The black line indicates where unrelated lanes were removed from the images. C ) and D ) P rogress curves from Tf coupled FOX assay of enterocyte cytosolic and membrane fractions prepared using alternative methods. Each symbol is the mean of two individual rats. In panels A, C & D, blanks are reaction mixes devoid of enzyme source (i.e. protein sample). (Raw data for Panel A, C, D of this figure courtesy of Dr. Ranganathan)

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94 Figure 4 3 Chemical properties of cytosolic FOX. A ) Effect of protein denaturants on enzyme activity. Data shown are reaction rates at 30 se c from 4 rats per treatment. ( ), no treatment. B ) Effect of 1 .5% Triton X 100 (TX) on cytosolic FOX activity Progress curves are shown for blank and cytosol samples with and without the addition of detergent. n=4 individual rats. p <0.05. Both panels: Me an SD is shown; *** p <0.0001 as compared to ( ). (The raw data from this figure courtesy of Dr. Ranganathan)

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95 Figure 4 4 Effect of copper deficiency on cytosolic FOX activity A) Western blot of Cp protein in rat serum. B) Western blot of Atp7a protein in duodenal enterocyte samples derived from experimental rats. 54 10 indicates the particular antiserum used. A) and B) Bottom row: The Ponceau S stained blots are shown below the immunoblots to exemplify equal loading and transfer of protein C) Cytosolic FOX activity assays in samples derived from experimental rats. Enzymatic rate (dA 460 /dt) is shown at various time points. Mean SD is shown. n=5. A) to C): Ctrl control rats, CuD copper deficient rats. (The raw data fro m Panel C of this figure courtesy of Dr. Ranganathan)

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96 Figure 4 5 FOX activity in mutant mouse models Wild type (WT) mice, sla mice (Heph sla/y ) and Cp knockouts ( Cp / ) were studied. A) Western blot for cytosolic Heph in three wt (+/y) and 3 mutant ( sla commercial source of the antibody. B) Western blot for Cp in 3 wt, 3 sla /y and 2 Cp / mice. MW, molecular weight markers. In A & B, the Ponceau S stained blots are shown below the immunob lots to exemplify equal loading and transfer of protein. Black lines indicate where unrelated lanes were removed from the images. C) and D) FOX activity assays are shown as mean SD. Enzymatic rate (dA 460 /dt) is shown at various time points. n = 3 for wt 6 for sla /y and 3 for Cp / (The raw data from Panel C & D of this figure courtesy of Dr. Ranganathan)

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97 Figure 4 6 Heph protein expression and FOX activity in enterocyte cytosol from Heph KO mice FOX activity was measured using two spectrophotometric methods, A) initial velocity Tf assay and B) end point F errozine (Fz) assay. Enzymatic rate (dA/dt) is shown at different time points for Tf assay, while A 570 progress curves are shown for Fz assay. Note that Fz assay is a su bstrate disappearance assay, in which lower numbers indicate higher enzyme activity. No statistical differences were noted between groups in either assay. n=4 for wt and 4 for Heph / y mice. C) Heph protein expression in both cytosol and membrane fractions of Heph /y and WT mice by D4 Ab. Ponceau S stained blots are shown below the immunoblots to exemplify equal loading and transfer of protein. (The raw data from panel A & B of this figure courtesy of Dr. Ranganathan)

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98 Figure 4 7 Hephaestin protein expression in enterocyte cytosol and membrane fractions from mice on different diets. A) Western blot analysis of enterocyte cytosol samples reacted with anti Heph antibody (called D4); Ponceau S stained blot to show comparable p rotein loading and transfer. B) Densitometry analysis of the film and blot in panel A. C) Western blot analysis of enterocyte membrane samples reacted with anti Heph antibody; Ponceau S stained blot to show comparable protein loading and transfer. D) Densi tometry analysis of the film and blot in panel C. B) and D) Different letters indicate statistical differences between groups (1 way ANOVA followed by Tukey multiple comparison test; p <0 .05). Results are expressed as mean SD.

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99 Figure 4 8 Cytosol and membrane ferroxidase (FOX) activity by Tf assay. Panel A) to D) showed FOX activity in A) enterocyte cytosol from Ctrl and FeD mice B) enterocyte cytosol from Ctrl and FeE mice C) enterocyte membrane from Ctrl and FeD mice D) enterocyte membrane from Ctrl and FeE mice. P <0.05, ** p <0.01 two tailed, two test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=4 for C trl, 4 f or FeD, and 5 for FeE

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100 Figure 4 9 Cytosol ferroxidase activity after incubat ion at room temperature or after heat treatment Cytosol samples were heated at 55 C for 20 min and then spun down to remove all precipitates T he resulting supernatants were subjected to Tf assay. Results are expressed as mean SD. N=4 for C trl, 4 for FeD, and 5 for FeE Figure 4 10 Serum Cp protein expression in Cp / and wt mice. Western blot of mouse serum s amples reacted with anti Cp antibody. Ponceau S stained blot showed comparable protein loading and transfer.

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101 Figure 4 11 Serum p PD assay for wt and Cp / mice. ** p <0.01, *** p <0.001, two tailed, two sample unequal varianc t test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=9 for wt and 8 for Cp / mice ; sera were pooled to 5 samples for analysis. Figure 4 12 Serum Fz assay for wt and Cp / mice. p <0.05, ** p <0.01, two tailed, two t test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=9 for wt and 8 for Cp / mice, sera were pooled to 5 samples for analysis.

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102 Figure 4 13 NaN 3 inhibition of serum p PD oxidase activity from wt and Cp / mice at 2 mM or 10 mM. Results are expressed as mean SD. N=8 for each group. Figure 4 14 DP inhibition of serum p PD oxidase activity from wt and Cp / mice. A) Serum p PD assay of wt mouse serum without and with DP. B) Serum p PD assay of Cp / mouse serum without and with DP. p <0.05, ** p <0.01, *** p <0.001, two tailed, two t test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=4 for wt, 3 for Cp /

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103 Figure 4 15 Cytosol and memb rane ferroxidase activity by Tf assay in wt and Cp / mice A) FOX activity of enterocyte cytosol from wt and Cp / mice. B) FOX activity of enterocyte membrane from wt and Cp / mice. Results are expressed as mean SD. N=3 for each group. Figure 4 16 Seru m p PD and Fz assays for wt and App / mice fed an FeD diet. A) Serum p PD assay. n=7 for each group. B) Serum Fz assay, n=5 for wt, 6 for App / Results are expressed as mean SD.

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104 Figure 4 17 Serum p PD assay with DP inhibition in wt and App / mice fed a n FeD diet. A) Serum p PD assay of wt mouse serum without and with DP. B) Serum p PD assay of App / mouse serum without and with DP. ** P <0.01, *** p <0.001, two tailed, two sample unequal t test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=5 for each group. Figure 4 18 Serum Fz assay with DP inhibition in wt and App / mice fed a n FeD diet. A) Serum Fz assay of wt mouse serum without and with DP. B) Serum Fz assay of App / mouse serum without and with DP. P <0.05, ** p <0.01, *** p <0.001, two tailed, two t test was utilized to compar e the values between two groups at the same time point Results are expressed as mean SD. N=5 for wt, 6 for App /

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105 Figure 4 19 Serum p PD and Fz assays for wt and App / mice fed a standard rodent diet A) Serum p PD a ssay. n=3 for each group. B) Serum Fz assay, n=3 for each group. Results are expressed as mean SD. Figure 4 20 Serum p PD assay with DP inhibition in wt and App / mice fed a standard rodent diet A) Serum p PD assay of w t mouse serum without and with DP. B) Serum p PD assay of App / mouse serum without and with DP. p <0.05, ** p <0.01, *** p <0.001, two tailed, two t test was utilized to compare the values between two groups at the same ti me point Results are expressed as mean SD. N=3 for each group.

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106 Figure 4 21 Serum Fz assay with DP inhibition in wt and App / mice fed a standard rodent diet A) Serum Fz assay of wt mouse serum without and with DP. B ) Serum Fz assay of App / mouse serum without and with DP. *** P <0.001, two tailed, two t test was utilized to compare the values between two groups at the same time point Results are expressed as mean SD. N=3 for each group. Figure 4 22 Enterocyte cytosolic and membrane FOX activity in wt and App / mice fed an FeD diet. A) FOX activity of enterocyte cytosol from wt and App / mice. B) FOX activity of enterocyte membrane from wt and A pp / mice. Results are expressed as mean SD. N=7 for each group.

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107 Figure 4 23 Cytosolic FOX activity in wt and App / mice fed a standard rodent diet Results are expressed as mean SD. N=4 for each group. Figure 4 24 Hephaestin protein expression in enterocyte cytosol and membrane fractions of wt and Atp7b / m ouse enterocytes. A) A representative Western blot of enterocyte cytosol samples reacted with anti Heph antibody; Ponceau S sta ined blot to show comparable protein loading and transfer. B) Densitometry analysis of the film and blot in panel A. C) A representative Western blot of enterocyte membrane samples reacted with anti Heph antibody; Ponceau S stained blot to show comparable protein loading and transfer. D) Densitometry analysis of the film and blot in panel C. B) and D) Results are expressed as mean SD. N=8~10 for each group, from two independent experiments.

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108 Figure 4 25 Enterocyte cytosol and membrane FOX activity from wt and Atp7b / mice by Tf assay. A) FOX activity of cytosol from wt and A tp7b / mice. B) FOX activity of enterocyte membrane from wt and A tp7b / mice. Results are expressed as mean SD. N=9 for wt, 10 for Atp7b / each g roup, combined from two independent experiments. Figure 4 26 Western blot analysis of Heph protein expression in enterocyte cytosol and membrane fractions from wt and Hep h /y mice. A) Western blot analysis of enterocyte c ytosol samples reacted with anti Heph antibody. B) Western blot analysis of enterocyte membrane samples reacted with anti Heph antibody. Ponceau S stained blot to show comparable protein loading and transfer.

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109 Figure 4 27 C ytosol and membrane enterocyte FOX activity in wt and Hep h /y mice. A) FOX activity of cytosol from wt and Hep h /y mice. B) FOX activity of enterocyte membrane from wt and Hep h /y mice. ** p<0.01, *** p <0.001, two tailed, two sample unequal variance Stude t test was utilized to compare the values between two groups at the same time point Resul ts are expressed as mean SD. N=7 for each group. Figure 4 28 Membrane Tf assay of native or heated samples. One time experim ent.

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110 Figure 4 29 FOX activity in enterocyte lysate with two different concentrations of Triton X 100 (Tx 100). DA 460 /dt for lysate at each time point was set as 1. Figure 4 30 Enterocyte cytosol and membrane FOX activities by Tf assay. A) FOX activity of cytosol from wt and Hep h /y mice. B) FOX activity of membrane from wt and Hep h /y mice. Results are expressed as mean SD. Enterocytes from 8 wt mice and 10 Hep h /y mice were pooled to 4 samples for each group for analysis.

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111 Figure 4 31 Ferritin protein expression in the enterocyte cytosol from Hep h /y and wt mice. A) A representative Western blot of enterocyte cytosol samples reacted with anti Ft antibody Ponceau S stained blot to show comparable protein loading and transfer. B) Densitometry analysis of Ft expression in 2 groups of mice. Results are expressed as mean SD. *** P t test. N=9 for wt mice, 10 for Hep h /y mice. Figure 4 32 Heph protein expression in enterocyte cytosol and membrane fractions of Heph Flox and Heph int/int mice. A) Western blot of cytosol samples reacted with anti Heph antibody. B) Western blot of membrane samples reacted with ant i Heph antibody. Ponceau S stained blot in A) and B) to show comparable protein loading and transfer.

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112 Figure 4 33 Enterocyte cytosol and membrane FOX activities from Heph Flox and Heph int/int mice by Tf assay. A) FOX act ivity of cytosol from wt and Heph int/int mice. N=4 for Heph Flox and 6 for Heph int/int mice B) FOX activity of membrane from wt and Heph int/int mice. N=5 for Heph Flox and 6 for Heph int/int mice Results are expressed as mean SD. P <0.05, two tailed, two sample unequal t test was utilized to compare the values between two groups at the same time point Figure 4 34 Ferritin protein expression in enterocyte cytosol from Heph Flox and Heph int/int mi ce. A) Western blot of cytosol samples reacted with anti Ft antibody. Ponceau S stained blot to show comparable protein loading and transfer. B) Densitometry analysis of Ft expression in panel A. Results are expressed as mean SD. N=6 for each group.

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113 Figure 4 35 Western blot analysis of serum Cp protein expression in wt and Heph /y /Cp / mice. Western blot of serum samples reacted with anti Cp antibody. Ponceau S stained blot to show comparable protein loading and transfer. F igure 4 36 Western blot analysis of Heph protein expression in enterocyte cytosol and membrane fractions of wt and Heph /y / Cp / mice. A) Western blot of cytosol samples reacted with anti Heph antibody. B) Western blot of cyt osol samples reacted with anti Heph antibody. Ponceau S stained blot of A) and B) to show comparable protein loading and transfer.

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114 Figure 4 37 Enterocyte cytosol and membrane FOX activities from wt and Heph /y / Cp / mice b y Tf assay. A) FOX activity of cytosol from wt and Heph /y / Cp / mice. B) FOX activity of membrane from wt and Heph /y / Cp / mice. N=5 for wt and 4 for Heph /y / Cp / Mean SD is shown. Figure 4 38 Ferritin protein expre ssion in enterocyte cytosol from wt and Heph /y / Cp / mice. A) Western blot of cytosol samples reacted with anti Ft antibody. Ponceau S stained blot to show comparable protein loading and transfer. B) Densitometry analysis of Ft expression in panel A. Resu lts are expressed as mean SD. *** P < 0.001 t test. N =4 for Heph /y / Cp / 5 for wt

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11 5 Table 4 1 Hematological parameters and mineral levels of experimental rodents a g/dL; b %; c, d ppm d wt mice used were the same genetic background (C57BL/6J) as Heph /y, s la and Cp / mice FeD iron deficient; CuD copper deficient; s la sex linked anemia; Cp ceruloplasmin Ctrl FeD p value Hemoglobin a 14. 82 1.77 (n=24) 3.92 1.18 (n=27) <0.0001 Hematocrit b 49.79 3.24 (n=24) 19.11 5.38 (n=27) <0.0001 Enterocyte Fe c 415.7 186.40 (n=20) 32.66 4.19 (n=18) <0.0001 Enterocyte Cu d 25.71 17.73 (n=18) 51.85 73.61 (n=18) 0.11 Ctrl CuD p Hemoglobin a 13.01 0.81 (n=5) 6.66 1.97 (n=5) <0.0001 Hematocrit b 44.36 2.56 (n=5) 24.54 7.33 (n=5) <0.0001 +/y d sla /y p p value Hemoglobin a 14.31 0.58 (n=7) 14.28 0.75 (n=6) 0.42 Hematocrit b 50.19 2.27 (n=7) 49.20 1.56 (n=6) 0.12 +/y d Heph /y p Hemoglobin a 14.31 0.58 (n=7) 13.49 0.30 (n=4) () (n=4)** < 0.01 Hematocrit b 50.19 2.27 (n=7) 46.98 1.68 (n=4) (n=4)** < 0.01 +/+ d Cp / p Hemoglobin a 14.31 0.58 (n=7) 10.59 0.85 (n=3) <0.0001 Hematocrit b 50.19 2.27 (n=7) 39.99 2.79 (n=3) < 0.0001

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116 Table 4 2 Hem atological parameters of wt and Cp / mice. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 9.41 0.42 148.18 5.19 0.46 0.018 49.33 0.99 15.90 0.17 322.36 5.43 Cp / 9.18 0.70 129.85 7.28*** 0.40 0.023*** 43.82 1.94*** 14.23 0.54*** 324.84 3.11 : *** P <0.001, Student t test. N=14 for wt, 11 for Cp /

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117 Table 4 3 Hematological parameter s of wt and App / mice fed FeD diet. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 10.29 0.94 134.88 7.28 0.42 0.064 41.01 2.73 13.03 1.13 317.25 7.44 App / 9.96 0.47 130.56 11.90 0.42 0.036 42.58 2.17 13.1 0.73 307.33 3.35 Results are expressed as mean S D. N= 9 for each group.

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118 Table 4 4 Hematological parameters of wt and App / mice fed standard rodent diet Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 9.53 0.43 150.00 7 .55 0.49 0.028 51.30 0.85 15.77 0.12 306.67 3.51 App / 8.03 2.18 128.17 33.52 0.41 0.11 51.37 0.65 15.97 0.21 311.50 5.68 Results are expressed as mean SD. N=3 for each group.

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119 Table 4 5 Hematologic al parameters of wt and Atp7b / mice. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 10.30 0.63 160.5 9.09 0.50 0.029 48.26 1.26 15.59 0.52 323.00 6.34 Atp7b / 6.33 0.65*** 101.34 11.68*** 0 .36 0.03*** 57.47 6.17*** 16.08 1.50 280.08 11.35*** : *** p< 0.0001, Student t test. Results are expressed as mean SD.

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120 Table 4 6 Hematological parameters of wt and Hep h /y mice in group 1. Groups/ Parameters RB C (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 9.60 0.53 153.57 5.68 0.48 0.016 50.53 1.83 16.03 0.34 317.14 6.23 Heph /y 10.75 0.43*** 142.29 14.91 0.46 0.03 42.76 2.85*** 13.23 1.35*** 309.14 12.63 : *** p< 0.0001, Student t test. Results are expressed as mean SD. N=7 for each group.

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121 Table 4 7 Hematological parameters of wt and Hep h /y mice in group 2. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 8.87 0.49 146.00 8.88 0.45 0.028 51.01 0.77 16.46 0.22 322.88 5.06 Heph /y 9.09 0.74 115.40 6.72*** 0.40 0.026*** 43.82 2.04*** 12.73 0.73*** 290.2 8.09*** : *** p< 0.0001, Student t test. Results are express ed as mean SD.

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122 Table 4 8 Hematological parameters of Heph Flox and Heph int/int mice. Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) Heph Flox 9.22 0.21 147.33 3.27 0.48 0.01 4 51.67 1.12 15.97 0.15 309.17 7.52 Heph int/int 9.61 1.23 136.17 17.29 0.44 0.060 45.85 2.54*** 14.20 0.78** 309.50 10.82 : ** p <0.01, *** p t test. Results are expressed as mean SD. N=6 for Heph Flox and 6 for Heph i nt/int

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123 Table 4 9 Hematological parameters for wt and Heph /y / Cp / mice Groups/ Parameters RBC (10 12 /L) Hb (g/L) Hct (L/L) MCV (fL) MCH (pg) MCHC (g/L) wt 9.24 0.20 147.40 2.07 0.46 0.010 50.08 1.36 15.96 0.23 318.80 7.85 Heph /y / Cp / 3.35 0.37*** 28.5 3.11*** 0.12 0.014*** 34.48 1.26*** 8.5 0.22*** 246.25 7.41*** : *** P <0.001. Results are expressed as mean SD. N=5 for wt mice and 4 for Heph /y / Cp / mice.

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124 CHAPTER 5 CYTOSOLIC HEPHA ESTIN Th e work in this chapter has been published and permission for reprint ing it has been obtained §§ Summary Discovered over a decade ago, hephaestin has been implicated as a ferroxidase vital for intestinal iron absorption. Stringent structural or kinet ic data derived from the purified, native protein is however lacking, leading to the hypothesis that an alternate, undiscovered form of hephaestin could exist in mammalian enterocytes. This possibility was tested using laboratory rodent and cell culture mo dels. Cytosolic and membrane fractions were obtained from rat enterocytes and purity of the fractions was assessed. Western blot analyses revealed hephaestin in cytosol obtained by three different methods, ruling out the possibility of a method induced art ifact being the major contributor to this observation. Absence of two different membrane proteins, ferroportin cytosolic samples tested by thin layer chromatography, elimin ated significant membrane contamination of cytosol. Further, immunohisto and immunocyto chemical analyses identified hephaestin in rat enterocytes and in two intestinal epithelial cell lines, IEC 6 and Caco 2, intracellularly. Additionally, cytosolic heph aestin increased upon iron deprivation but more important, decreased significantly upon copper deprivation, mimicking the response of membrane bound hephaestin. Moreover, ferroxidase activity §§ This research was originally published in Biometals Ranganathan PN, Lu Y ** Fuqua BK, Collins JF. Immunoreactive hepha estin and ferroxidase activity are present in the cytosolic fraction of rat enterocytes. Biometals 2012 Aug;25(4):687 95 Springer ** co first author.

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125 was present in rat cytosol, and was partly inhibited by anti hep haestin antibody. Finally, lack of immunodetectable ceruloplasmin by western blot precluded ceruloplasmin as an underlying cause of this activity. These data demonstrate that rat enterocytes contain a soluble/cytosolic form of hephaestin possibly contribut ing to the observed ferroxidase activity. Introduction Hephaestin (Heph) and ceruloplasmin (Cp) typify mammalian cupro enzymes deemed necessary for the oxidation of iron to facilitate intestinal iron absorption (Heph), and release from sites of storage inc luding macrophages of the reticuloendothelial system (Cp). Cp has been extensively investigated since its discovery (64) at biochemical, kinetic and structural levels including by X ray crystallography (8) Conversely, Heph, first identified as the mutant gene in the in the sex li nked anemia ( sla ) mouse (53, 138) has not been thoroughly investigated at structural or functional levels. With no unequivocal structural/biophysical evidence, the protein was established to be membrane inserted an d considered to be the vital ferroxidase (FOX) essential for Fe oxidation in the intestine (61, 77) Several important caveats to these assumptions should be considered: 1) wild type Heph was never purified to homog eneity from natural sources; 2) neither partial amino acid (AA) sequence nor an experimentally derived AA homology modeling compared to Cp (which is a soluble protein, deri ved from a different gene, synthesized at a different site, and found in different locations). This is further complicated because the AA sequence of Heph is a derived one (from cDNA), which is hypothetical; 4) western blot analyses were based on total cel l/tissue lysates, the major constituent of which is the cytosol; 5) the same ambiguity applies to FOX activity

PAGE 126

126 measurements since they were also done in total lysates; 6) elegant work (54) demonstrated FOX activity in a recombinant Heph devoid o f the membrane spanning segment (i.e. soluble). Such a lack of experimental structural/functional data on intestinal Heph raises questions about its site of function in enterocytes, particularly a ( Frazer et al 2001 ; Kuo et al. 2004 ) and in endosomes ( Kuo et al. 2004 ) of intestinal epi thelial cells. Previous studies determined that the cytosolic/soluble fraction of rat and mouse enterocytes contains a functional ferroxidase (108) Given the above delineated ambiguities regarding various aspects of Heph, the possibility that an alternative form of Heph could exist was considered. Experiments described in this manuscript indeed provide evidence for a cytosolic/soluble Heph expressed in rat enterocytes, possibly contributing to the noted FOX activit y in this cellular fraction. Results and discussion Heph Expression in Rat Intestinal Cytosol and Fraction Purity To eliminate chances of a method induced artifact being a major contributor to the observed outcome, at the outset, cytosolic and membrane portions were obtained from enterocytes lysed by three different methods, ie., grinding, hypotonic lysis & freeze thaw and results from two representative samples from each method are shown. The membrane fractions contained the expected immuno reactive He ph in all six samples (Fig. 1a, bottom row ). Interestingly, the cytosolic fraction also clearly revealed Heph in all six samples (Fig. 1a, top row ). As there are no published reports of a soluble/cytosolic Heph in rodent enterocytes, additional experiments were done to rule out membrane contamination of cytosol. Cytosolic nature was first ascertained by the

PAGE 127

127 presence of lactate dehydrogenase (LDH, marker enzyme) activity (108) Experiments performed to assess fraction purity by employing immunoblotting against a dilution series of a well documented membrane protein (Atp7a) spiked cytosol, revealed > 95% purity (Fig. 1b), while detecting strong bands in membrane, as expected. To reconfirm this data, western blot analysi s was repeated using antibody against a second membrane protein, ferroportin 1 (Fpn1) ; no Fpn1 was detected in either cytosol while it was present as expected in both membrane fractions, KNRK cell lysate serving as a positive control (Fig. 1c). Finally a s a third precautionary step, two representative cytosolic and membrane fractions were also analyzed by 1 dimensional TLC. As shown in Fig. 1d, both as a [ + ] control) while both cytoso ls were negative, further supporting the results obtained by im m unoblotting. Thus, the results from these four different experimental approaches not only validate the authenticity of the cytosolic fraction, but also remove any doubt that the observed locat ion of Heph could have resulted from either a method induced artifact or from contamination by membrane. The p resence of two versions of the same protein is not unique. For example, a GPI anchored form of Cp exists in mammalian astrocytes ( Patel and David 1997 ) and a truncated transferrin receptor has been described in sheep reticulocytes (3, 102) Detection of Heph by I mmunoflourescence Heph was detected by SDS based immunoblot analysis which is a denaturing technique, while protein recognition occurs in a native conformation in nature. Therefore, two common, relatively non denaturing (unlike SDS PAGE), in situ techniques, namely immunohistochemistry (IHC) and immunoc ytochemistry (ICC) were also performed using the same anti Heph (D4) antibody. In rat intestinal sections, a

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128 specific signal was detected in the basolateral membrane of enterocytes in addition to a nondescript staining pattern inside the cells (Fig. 2a). S econdary antibody alone did not lead to a noticeable signal (panel b), attesting to the specificity of the staining in panel A. Moreover, the antibody produced a similar, intracellular punctuate staining pattern in IEC 6 (panel c) as well as Caco 2 cells ( panel d). Similar results were seen by anti Heph (GeneTex) antibody (data not shown). Since (i) Heph was also detected in a native state and (ii) in all three diffe rent biological sources tested ( i e. rat intestine and rat and human intestine derived cell lines ) this set of data provides further support for the existence of a putative, soluble / intra cellular form of Heph. It is worth noting that Heph has been documented in a supranuclear location ( Frazer 2001 ; Kuo et al. 2004 ) and in mouse endosomes (44, 77) Heph Expression in Response to Iron and Copper Deficiency Since membrane Heph is known to respond differently when rodents are subjected to deprivation of dietary iron or copper (Chen et al. 2004), the next logical step was to test if this putative, soluble Heph responded likewise. Rats weaned onto the deficient diets had drastic reductions in serum hemoglobin an d hematocrit levels (>80% in the FeD group and >50% in the CuD group for both parameters) indicating the effectiveness of the dietary regime (data not shown). Denaturing western data from two representative samples using anti Heph (D4) antibody showed an i ncrease in Heph protein upon Fe withdrawal compared to control rats, similar to the membrane bound form (Fig. 3a). Notably, repeating the immunoblotting under native conditions yielded very similar results (Fig. 3b), indirectly supporting results shown in Fig. 3a as well as in Fig. 2. Further, being a multi Cu FOX, membrane Heph is known to decrease in low Cu conditions, presumably due to the requirement of Cu as the prosthetic group. As shown

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129 in Fig. 3c, a clear decrease in cytosolic Heph upon Cu withdrawa l was noted as compared to control rats, again mimicking the membrane bound form. To obtain further validity, the experiment was repeated with three representative samples using a different anti Heph antibody (GeneTex), which also yielded identical results in all three samples (Fig. 3d). Thus, results shown in Fig.3, representing samples obtained from four (for Ctrl & FeD) or ten (for Ctrl & CuD) individual rats and tested with two anti Heph antibodies, showing parallel response to withdrawal of two physiol ogically relevant metal ions, Fe and Cu, strengthens the link with membrane Heph. Ferroxidase ( FOX ) Activity in Rat Enterocyte Cytosol Since catalytic activity is the ultimate proof for an enzyme, FOX activity was tested in cytosol using apo transferrin in a coupled assay, with diferric transferrin being the end product Rat cytosol clearly exhibited FOX activity, as evidenced by the progress curve shown in Fig 4a. This enzymatic activity was reduced by >30% by anti Heph antibody (Fig. 4b), suggesting that at least part of this activity could be attributed to a soluble/cytosolic Heph or an immunologically related protein. These data raise two important points: 1) A cytosolic FOX would only be of physiologic relevance if its substrate (Fe 2+ ) was also present ; and 2) A soluble FOX could function within intracellular, iron containing membrane bound structures. With regard to the initial point, several lines of evidence support the presence of free iron in the cytosol, existing predominantly as Fe 2+ the substra te for FOX. First and most important, the cytosolic environment is reducing, favoring any iron present as Fe 2+ (63, 144) Millero (1985) has shown that Cl and CO 3 2 which are physiologic anions, retard oxidation. Moreover, a cytosolic pool of free iron has been proposed, constituting ~5% of the total (5) being buffered as glutathione complexed iron (Fe 2+ GS) due to

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130 cytoplasmic glutathione (GSH). Moreover, cytosolic electrode potential (E 0 the potential difference between the metal and the solution, in V) is considered to favor Fe 2+ o ver Fe 3+ (91) I n addition, ~80% of the iron pool in K562 ( human, myelogenous leukemia) cells is known to exist as F e 2+ (10) These facts delineate how an intracellular environment can support the existence of Fe 2+ the substrate for FOX. Although the cytosolic environment favors Fe 2+ oxidized iron could interact with other proteins /biological molecules that could stabilize it in the ferric form. Regarding the second point mention ed above cytosolic FOX is active at pH 5.0 which is consistent with the pH of acidified, intracellular vesicles. Interestingly, Heph has been localized in endosomes, presumably within the membrane (77) Ferroporti n 1 (Fpn1) the only intestinal iron exporter has also been observed within intracellular compartments (150) Cytosolic FOX could thus be located within Fe 2+ containing vesicles, perhaps participating in the proposed trans cytosis of dietary iron (83) Thus, data presented so far provide preliminary ratio nal, immunological and enzymatic support for a cytosol based FOX activity similar to membrane FOX activity, presumably contributed at least in part by a soluble form of Heph. Could Intestinal Ceruloplasmin ( Cp ) Explain Cytosolic FOX Activity? Intestinal Cp has been noted in mice (21) and if it is true in rats, at least part of the anti Heph Ab resistant activity ( > 70%) could be attributed to Cp. To test this possibility, enterocyte cytosols were immunoprobed with a well established anti Cp antibody. Western blot data from a representative sample shown in Fig. 5 clearly demonstrates that no Cp could be detected in rat cytosol ( an additional experiment confirmed this observation; not shown). Presence of the expected b and in serum

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131 ( positive control) and its absence in membrane (negative control) in addition to authenticating the data, suggests that Cp may not contribute to this activity. Conclusion s The objective of this study was to test the hypothesis that a n alternat ive form of Heph exists in rat enterocytes This supposition was precipitated by a lack of rigorous and cohesive structural and kinetic data pertaining to the presumed membrane bound form of Heph To th is end, after establishing the purity of rat enterocyt e derived cytosolic preparations by six different approaches, data presented in this manuscript show the presence of cytosolic Heph by: 1) immunolocalization in a native form from three different (1 animal and 2 cell lines) sources/samples; 2) clear presen ce of the expected (~130 kD) band not only by standard denaturing (SDS) PAGE but also under native conditions, thereby also supporting the IHC and ICC data; 3) responding to two different dietary conditions, increasing during iron deficiency and decreasing during copper deficiency, similar to membrane bound Heph; 4) most important, displaying FOX enzymatic activity in all rat samples studied consistent with the presence of immunoreactive Heph; and finally, 5) indicating the lac k of involvement of ceruloplas min in this phenomenon. The data presented here when considered in its entirety suggest that a cytosolic / soluble form of Heph exists in rat enterocytes. From a functional standpoint, given the low catalytic efficiency of ferroxidases in general and rode nt FOX in particular, the cytosolic ferroxidase envisaged here could augment the limited iron oxidizing capacity of enterocytes.

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132 Figures Figure 5 1 Presence of immunoreactive Heph in cytosolic and membrane fractions and fr action purity A) Western blot of Heph protein in cytosolic and membrane fractions of rat duodenal enterocytes prepared using three different methods. D4 indicates the particular anti Heph Ab used. B) Western blot of Atp7a protein in 60 g protein samples containing various ratios of membrane to cytosolic proteins. 54 10 indicates the particular anti Atp7a Ab used. C) Western blot of FPN protein in rat duodenal enterocytes and KNRK (a transformed rat kidney [NRK] cell line) lysate (positive control). Resul ts from two individual rats are shown. Stained blots in panels B & C exemplify proper loading and transfer of proteins. The blank space and black line in panel C indicate removal of unrelated lanes. D) Thin Layer Chromatography (TLC) of water, buffers, ent erocyte cytosol and membrane fractions, and P lipid (phospho lipid, used as a positive control). (Panel D of this figure courtesy of Dr. Ranganathan)

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133 Figure 5 2 Immunofluorescence analysis of rat duodenum, IEC 6 cells and C aco 2 cells A) and B) Immunohistochemical analysis of FeD rat duodenal sections, A) with and B) without the D4 anti Heph antibody. C) and D) Immunocytochemical analysis of pre confluent IEC 6 and Caco 2 cells. Data in all panels are representative of thre e independent experiments performed. FeD, iron deficient.

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134 Figure 5 3 Effects of iron and copper deprivation on Heph protein expression. A) denaturing and B) native western blot analysis of Heph in cytosolic and membrane fr actions of Ctrl and FeD rat enterocytes. Data in panel A are representative of experiments done with 12 individual rats per group. C) and D) Denatured western blot analysis of Heph in cytosolic and membrane fractions of Ctrl and CuD rat enterocytes by (C) D4 and (D) GeneTex anti Heph Abs. Stained blots below show proper loading and transfer of protein samples. In panels A C, data from two rats per group are shown, and in panel D data from 3 rats per group are shown. Ctrl: Control, FeD: iron deficient, CuD : copper deficient. Figure 5 4 FOX activity of rat duodenal enterocyte cytosol and antibody inhibition A ) the enzyme progress curve of rat cytosol FOX activity by Tf assay. Data represent three individual rats; mean SD i s shown. B ) e ffect of anti Heph (D4) and anti Alox15 (against an unrelated protein ) Abs on cytosolic FOX activity by Tf assay. n=2 individual rats; mean is shown. (The raw data from this figure courtesy of Dr. Ranganathan)

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135 Figure 5 5 Ceruloplasmin expression in rat duodenal enterocyte fractions and serum. Western blot analysis for Cp in enterocyte cytosol and membrane and in serum of control rats. Stained blot below exemplifies proper loading and transfer of protein samples. Data are representative of two independent experiments that were performed

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136 CHAPTER 6 CONCLUSIONS Ceruloplamin Protein Expression and Activity Serum Cp protein expression and activity was investigated in rats fed control or low iron diets containing low, normal or high copper levels. Hepatic copper levels increased with iron deficiency and in copper extra groups. Cp protein expression increased during iron deprivation and with higher copper intake. However, hepatic Cp mRNA expression was not responsi ve to dietary treatments. Both in gel and spectrophotometric amine oxidase ( p PD) and ferroxidase (Ferrozine) assays indicated that Cp enzymatic activity was enhanced upon hepatic copper loading. Furthermore, c orrelation analysis suggested that liver copper levels ultimately determine Cp protein expression and activity. These data suggested that the increase of Cp activity is likely due to a post transcriptional mechanism, which increased metallation of Cp protein, resulting in a higher level of serum holo Cp (the functional form of the enzyme). However, further dietary treatment studies in mice did not achieve the same results as in the rat studies. This may be due to the fact that mice are resistant to diet induced iron deficiency. As Cp expression and act ivity are also enhanced in iron deficient humans (137) this leads me to question the use of mice for iron homeostasis related investigations. Hephaestin Prote in Expression and Activity Heph protein expression and activity was investigated in rats fed FeD or CuD diets. In order to enrich membrane protein (where Heph was presumably anchored), ultracentrifugation was utilized to separate soluble/cytosolic and memb rane proteins. In

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137 contrast to previously published methods, a spectrophotometric based apo Tf coupled ferroxidase assay was used. In iron deficient rats, enhanced Heph protein expression was noted in enterocyte membrane fractions, but FOX activity was not different from controls. Surprisingly, robust immunoreactive Heph protein levels were detected in enterocyte cyto sol, and FOX activity increased ~30% upon iron deprivation. Both membrane bound Heph and cytosolic ( sol uble) Heph (sHeph) were responsive to c opper deprivation, as evidenced by decreased protein expression. However, cytosolic FOX activity was unchanged. Additional experiments validated the purity of the preps and ruled out the possibility that sHeph could result from a method induced artifact. I t is also confirmed that the cytosolic FOX activity was indeed protein mediated. Thus, r esults from the rat studies suggested that in addition to sHeph, there is another FOX contributing to total cytosolic activity, which increases during iron deficiency b ut was unaffected by copper deficiency. Subsequent studies performed in Heph /y mice showed that, despite the absence of detectable Heph protein in enterocyte cytosol, FOX activity was still present, suggestin g the existence of a novel FOX (named cytoFOX). Possible Identities for CytoFOX The possible identities for the novel cytosolic/soluble ferroxidase (cytoFOX) were investigated in various mutant mouse models plus in iron overloaded wt mice. Possible contributions of cytosolic FOX activity by H Ft were s tudied in wt mice fed an iron overload diet. No FOX activity was showed in samples that had been heated moderately, where H Ft should remain soluble and active; this suggested that H Ft did not contribute to FOX activity under the current Tf assay conditio ns. Whether Cp or App were playing a role in the cytosol as ferroxidases was investigated in Cp / and App / mice. Cytosolic FOX activity revealed no differences between wt and KO mice,

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138 indicating that those proteins are unlikely to contribute to the acti vity. Moreover, results from Heph /y mice validated previous conclusions that Heph alone is not adequate, as the cytosolic fractions still exhibit ~65% of wt cytosolic FOX activity. Heph /y / Cp / mice showed similar cytosolic FOX activity to wt mice. Overa ll, my studies suggested that the novel c ytoFOX may serve as a backup for Cp, and for both cytosolic and membrane bound Heph.

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139 APPENDIX MULTIPLE MENKES COPPER ATPASE (ATP7A) TRANSCRIPT AND PROTEIN VARIANTS ARE INDUCED BY IRON DEFICIENCY IN RAT DOUDENAL ENTEROCYTES The work in this appendix has been published and permission for reprint ing it has been obtained *** Summary The Menkes copper ATPase (Atp7a) pumps copper into the trans Golgi for cuproenzyme synthesis, and translocates to the basolateral membran e of enterocytes Atp7a transcript exist in rat duodenum, all of which are strongly induced during iron deprivation. To explore a possible role for Atp7a (and copper) i n intestinal iron absorption, the current studies were undertaken to test the hypothesis that multiple Atp7a transcript and protein variants exist in intestinal epithelial cells. Northern blot analyses using probes generated from the full length Atp7a cDNA revealed several specific hybridization bands, all of which were more intense in RNA samples extracted from duodenal enterocytes isolated from iron deficient rats. A PCR based approach, ants and a reverse primer in exon 23, demonstrated that 3 full length transcripts exist in rat IEC 6 cells. To identify possible Atp7a protein variants, three distinct polyclonal antisera were utilized. The specificity of the antisera was first established by western blotting and immune precipitation studies using samples derived from isolated rat enterocytes and Atp7a knockdown IEC 6 cells. Several specific immunoreactive bands were documented, and *** This work was originally published in J Trace Elem Med Biol Lu Y, Kim C, Collins JF. Multiple Menk es copper ATPase (Atp7a) transcript and protein variants are induced by iron deficiency in rat duodenal enterocytes. J Trace Elem Med Biol 2012 Jun;26(2 3):109 14 Elsevier GmbH

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140 a unique Atp7a protein distribution in cytosolic vesicle l ike structures was noted. In conclusion, multiple Atp7a transcript and protein variants exist in rodent intestinal epithelial cells and are induced by dietary iron deprivation. Further studies will be designed to determine the subcellular distribution of A tp7a protein variants and possible unique functions of each. Introduction important for intestinal copper absorption. Under basal conditions, Atp7a is localized to the trans gol gi apparatus of enterocytes where it provides copper to newly synthesized cupro proteins. However, when intracellular copper is elevated, Atp7a translocates to the basolateral membrane and facilitates copper export into the blood stream. Mutations in ATP7A copper accumulation and systemic copper deficiency. Previously, induction of Atp7a mRNA and protein expression was noted in rodent enterocytes during iron deficiency along with incre ased copper transport and liver copper accumulation (25, 111) Subsequently, an in vitro model (i.e. IEC 6 cells) was developed to further investigate the molecular mechanism underlying this observation. Actinomyci n D inhibition of Atp7a mRNA induction suggested that this process was conducted to begin initial characterization of the gene promoter, and unexpectedly, three different 5 discovered exon), 2 and 3; the second contained exons 1, 2 and 3; and the third had exon 1 spliced to exon 3. All of these variants were strongly induced during iron deficiency (28)

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141 To further examine the potential role of Atp7a in intestinal iron homeostasis, the present investigation was undertaken to identify Atp7a transcript and protein variants in rat enterocytes and IEC 6 cells and to q uantify their expression during dietary iron deprivation. Northern blot and RT PCR based approaches were utilized to identify transcript variants while three different anti Atp7a antibodies were utilized to identify protein variants. The data presented her ein supports the existence of multiple splice and immunoreactive protein variants in intestinal epithelial cells, many of which are strongly induced during iron deficiency. Methods IEC cells were obtained from ATCC and maintained at 37 o C in a humidified i ncubator with 5% CO 2 in high glucose DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution and 0.1% insulin (Sigma). The cells were used between passages ~18 25. Three gene specific shRNA entry vectors (BLOCK iT) targeti ng the rat Atp7a transcript (GenBank accession # NM_052803) were designed and produced by Invitrogen. The shRNAs were designed to hybridize to regions of the mRNA near bps in to IEC 6 cells by antibiotic selection essentially according to the manufacturer's instructions. Trial experiments determined that cells expressing all three Atp7a specific shRNAs simultaneously showed more significant inhibitory effects than individual sh RNA expressing cells. Thus, cells transfected with all three shRNA vectors (called

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142 All animal studies were approved by the University of F lorida IACUC. Weanling, male Sprague Dawley rats (Harlan, Indianapolis, IN) were housed in overhanging, wire mesh bottom cages for 35 days until sacrifice. The rats had food and iron free water ad libitum The diets were based on the AIN 93G formulation ( Dyets Inc., Bethlehem, PA) with ~200 ppm Fe (control; Ctrl) or ~3 ppm Fe (iron deficient; FeD). Although the AIN 93G formulation includes 40 ppm iron, the authors have used this control diet modeled after standard rodent chows (with ~200 ppm Fe) for severa l years. This diet is not adverse effects (as is also noted with rodents on standard chow) and further, the iron is present in a less bioavailable form in the diet (ferric citrate). Except for iron l evels, the diets were otherwise identical. Previous protocols were followed for tissue collection and enterocyte isolation. Confluent IEC 6 cells and isolated enterocytes were processed into cytosol and membrane fractions as previously described or lysed by hypotonic buffer plus 0.25% (v/v) NP Total RNA was extracted from IEC 6 cells or rat enterocytes using TRIzol first strand cDNA synthesis system (Invitrogen) following the manufacturer's recommendations. To amplify full length Atp7a transcripts, gene specific primers were designed tggaatcctagacagaatctactat cctggaatcctaggaatgtaaagaca cctggaatcctacctttccctagaa acataatgccaggttcagagctcc

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143 was accomplished using Ex Taq DNA polymerase (Takara), essentially according to the Total RNA was isola ted from IEC 6 cells or rat enterocytes and loaded onto denaturing 1% agarose gels for separation and verification of integrity. RNA was subsequently transferred onto nylon membranes and cross linked under UV light. The full length Atp7a cDNA (containing e xons 1 23) was amplified by RT PCR, cloned into a plasmid vector and verified by sequencing. The cDNA insert was subsequently excised from the vector, gel purified and biotin labeled DNA probes were generated (DIG High Prime DNA labeling and Detection Star ter Kit II, Roche). Blots were hybridized and instructions. buffer (25 mM Tris HCl, 140 mM NaCl, pH 7.4) in the presence of 1% NP protein A sepharose (Sigma) was added to the mixture and the sample was mixed for 60 min at 4 C. The sepharose beads were next pelleted at 2,500 g for 3 min. Supernatants were removed and the pellets were washed with 25 mM Tris HCl, 500 buffer were added to the final pellets, which were then heated at 78 C for 15 min to release the protein antibody complex bound to the protein A sepharose. For blocking mixture and incubated for 2 hrs at room temp with occasional gentle mixing or overnight at 4 C. A control reaction with the same volume of Ab in 1X PBS (minus the peptide[s])

PAGE 144

144 was processed identically. loaded onto 7.5% SDS/PAGE g els for electrophoretic separation. Proteins were transferred to PVDF membranes and reacted with different anti Atp7a antibodies, by standard methods. One Atp7a antibody (called 54 10) was a polyclonal anti serum from rabbits that were injected with two pe ptides (NH 2 pkkdrsanhldhkre COOH and NH 2 khsllvgdfredddttl antibody was generated by injecting one 37 amino acid peptide from the N terminal region (NH 2 rtieqqigkvngvhhikvsleeksatviynpklqt pk COOH) into two rabbits; the reagent used herein was the affinity purified product (Open Biosystems, Huntsville, AL). A well validated Atp7a (R17) antibody was a kind gift from Dr. Julian Mercer, Australia. This antibody was raised in sheep against the 590 N terminal region of Atp7a containing the six Cu binding domains fused to a (His) 6 tag at the N terminus. HRP conjugated anti rabbit and anti sheep secondary antibodies were from Bethyl laboratories. For Ab blocking experiments, Ab was mixed with pepti des as mentioned in the IP section prior to adding it to the blocking buffer (5% non fat dry milk in 1X TBST). IEC 6 cells were seeded onto poly D lysine coated coverslips in 6 well cell Fluor 647 secondary antibody (Invitrogen) were used at a 1:1000 dilution along with confocal microscopic imaging. Results and Discussion The iron status of rats was determined by measuring hemoglobin (Hb) and hematocrit (Hct) levels in bl ood removed at sacrifice. Significantly decreased Hb and

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145 Hct (>60%) was observed in all rats consuming the FeD diet (data not shown), consistent with previously published studies (25, 26, 70) Northern blot analysi s of RNA extracted from duodenal enterocytes of Ctrl and FeD rats using biotin labeled full length Atp7a specific cDNA probes revealed at least four transcripts (Fig ure A 1A). Moreover, all transcripts were induced upon iron deprivation. This supports pre vious investigations which documented transcriptional regulation of Atp7a (148) and induction during iron deprivation (25, 28) To date, to our knowledge, this is the first study that used the entire Atp7a cDNA sequence (exons 1 23; >4 kb) as a template to generate probes for Northern blot analysis. The exact nature of the different bands is not known; however, the band at ~4.3 kb could represent the script, as this size is consistent with the longest cDNA clone deposited in GenBank (accession # NM_052803). Previously, Reddy et al. (112) used a 530 bp oligonucleotide homologous to exon 23 as a probe and identif ied only one hybridization band at ~5.5 kb in human cells. Moreover, Ackland et al. (2) generated hybridization band at ~8.5 kb in human breast cancer cells. In the current study, full length Atp7a probes also hybridized with transcripts in RNA isolated from rat IEC 6 cells (Fig ure A 2B). Longer gel runs revealed that the major hybridization band at ~4.3 kb was actually a doublet, po ssibly revealing putative splice variants of very similar size. Furthermore, consistent with the Northern blot data, multiple, potential alternative splice variants were noted by RT PCR using exon 1 forward and exon 23 reverse PCR primers (Fig ure A 1C).

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146 P (28) based d splice variants were conjoined with was specific for exon 23. A ~4.6 kb band was amplified using all three primer combinations (Fig ure A 2A), demonstrating the existence of alternative transcripts. The exon 1/1A and 1/2 splice variants would presumably be translated from a start codon in exon 2, whereas the exon 1/3 splice variant would use an alternative, in frame start codon in exon 3 (28) Since multiple Atp7a transcript variants were identified in rat enterocytes and IEC 6 cells, the next objective was to investigate whether Atp7a prot ein variants existed as well. This issue was addressed using three anti Atp7a antisera and an array of immunochemical techniques. First, the validity and specificity of the antibodies was assessed by immunoprecipitation (IP) studies combined with western b lot analysis. Immunoreactive proteins of similar m.w. were detected by all 3 reagents on western blots (Fig ure A 3, all panels, left most lanes), and in most cases, more than one band was present. The specificity of the 54 10 antiserum was shown by IP wit h and without pre incubation with the immunogenic peptides followed by immunoblotting (Fig ure A 3A). IP with the Long and R17 antisera also pulled down a protein that was recognized by the 54 10 antiserum, at a consistent m.w. as by western blot (Fig ure A 3C). The Long antiserum revealed a protein of consistent m.w. by IP followed by immunblotting and by western blot alone (Fig ure A 3B). Lastly, IP with all 3 antisera pulled down proteins recognized by the R17 reagent. All in all, these experiments

PAGE 147

147 sugge st that all three of these reagents are specific for the Atp7a protein in IEC 6 cell extract and membrane preps. To further address the specificity of these reagents for the rat Atp7a protein, shRNA knockdown cells were established. Quantitative RT PCR de monstrated that IEC 6 cells expressing a combination of three Atp7a specific shRNAs showed a significant reduction in Atp7a transcript levels (>50%), as compared to cells expressing a negative control shRNA (data not shown). Immunoblot analysis using all t hree antisera showed that the Atp7a shRNAs significantly reduced levels of a potential full size (~190 kDa) Atp7a protein (Fig ure 4A and B), providing strong evidence of specificity. Furthermore, the Long antiserum detected multiple immunoreactive bands, a ll of which were attenuated in Atp7a shRNA expressing cells (Fi g ure A 4C). Multiple bands could also be attenuated by pre incubation of the Long antiserum with the immunogenic peptide (F i g ure A 4D). These experiments prove specificity of the different a ntisera for the Atp7a protein and provide strong evidence supporting the existence of multiple protein variants in rat intestinal epithelial cells. during iron deficiency, it wa s important to investigate whether any of these putative protein variants were also induced in the duodenum of iron deficient rats. Thus, cytosol and membrane preps from isolated duodenal enterocytes of Ctrl and FeD rats were reacted with the 54 10, Long a nd R17 Abs. Multiple immunoreactive bands detected by the 54 10 Ab were greatly induced in iron deficient rats (Fig ure A 5A), consistent with previous observations (111) In a shorter exposure, a doublet was clear ly visible,

PAGE 148

148 above. Moreover, immunoreactive bands detected by the Long and R17 antisera were also increased in the samples derived from iron deficient rats (Fig ure A 5 B). Lastly, the intracellular localization of immunoreactive protein(s) detected by the Long antiserum was investigated. It is important to note here that co localization of Atp7a with a trans Golgi specific marker was noted previously using the 54 10 ant iserum (28) Using the Long antiserum in immunocytochemistry studies in IEC 6 cells, intracellular staining of widely dispersed vesicle like structures was noted (Fig ure A the subject of further investigation. Conclusions Multiple Atp7a transcripts were detected in IEC 6 cells and rat duodenum, with most showing strong induction by dietary iron deprivation. Several Atp7a protein variants also exist in IEC 6 cells and rat duodenal enterocytes and are induced during iron deficiency. The extensive validation of the immune reagents used via peptide blocking experiments, IP studies and shRNA mediated Atp7a knockdown, strengthens and va lidates these observations. Further studies will address sub cellular localization and potentially novel iron/copper related physiologic functions of these Atp7a protein v ariants.

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149 Figures Figure A 1 Northern blot analysis u sing full length cDNA probes with RNA extracted from duodenal enterocytes of Ctrl and FeD rats and IEC 6 cells. RNA gels shown in A ) and B ) exemplify comparable loading and RNA quality. C ) RT PCR amplification of Atp7a cDNA from IEC 6 cells using exon 1 fo rward and exon 23 reverse primers. (Data from this figure courtesy of Changae Kim)

PAGE 150

150 Figure A 2 Full end splice variants. A ) PCR amplification of cDNA usin g different exon spanning forward and exon 23 reverse primers. B ) splice variants, with relative locations of primers noted by arrows (Panel A of t his figure courtesy of Changae Kim)

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151 Figure A 3 IP of IEC 6 cell extract and membrane preps using three different anti Atp7a antibodies A ) IP by 54 10 or peptide blocked 54 10 Ab followed by probing with the same Ab B ) IP by Long Ab followed by probing with the same Ab C ) IP by Long or R17 Ab followed b y probing with 54 10 Ab D ) IP by 54 10, Long or R17 Abs followed by probing with R17 Ab. In all panels, the in B, C & D indicate where non related lanes were removed from image s. Memb, membrane. Representative experiments are shown.

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152 Figure A 4 I mmunoreactive protein expression in Atp7a specific shRNA expressing 6 cells. Cell extracts were pro bed with 54 10, Long and R17 Abs. A) Cytosol and membrane fractions were probed with 54 10 in B) or Long in C) Abs. Cytosol is shown to demonstrate the relative purity of the preps. Stained blots in A, B & C exemplify equal protein loading and efficient tr ansfer; D ) Cell extract was probed with Long or peptide blocked Long Ab. The line indicates where unrelated lanes were removed from the image. Representative images from several experiments are shown.

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153 Figure A 5 Atp7a pro tein expression in cytosol and membrane fractions of isolated enterocytes from contro l and iron deficient (FeD) rat s A ) Cytosol and membrane fractions were probed with 54 10 Ab. Two different length exposures of the same blot are shown; B ) Cytosol and mem brane fractions were probed with Long and R17 Abs. Stained blots shown in A & B exemplify equal protein loading and efficient transfer. The line in panel A indicates where unrelated lanes were removed from the image. A representative experiment is shown

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154 Figure A 6 Immunocytochemical anal ysis of IEC 6 cells by Long Ab. A) Pre confluent IEC 6 c ells were reacted with Long Ab. B) A higher magnification view of IEC 6 cells stained with the Long Ab. A representative experiment is shown.

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167 BIOGRAPHICAL SKETCH Yan Lu was born in Chengdu, China. She received her Bachelor of Medicine in Preventive Medicine from the West China School of Public Health, Sichuan University, in 2007 In the same year, she also obtained her Bachelor of Arts in English from the College of Foreign Language s and Cultures in the same university. After graduation, she came to the United States to pursue a higher degree in nutrition. From 2007 to 2008, she was a m of New York at Buffalo and jo transferred to the University of Florida with Dr. Collins and became a Ph.D. student in the Food Science and Human Nutrition Department. Her major research focus is ferroxidases in iron absorption and met abolism. From May to August of 2012, she visited the Iron Metabolism Laboratory in Queensland Institute of Medical Research in Brisbane, Australia and trained under the guidance of Dr. Greg ory J. Anderson. During the past three years, she received various travel awards to attend Experimental Biology conferences and presented her research as posters. Yan is always interested in disease prevention related research from nutritional perspectives. After graduating in December 2012, she intends to engage in trans lational research.