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Zinc Transport and Metabolism in the Exocrine Pancreas

Permanent Link: http://ufdc.ufl.edu/UFE0041021/00001

Material Information

Title: Zinc Transport and Metabolism in the Exocrine Pancreas
Physical Description: 1 online resource (143 p.)
Language: english
Creator: Guo, Liang
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acinar, ar42j, cancer, deficiency, dexamethasone, efflux, element, excretion, exocrine, gene, glucocorticoids, gr, granule, homeostasis, metabolism, metallothionein, mre, mtf, nutrient, nutrition, pancreas, pancreatic, regulation, secretion, slc30a1, slc30a2, stat5, trace, transcription, transporter, zinc, znt1, znt2, zymogen
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: Introduction: Zinc deficiency affects more than one billion children under 5 years old and is responsible for 400,000 deaths annually worldwide. Secretion from the exocrine pancreas is a major route of endogenous zinc loss. Thus it plays an important role in the maintenance of zinc homeostasis. The molecular mechanisms, pathways, and the transporters for pancreatic zinc secretion are still not clear. Glucocorticoid therapy increases zinc excretion, and causes a rapid depression of serum zinc. So far, no zinc transporter has been found to be regulated by glucocorticoid hormones. Objectives: The major aims were to identify the zinc transporters in exocrine pancreas and evaluate their role in zinc secretion and to define physiological effects of zinc transporter expression in pancreatic acinar cells. Results: Two highly expressed zinc transporters, ZnT1 and ZnT2 were identified in acinar cells, the major cell type in the exocrine pancreas. Immunofluorescence localized ZnT1 and ZnT2 to the plasma membrane and zymogen granules, respectively. Dietary zinc restriction significantly decreased the zinc concentration over 50% in both pancreatic cell cytoplasm and in zymogen granules and was correlated with decreased expression of ZnT1 and ZnT2. In contrast, with an up-regulated ZnT1 and ZnT2 observed after oral zinc administration produced an increase in pancreatic zinc content. Zinc stimulated ZnT1 expression in a dose-dependent manner in rat pancreatic AR42J cells. ZnT2 was stimulated by 100 nM dexamethasone during AR42J differentiation. In agreement, 10 mg/kg body weight dexamethasone administered to mice induced ZnT2 expression and resulted in a reduction in pancreatic zinc content. ZnT2 siRNA in AR42J cells caused an increase in cytoplasmic zinc and decreased zymogen granule zinc, which further shows that ZnT2 mediates the sequestration of zinc into zymogen granules. A crucial metal response element was found in the ZnT2 promoter that confers the responsiveness to zinc. Both glucocorticoid receptor and Stat5 signaling were required in dexamethasone induced ZnT2 expression. Conclusions: ZnT1 controls zinc efflux directly across the apical membrane in a zinc-dependent manner, whereas ZnT2 participates in zinc sequestration into secretory granules. The two transporters appear to function closely in acinar cell zinc secretion and constitute an important component of the entero-pancreatic zinc circulation. Significance: Understanding of the molecular mechanisms and regulation of endogenous zinc loss via pancreatic secretion is an important part of zinc homeostasis. The results of this study suggest that normal pancreatic acinar cell function is an essential physiologic component that influences body zinc retention and prevents zinc deficiency.
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 Liang Guo.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Cousins, Robert J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041021:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041021/00001

Material Information

Title: Zinc Transport and Metabolism in the Exocrine Pancreas
Physical Description: 1 online resource (143 p.)
Language: english
Creator: Guo, Liang
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acinar, ar42j, cancer, deficiency, dexamethasone, efflux, element, excretion, exocrine, gene, glucocorticoids, gr, granule, homeostasis, metabolism, metallothionein, mre, mtf, nutrient, nutrition, pancreas, pancreatic, regulation, secretion, slc30a1, slc30a2, stat5, trace, transcription, transporter, zinc, znt1, znt2, zymogen
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: Introduction: Zinc deficiency affects more than one billion children under 5 years old and is responsible for 400,000 deaths annually worldwide. Secretion from the exocrine pancreas is a major route of endogenous zinc loss. Thus it plays an important role in the maintenance of zinc homeostasis. The molecular mechanisms, pathways, and the transporters for pancreatic zinc secretion are still not clear. Glucocorticoid therapy increases zinc excretion, and causes a rapid depression of serum zinc. So far, no zinc transporter has been found to be regulated by glucocorticoid hormones. Objectives: The major aims were to identify the zinc transporters in exocrine pancreas and evaluate their role in zinc secretion and to define physiological effects of zinc transporter expression in pancreatic acinar cells. Results: Two highly expressed zinc transporters, ZnT1 and ZnT2 were identified in acinar cells, the major cell type in the exocrine pancreas. Immunofluorescence localized ZnT1 and ZnT2 to the plasma membrane and zymogen granules, respectively. Dietary zinc restriction significantly decreased the zinc concentration over 50% in both pancreatic cell cytoplasm and in zymogen granules and was correlated with decreased expression of ZnT1 and ZnT2. In contrast, with an up-regulated ZnT1 and ZnT2 observed after oral zinc administration produced an increase in pancreatic zinc content. Zinc stimulated ZnT1 expression in a dose-dependent manner in rat pancreatic AR42J cells. ZnT2 was stimulated by 100 nM dexamethasone during AR42J differentiation. In agreement, 10 mg/kg body weight dexamethasone administered to mice induced ZnT2 expression and resulted in a reduction in pancreatic zinc content. ZnT2 siRNA in AR42J cells caused an increase in cytoplasmic zinc and decreased zymogen granule zinc, which further shows that ZnT2 mediates the sequestration of zinc into zymogen granules. A crucial metal response element was found in the ZnT2 promoter that confers the responsiveness to zinc. Both glucocorticoid receptor and Stat5 signaling were required in dexamethasone induced ZnT2 expression. Conclusions: ZnT1 controls zinc efflux directly across the apical membrane in a zinc-dependent manner, whereas ZnT2 participates in zinc sequestration into secretory granules. The two transporters appear to function closely in acinar cell zinc secretion and constitute an important component of the entero-pancreatic zinc circulation. Significance: Understanding of the molecular mechanisms and regulation of endogenous zinc loss via pancreatic secretion is an important part of zinc homeostasis. The results of this study suggest that normal pancreatic acinar cell function is an essential physiologic component that influences body zinc retention and prevents zinc deficiency.
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 Liang Guo.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Cousins, Robert J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041021:00001


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1 ZINC TRANSPORT AND METABOLISM IN THE EXOCRINE PANCREAS By LIANG GUO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Liang Guo

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3 To my parents Zhisheng Guo and Anrong Jin my fiance, Xin Deng

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4 ACKNOWLEDGMENTS The last four years have been the most rewarding and memorable time to me. The completion of this dissertation is a result of the amazing support and encouragement from many people to whom I will be grateful forever. I would like to acknowledge my mentor, Dr. Robert J. Cousins. During the past four years, I have been learning how to be a successf ul scientist from all perspectives Your hard work and dedication to the research always encourage me to do my best and accomplish the best. I have been impres sed by your willingness to acquire new knowledge and try new techniques all the time. Science and technology never stops; neither do we. I thank you for your encouragement when I was facing difficulties. I thank you f or helping me polish my thinking when new evidence appeared. Without your guidance, intuition, and inspiration this would not have been possible. I would like to thank each of my committee members : Dr. Harry S. Sitren, Dr. Mitchell D. Knutson, and Dr. Don A Samuelson, for your thoughtfulness knowledge, and inspiration s, which I received from each conversation I have had with you. I am r eally grateful for your significant, generous time contributions and guidance through my graduate study. I thank Dr. Juan P. Liuzzi and Dr. Fudi Wang for guiding me through the experiments and always being good role model s. Thanks for your thought provoking support motivation and enthusiasm that helped me become a mature researcher. The time you spent in Dr. Cousins lab will always be the highlights of my experience with you. I would like to thank Dr. Louis A. Lichten, MoonSuhn Ryu, Dr. ShouMei Chang, Tolunay B. Aydemir, and Alyssa Maki for your friendship and support. The time I have spent with you in the lab has been the best time and unforgettable experience for me I

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5 would like to acknowledge Virginia Mauldin and Carrie Guzman for your excellent edi ting and document preparation. Finally, to my fiance, Xin; my sunshine, my best friend, y ou are the reason I can be what I am.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Zinc is an Essential Nutrient ................................................................................... 17 Cellular Functions of Zinc ....................................................................................... 18 Zinc Homeostasis ................................................................................................... 19 Cellular Zinc Homeostasis ................................................................................ 19 Zinc M etabolism and the Pancreas .................................................................. 20 Zinc Absorption ................................................................................................ 21 Zinc Excretion ................................................................................................... 22 Zinc Metabolism and Pancreatic Metallothionein ............................................. 22 Zinc Deficiency ....................................................................................................... 23 Zinc Toxicity ............................................................................................................ 25 Zinc Transporter Families ....................................................................................... 25 2 MATERIALS AND METHODS ................................................................................ 28 Animal E xperiments ................................................................................................ 28 RNA Extraction and Quantitative Real Time PCR .................................................. 28 Isolation and Culture of Pancreatic Acinar Cells ..................................................... 29 Zymogen Granules Isolation ................................................................................... 30 FluoZi n 3 AM Labile Zinc Quantification ................................................................. 30 Immunoblotting and Immunohistochemistry ............................................................ 31 65Zn Uptake and Efflux Assay ................................................................................. 31 siRNA Mediated Knock down ................................................................................. 31 Cultur e of Rat AR42J Acinar Cells .......................................................................... 32 Atomic Absorption Spectrophotometry .................................................................... 33 Transfection and Luciferase Assay ......................................................................... 33 Statistical Analysis .................................................................................................. 34

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7 3 ZINC REGUL ATES EXPRESSION OF ZINC TRANSPORTERS ZnT1 AND ZnT2 IN PANCREATIC ACINAR CELLS ................................................................ 35 Introduction ............................................................................................................. 35 Results .................................................................................................................... 38 Discussion .............................................................................................................. 43 4 GLUCOCORTICOIDS REGULATE ZnT2 EXPRESSION IN PANCREATIC ACINAR CELLS ...................................................................................................... 72 Introduction ............................................................................................................. 72 Results .................................................................................................................... 75 Discussion .............................................................................................................. 79 5 ZINC TRANSPORTERS IN PANCREATIC ACINAR CELLS UNDER STRESS CONDITIONS ....................................................................................................... 108 Introduction ........................................................................................................... 108 Methods and Results ............................................................................................ 109 Discussion ............................................................................................................ 110 6 ZINC TRANSPORTERS IN HUMAN PANCREATIC CANCER ............................ 116 Introduction ........................................................................................................... 116 Methods ................................................................................................................ 116 Results .................................................................................................................. 116 Discussion ............................................................................................................ 118 7 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 123 Conclusions .......................................................................................................... 123 Future Directions .................................................................................................. 126 LIST OF REFERENCES ............................................................................................. 131 BIOGRAPHICAL SKETCH .......................................................................................... 143

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8 LIST OF FIGURES Figure page 3 1 ZnT1, ZnT2 and MT mRNA expression in mouse pancreas ............................... 47 3 2 Zinc concentrations in serum, pancreas and liver from mice fed with zinc adequate or deficient diet ................................................................................... 48 3 3 Zip5 expression in mouse pancreas after 3 wks dietary zinc restriction or a zinc adequate diet .............................................................................................. 49 3 4 ZnT1 and ZnT2 western blotting in mouse pancreas after 3 wks dietary zinc restriction ............................................................................................................ 50 3 5 ZnT1 western blotting in mouse pancreas after 3 wks dietary zinc restriction .... 51 3 6 ZnT1 western blotting in mouse pancreas after 3 wks dietary zinc restriction .... 5 2 3 7 Confocal immunofluorescence analysis of ZnT2 in purified zymogen granules isolated from mouse pancreas. ........................................................................... 53 3 8 Zinc concentrations in the brain, pancreas and liver of mouse 3 h after a zinc gavage ................................................................................................................ 54 3 9 Zinc concentrations in the pancreas, liver and serum in mouse 3h and 8h after zinc gavage ................................................................................................ 55 3 10 ZnT zinc transporter family mRNA expression in the pancreas from mice 3 h and 8 h after zinc gavage ................................................................................... 56 3 11 Zip zinc transporter family mRNA expression in the pancreas from mice 3 h and 8 h after zinc gavage ................................................................................... 57 3 12 Zinc influences ZnT1 mRNA levels in a dose dependant manner in AR42J cells .................................................................................................................... 58 3 13 Zinc influences MT1, and ZnT1 mRNA levels in a dose dependant manner in AR42J cells ......................................................................................................... 59 3 14 Zinc influences MT and ZnT1 mRNA levels in a timedependant manner AR42J cells ......................................................................................................... 60 3 15 Influence of zinc on ZnT1 in AR42J cells ............................................................ 61 3 16 Influence of zinc and ZnT1 siRNA on ZnT1 expression ...................................... 62 3 17 MT, ZnT1 and ZnT2 mRNA abundance in AR42J cells incubated with low zinc medium ....................................................................................................... 63

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9 3 18 Zinc chelators influences ZnT1, ZnT2 and MT mRNA levels and their protein expression .......................................................................................................... 64 3 19 Low zinc medium influences ZnT1 and ZnT2 expression in AR42J cells ........... 65 3 20 Zinc chelating reagents influence ZnT1 expression in AR42J cells .................... 66 3 21 Western analy sis of mZnT1 over expression in HEK 293 cells .......................... 67 3 22 IF analysis of mZnT1 over expression in HEK 293 cells .................................... 68 3 23 Western analysis of ZnT2 ................................................................................... 69 3 24 Western analysis of N glycosation in rat ZnT1, ZnT2, ZnT4 in AR42J cell membrane fraction, and human ZnT1 in HEK 293 cell fraction by PNGase F digestion ............................................................................................................. 70 3 25 Western analysis of ZnT2 in mouse and rat pancreas ........................................ 71 4 1 Dexamethasone regulates MT, and ZnT2 mRNA levels in AR42J cells ............. 84 4 2 The responsiveness of ZnT1 and ZnT2 expression to Zinc and/or dexamethasone in AR42J cells .......................................................................... 85 4 3 The responsiveness of MT and ZnT1 expression to zinc and/or dexamethasone in AR42J cells .......................................................................... 86 4 4 Dexamethasone influences ZnT2 expression in pancreatic acinar cells ............. 87 4 5 Zinc concentration in the pancreas and serum in mouse 3 h after dexamethasone i.p. inj ection .............................................................................. 88 4 6 Dexamethasone influences zinc concentration in the pancreas, liver, and serum in mice 8 h after dexamethasone i.p. inj ection ......................................... 89 4 7 MT (A) and ZnT2 (B) mRNA expression in CD1 mice injected i.p. with dexamethasone .................................................................................................. 90 4 8 ZnT2 knock down in AR42J cells by ZnT2 siRNA .............................................. 91 4 9 Zinc concentrations in cytoplasm and ZG fractions after ZnT2 knock down by ZnT2 siRNA in AR42J cells ................................................................................ 92 4 10 ZnT2 siRNA knockdown and dexamethasone influences ZnT1 and ZnT2 expression in AR42J cells .................................................................................. 93 4 11 ZnT2 siRNA knockdown influences MT mRNA in AR42J cells .......................... 94 4 12 ZnT2 overexpression increases zinc efflux in HeLa cells ................................... 95

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10 4 13 The effects of dexamethasone and glucocorticoid modulators, RU486 and CpdA, on the MT and ZnT2 mRNA expression in AR42J cells ........................... 96 4 14 Transcription factor binding site analysis in the proximal region of mouse ZnT2 promoter .................................................................................................... 97 4 15 Possible GR and Stat5 synergistic signaling pathway ........................................ 98 4 16 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the MT mRNA expressions in AR42J cells ......................................................................................................... 99 4 17 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the ZnT2 mRNA expressions in AR42J cells ....................................................................................................... 100 4 18 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the MT and ZnT2 mRNA expressions in AR42J cells ............................................................................... 101 4 19 Conserved MRE sequences in the promoter sequence alignment of multiple species ............................................................................................................. 102 4 20 ZnT2 promoter activity in transfected HEK 293 and HeLa cells in response to zinc ................................................................................................................... 103 4 21 ZnT2 promoter activity in transfected HeLa cells in response to zinc ............... 104 4 22 pGL3Basic and pGL4 vector luciferase activities under DEX treatment in HeLa cells ......................................................................................................... 105 4 23 CpG island analysis in ZnT2 gene. ................................................................... 106 4 24 Predicted human ZnT2 protein structure (A) and zinc binding sites (B). ........... 107 5 1 MT and ZnT1/2 mRNA expression in AR42J cells stimulated with cerulein ..... 112 5 2 Supreamaximal CCK8 stimulation decreased the expression of ZnT1 in AR42J cells ....................................................................................................... 113 5 3 MT, and ZnT1/2 mRNA expressions in AR42J cells stimulated with supreamaximal CCK8 ....................................................................................... 114 5 4 MT and ZnT1/2 mRNA expressions in AR42J cells stimulated with ethanol .... 115 6 1 mRNA abundance panel of Zip family in human pancreatic cancer. ................ 120 6 2 mRNA abundance panel of ZnT family in human pancreatic cancer. ............... 121

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11 6 3 MT mRNA abundance in normal human pancreas and pancreatic cancer. ...... 122 7 1 Hypothetical schematic model of zinc transport and secretion in pancreatic acinar cells ........................................................................................................ 130

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12 LIST OF ABBREVIATIONS Ab Antibody ADx A drenalectomy AE Acrodermatits enteropathica AP Affinity purified antibody AP1 A ctivator protein 1 BSA Bovine serum albumin ChIP Chromatin Immunoprecipitation CDF Cat ion Diffusion Facilitator cDNA complementary DNA CNH Chromonebased nicotinyl h ydrazone CpdA Compound A ( an analog of a hydroxyphenyl aziridine precursor ) CP C ytoplasm DEX Dexamethasone DNA Deo xyribonucleic acid DPA Dip icolinic acid DTPA Diethylene triamine pentaacetic acid EST Expressed sequence tag ER E ndoplasmic r eticulum or estrogen receptor GAS Gamma interferon activation site GC Glucocorticoid GFP Green fusion protein GI G astrointestinal GR Glucocorticoid receptor GRE Glucocorticoid response element

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13 HEK Human embryonic kidney 293 cells ICC I mmunocytochemistry IF Immunofluorescence IHC Immunohistochemistry JAK2 Janus kinase 2 LZT LIV 1 subfamily of ZIP zinc transporters MRE Metal response element mRNA Messenger RNA MT Metallothionein MTF1 Metal response element binding transcription factor 1 NF B N uclear factor PBS Phosphate buffered saline PKC Protein kinase C PNG Peptide N g lycosidase qPCR Quantitaive polymerase chain reaction rER Ro ugh endoplasmic r eticulum RNA Ribonucleic acid siRNA Small interfering RNA shRNA Small hairpin RNA SNP Single nucleotide polymorphism SLC solute carrier STAT5 Signal transducer and activator of transcription 5 STAT5 RE Signal transducer and activator of transcription 5 response element TBS Tris buffered saline TBST Tris buffered saline with Tween 20

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14 TGN Transgolgi network TSS Transcription start site TMD Transmembrane domain TPEN N,N,N,NTetrakis (2 pyridylmethyl) e thylenediamine TRAIL TNF related apoptosis inducing ligand VDAC Voltagedependent anion channel ZG Zymogen granule ZIP Zrt Irt like zinc transporter SLC39A superfamily ZnA Zinc adequate ZnD Zinc deficient ZnT Zinc transporter SLC30A superfamily

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ZINC TRANSPORT AND METABOLISM IN THE EXOCRINE PANCREAS By Liang Guo December 2009 Chair: Robert J. Cousins Major: Nutritional Science s Introduction: Zinc deficiency affects more than one billion children under 5 years old and is responsible for 400,000 deaths annually worldwide. Secretion from the exocrine pancreas is a major route of endogenous zinc loss T hus it plays an important role in the maintenance of zinc homeostasis. The molecular mechanisms, pathways, and the transporters for pancreatic zinc secretion are still not clear. Glucocorticoid therapy increases zinc excretion, and causes a rapid depression of serum zinc. So far, no zinc transporter has been found to be regulated by glucocorticoid hormones. Objectives: The major aims were to identify the zinc transporters in exocrine pancreas and evaluate their role in zinc secretion and to define physiologic al effects of zinc transporter expression in pancreatic acinar cells. Results: T wo highly expressed zinc transporters ZnT1 and ZnT2 were identified in acinar cells the major cell type in the exocrine pancreas Immunofluorescence localized ZnT1 and ZnT2 t o the plasma membrane and zymogen granules, respectively. Dietary zinc restriction significantly decreased the zinc concentration over 50% in both pancreatic cell cytoplasm and in zymogen granules and was correlated with decreased expression of ZnT1 and ZnT2. In contrast, with an upregulated ZnT1 and ZnT2

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16 observed after oral zinc administration produced an increase in pancreatic zinc content Zinc stimulated ZnT1 expression in a dose dependent manner in rat pancreatic AR42J cells. ZnT2 was stimulated by 100 nM dexamethasone during AR42J differentiation. In agreement, 10 mg/kg body weight dexamethasone administered to mice induced ZnT2 expression and resulted in a reduction in pancreatic zinc content. ZnT2 siRNA in AR42J cells caused an increase in cytoplasmic zinc and decreased zymogen granule zinc, which further shows that ZnT2 mediates the sequestration of zinc into zymogen granules. A crucial metal response element was found in the ZnT2 promoter that confers the responsiveness to zinc. Both glucocortico id receptor and Stat5 signaling were required in dexamethasone induced ZnT2 expression. Conclusions: ZnT1 controls zinc efflux directly across the apical membrane in a zinc dependent manner, whereas ZnT2 participates in zinc sequestration into secretory gr anules. The two transporters appear to function closely in acinar cell zinc secretion and constitute an important component of the enteropancreatic zinc circulation. Significance: Understanding of the molecular mechanisms and regulation of endogenous zinc loss via pancreatic secretion is an important part of zinc homeostasis. The results of this study suggest that normal pancreatic acinar cell function is an essential physiologic component that influences body zinc retention and prevents zinc deficiency.

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17 CHAPTER 1 INTRODUCTION Zinc is an Essential Nutrient Zinc is an es sential micronutrient in humans. There are about 2 3 g of zinc in our bodies which is the second largest quantity, after 4 5 g of body iron. Zinc deficiency affects more than one billion children under 5 years old and is responsible for 400,000 deaths annually worldwide (1) Zinc deficiency is a type II nutrient deficiency, fundamentally differ ent from iron deficiency, which is type I nutrient def iciency (2) During iron deficiency, the concentration of iron in the tissues is reduced and specific defects develop, most commonly anemia. But there is no effect on growth and body weight until anemia and other complications develop. However, if dietary zinc is low in children, an immediate growth cessation occurs, whi le most tissue zinc contents are normal. In other words, in both iron and zinc deficiency in growing animals the absolute amount of the nutrient within the body is less than normal. However, the reduced amount of iron is in a normal sized body, whereas with zinc there is a restriction in body size to maintain the cellular zinc concentration. Therefore, growth retardat ion delayed sexual maturation, and hypogonadism are the most common symptoms associated with severe zinc deficiency in children. Current k nowledge shows zinc has a significant biological role in catalytic, structura l, and regulatory functions in hundreds of proteins (3 5) These properties explain why zinc is of such importance to maintain ing normal cellular functions and why zinc deficiency would produces a nutrient deficiency. The details of the cellular functions of zinc are reviewed in the following paragraph. Compar ed to iron, zinc overload is rare, and zinc is considered to be relatively nontoxic, particularly if tak en orally. However, in

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18 vitro studies have showed excessive zinc in the cell is toxic causing apoptosis and necrosis (6) Therefore, the result of excessive cellular zinc is more acute and severe than the free radical damage by iron overload. Since both excessive cellular zinc and zinc deficiency cause deleterious effects, the zinc concentration in the cell, especially the labile zinc pool must be tightly regulated. Body zinc homeostasis is achieved through balancing the zinc absorption, tissue utilization, and excretion. D ysregulation of zinc homeostasis contributes not only to systemic zinc deficiency and toxicity, but also to the pathogenesis of several diseases, including diarrhea, cancer lower immunity, diabetes, Alzheimers disease, a crodermatit i s enteropathica fume fever, and a gerelated m acular d egeneration (7 11) The mechanism of zinc mediated cytoxicity is still not very clear. Protein and nucleic acids synthesis inhibition, calcium signaling interference and glutathione oxidation have been proposed to be the del eterious effects of elevated cellular zinc. I f an extreme ly large amount of zinc is taken, toxicity symptoms will occur, including nausea, vomiting, epigastric pain, lethargy, and fatigue. Long term high zinc diets will also interfere with copper absorption, resulting in severe copper deficiency (12) Cellular Functions of Zinc Zinc is an important catalytic and structural component of more than 300 zinc metalloenzymes in more than 50 different enzyme categories. In some case s, depletion of zinc will result in a lower enzyme activity. Here are some examples of zinc enzymes: n icotinamide adenine dinucleotide dehydrogenase (NADH) RNA polymerase DNA polymerase, a lkaline phosphatase s uperoxide dismutase c arbonic anhydrase g lut amic dehydrogenase l actate dehydrogenase and m alate dehydrogenase However, zinc has also been found to play an inhibitory role in certain enzymes. In other words, removal of

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19 zinc from an inhibitory, zinc specific enzymatic site results in a marked increase of enzyme activity (> 10fold increase) These are some examples of these enzymes, caspase 3 fructose 1,6diphosphatase g lyceraldehyde 3phosphate dehydrogenase a ldehyde dehydrogenase and t yrosine phosphatase There are also pancreatic digestive enzymes associated with zinc: carboxypeptidase A1 and A2 a minopeptidase carboxypeptidase B a mylase chymotrypsinogen B1, l ipase trypsin 4. Zinc Homeostasis Cellular Zinc Homeostasis Cellular zinc homeostasis requires tight control of zinc uptake/influx storage, and export/efflux. Intracellular zinc compartmentation is an important source of cellular zinc storage. The vesicular zinc exchange with the cytoplasm pool plays a critical role in the cellular phy siological functions of zinc Diffe rent tissues have their own zinc metallo protein profiles to undertake unique functions, thus the cellular zinc homeostasis control is a complex system requiring precise regulation. Zinc concentration in the plasma is about 915 and most of the extracellular z inc car ried in the plasma is bound to albumin (7585%) (13, 14) The rest are bound to some other proteins such as 2 macroglobin (13) Serum zinc concentration falls with the hypoalbuminemia that accompanies aging (15) Two zinc transporter families, SLC30 A and SLC39 A of a total of 24 zinc transporters so far have been found to function in this homeostasis system. Each one of them has its own unique tissue profile, expression regulation, intracellular localization, and transport activity (3, 5). Pancreatic intracellular zinc homeostasis is very sensitive to body zinc status. Homeostasis is achieved through the coordinated regulation of zinc transporters. So far, there are two zinc transporter families (Zips and

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20 ZnTs) involved in zinc influx, efflux and intracellular compartmentalization within pancreatic acinar cells (3, 16, 17) Zinc Metabolism and the Pancreas Zinc homeostasis is a balance between gastrointestinal absorption, and tissue storage and secretion into the intestinal lumen through intestine cells and via the pancreatic duct. Under normal conditions, a considerable amount of zinc is released from the exocrine pancreas. The exocrine pancreas plays an important role in zinc homeostasis (18, 19) During peri ods of zinc deficiency, zinc output is decreased. Similarly, during excessive zinc intake there is a mar ked increase in pancreatic out put of zinc. S erum zinc increase s with decreasing exocrine pancreatic function (20) P ancreatectomy increases serum zinc (21) and zinc deficiency decreases the pancreatic secretory response (22) Alterations in zinc metabolism have been reported in patients with pancreatic insufficiency. It was reported that a decrease in zinc output in duodenal aspirates occurs after secretion stimulation in pancreatic insufficient patients (23) The amount of zinc in pancreatic biliary secreti ons is also dependent on body zinc status (19) In addition to zinc secretion, the pancreas and liver have been shown to be the most responsive organs to Zninduced MT synthesis. Pancreatic zinc output is also found to be correlated significantly w ith enzyme and bicarbonate output, indicating that pancreatic zinc output could be a simple and accurate method for evaluation of the exocrine pancreatic function (24) Therefore, the adaptive mechanism of zinc metabolism in the human body will efficiently maintain zinc homeostasis under the conditions of varying dietary intake and physiological requir ements. The role of bile secretion in zinc metabolism is relatively less clearer. In my dissertation, special focus

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21 will be given to the upand downregulation of zinc transporters inv olved pancreatic zinc secretion. Zinc is also an especially important mediator in insulin synthesis, storage and secretion in pancreatic cells. The 3:1 molar ratio of insulin to zinc reveals that insulin is stored as a hexamer with two zinc ions in the c enter (25) Therefore, z in c is secreted cells Recently, a systemic search for the genetic variants in type 2 diabetes mellitus was conducted using highdensity array, which resulted in the genotyping of 392,935 singlenucleotide polymorphis ms. A nonsynonymous polymorphism in the zinc transporter 8, ZnT8 (SLC30A8), was identified among the four loci containing variants that confer type 2 diabetes risk (26) A considerable amount of zinc is secreted in pancreatic biliary secretions, and the actual amount depends on the body zinc status. Carboxypeptidase activity is lowered in zinc deficiency, and then returns to normal when given zinc supplementation (27) Zinc Absorption The primary site of zinc absorption is the proximal segment of the small intestine, including duodenum and proximal jejunum. There are many factors influencing zinc absorption. Dietary intake of zinc is one of t he major factors, especially longterm, chronic zinc intake, which can influenc e the zinc absorption rate. Other dietary factors can also influence zinc absorption, such as phytate, as an inhibitor Hence, zinc transporter expression is affected as well. Pancreatic zinc output was also found to be correlated significantly wit h enzyme and bicarbonate output, indicating that pancreatic zinc output could be a simple and accurate method for evaluation of the exocrine pancreatic function (24)

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22 Zinc Exc retion The gastrointestinal tract is the primary site of maintaining zinc homeostasis; both zinc absorption and excretion are regulated in the GI tract. Endogenous zinc can be lost through many different routes. Fecal zinc loss is the pr imary zinc loss route, and pancreatic zinc secretion is believed to be the most abundant source of fecal zinc loss Zinc can also be found in urinary excretions, integument loss, and in milk during lactation. Zinc Metabolism and Pancreatic Metallothionein The expression of m etallothionein ( MT ) induced by zinc through the MTF1 transcription factor has been well studied. The most responsive organs of MT induction by zinc treatment are the liver and pancreas, suggesting the importance of these organs in the z inc metabolism and homeostasis regulation. We and other groups found that the MT expression is dramatically and rapidly increased within 3 h after zinc injections or gavage. However, the induction returns to normal level 3 h after these treatment i n agr eement with the finding that a new steady state is quickly reached. MT is considered an important component of intracellular zinc homeostasis regulation. MT knock out mice display zinc homeostasis dysregulation and higher endogenous zinc loss into the gut because of relative ly lower zinc retention in the pancreas. In combination with the changes we have seen in the zinc transporter regulation, we can conclude that the zinc transporters work closely with MT in regulating the zinc secretion from the pancreas. On the other hand, MT might also modulate zinc homeostasis and metabolism in pancreatic islet cells and pancreatic endocrine neoplasms as those tissues contain matrix metalloproteinases, which require zinc in the catalytic domain (28)

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23 M etallothionein is an important component for reducing the efficiency of zinc absorption at elevated zinc intakes (29) The transgenic mice with multiple copies of the MT 1 gene, showed 10to 20fold greater MT protein levels in the pancreas (30) This over expression resulted in 300% more zinc in the pancreas. In contrast, MT levels decreased markedly when the mice were fed a Zndeficient diet, whereas MT in other organs decreased only moderately (31, 32) In MT knock out (MT / ) mice, the zinc secretion from the pancreas of subcutaneously administered 65Zn is more than twice that of wild type mice, and the absence of MT in the pancreas has been strongly implicated in causing this increase (33) The zinc concentration of the pancreas is lower in the MT knock out mice, which indicates that less zinc is sequestered with in the pancreas under steady state conditions resulting in a higher rate of endogenous zinc secretion from the exocrine pancreas (33, 34) M T 2 has also been found to be present in the pancreatic secretions (32) and this suggests that the MT2 isoform, which is more resist ant to degradation, may commit some pancreatic zinc to excretion (35) With adequate zinc intake, this difference in the handling of zinc in pancreas between MT knock out and wildtype mice does not seem to be detrimental. However, during zinc restriction or deficiency the decreased ability to limit secretion of zinc could be deleterious and may be one of the reasons why MT knock out mice are less able to withstand zinc deficiency (36) Zinc Deficiency Acrodermatitis enteropathica (AE) is a rare inherited autosomal recessive disease caused by severe zinc deficiency. The typical symptoms are periorificial and acral dermatitis, alopecia, and diarrhea in infants Two groups identified and described the SLC39A4 gene mutation in the AE patients (37, 38) There are m any different types of

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24 mutations spread over the entire gene. M ost of the patients with AE have homozygous or c ompound heterozygous mutations. However, some of them have eit her no mutation in Zip4 or mutation in only one allele. The molecular basis of AE seems to be a complex genetic defect therefore, there could be other molecular causes of dysregulation of t he SLC39A4 gene transcription. There are some other symptoms assoc iated with zinc deficiency not limited to AE, including hypogonadism, alopecia, impaired immunity, anorex ia, dermatitis and impaired wound healing. Dietary zinc deficiency is unlikely in a healthy wellnourished population. However, t here are also many ot her conditions and diseases caus ing secondary zinc deficiency. During zinc deficiency, p soriasiform dermatitis develops around the eyes, nose, and mouth, as well as on the buttocks. There are other symptoms including hair loss, low immunity, and recurrent infection, growth retardation, and diarrhea. Some other conditions related to zinc deficiency are malabsorption, d iabetes mellitus and stress (sepsis, burns, and head injury) causing acute phase response h epatic insufficiency, diuretics, s ickle cell disease and chronic renal failure. The e lderly institutionalized and homebound patients are also at risk of zinc deficiency (39) Cell Biology of Zinc Chelation. There are many types of zinc ion chelators being used in cell biology studies ( e.g. cell membrane permeable chelator, TPEN, and cell membrane nonpermeable chelators, DTPA, DPA etc ) These chelators show different zinc ion binding affinities, and specificities. For example, TPEN, has a high zinc ion binding specificity, but a low zinc binding affinity. Zinc chelation causes cell apoptosis, but the molecular mechanisms are not clear. A recent study shows that zinc chelation by TPEN can induce rapid depletion of the X linked inhibitor of apoptosis, which is the

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25 most potent member of the inhibitor of apoptosis protein. Thus, it sens itizes prostate cancer cells to TRAIL (TNFrel ated apoptosis inducing ligand) mediated apoptosis (40) There is future pot ential for zinc chelation to be applied as a new approach to cancer therapy. Zinc Toxicity The upper limit of recommended dietary intake for zinc is 40 mg/day. Prolonged high zinc intake will cause copper deficiency at a level of 100 150 mg/day for 6 8 weeks. That is due to the interference of copper absorption by high concentration of zinc in the lumen of the small intestine. The induced copper deficiency causes red blood cell microcytosis, neutropenia, and impaired immunity. Quantities of 200 800 m g/day can cause more acute and severe symptoms, including anorexia, vomiting, and diarrhea (41) Damage to the pancreas has also been well documented in birds and chickens (42, 43) Inhaling zinc oxide fumes in industrial workers is causing metal fume fever (also called brass founders ague or zinc shakes), and severe neurologic damage. A recent study suggests a novel pat hway, whereby zinc activates damagesensing TRPA1 ion channels in the nociceptive somatosensory neurons (44) Zinc Transporter Families There are two zinc tr ansporter families in mammalian cells, ZnT and Zip. ZnT (SLC30A) family is a group of zinc exporter, transporting zinc out of cellular cytoplasm to the extracellular space or intra cellular compartments and organelles. So far, there are ten different mem bers have been found in mouse and human, named from ZnT1 ZnT10 ( 3 5) Due to the expression level and their responsiveness to the zinc status, the two members of Z nT family, ZnT1 and ZnT2, will be the major focus of this study.

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26 The ZnT (SLC30) Family The members of ZnT family share certain similarity in their protein structure and membrane topology. Their amino acid sequences share high similarity. For example, they are predicted to have six transmembrane domains (TMDs), and a histidinerich loop (HX)3 6 between TMD IV and V, except for ZnT6, which has a serinerich loop in place of histidinerich loop. The histidi nerich loop is essential for ZnT5 and ZnT6 heter ooligomeric structure (45) The expression of most zinc transporter s show high tissue specificity. ZnT1 is the first discovered mammalian zinc transporter (46) ZnT2 was first cloned from a rat kidney cDNA library by complementation and was found to be localized to acidic vesicles and to facilitate zinc sequestration in intracellular compartments (47) ZnT2 expression is limited primarily to the pancreas, mammary gland, prostate, small intestine, kidney, and place nta (48 50) When dietary zinc intake is low, ZnT2 expression decreases in some tissues e.g. pancreas (51) Previously we found ZnT1 is regulated by dietary zinc in both small intestine and liver (52) A mis sense mutation (H54R ) in ZnT2 in women produces low breast milk zinc content, and results in neonatal zinc deficiency in breast fed infants (53) That finding suggests an important role of ZnT2 in secretory pathways for zinc in mammary gland. I n human fibroblastoid cells, ZnT2 can facilitate vesicular z inc accumulation independently of AP 3, which might regulate the trafficking of ZnT family members to late endosomes and/or lysosomes (54) The major objectives of the study on the zinc transport and metabolism in the exocrine pancreas :

PAGE 27

27 1) Identifying zinc transporter(s) regulated by dietary zinc intake in the pancreas. And determining the m olecular mechanism of the regulation of zinc transporter expression by dietary zinc. 2) Determining the signaling pathway involved in glucocorticoids induced the expression of ZnT2 in the pancreatic acinar cells. 3) Localization and functional characterization of ZnT2 in the pancreatic acinar cells. 4) Preliminary study on the expression of zinc transporter in pancreatitis and pancreatic cancer.

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28 CHAPTER 2 MATERIALS AND METHODS Animal Experiments CD1 male mice, 2530g ( about 2 3 mo old, Charles River) were individually housed and fed a AIN76based diet (Research Diets) formulated with egg white protein containing 0.85 mg Zn/kg diet or 30 mg Zn/kg diet for 21 days Body weight and food consumption were measured every 7 days. Mice were killed on day 21. Blood was drawn by cardiac punct ure and serum was isolated. These methods hav e been described previously (51) Liver and pancr eata were removed, and excised pieces kept in RNA later (Applied Biosystems) at 4 C. The remainder of the pancreata were kept in ice cold phosphatebuffered saline until all the pancreata were collected. Crude nuclei mitoch ondria, zymogen granules, golgi and plasm a membranes and cytosol fractions were isolated as described in detail below In some experiments, DEX phosphate was injected interperitoneally at 10mg/kg body weight Alternatively, z inc sulfate dissolved in 0.9% NaCl was given orally ( g body weight or 0.9% NaCl alone) to the mice. Mice were sacrificed at 3 and 8 h after the gavage. Blood was drawn and tissues were collected for RNA isolation and zinc analysis. The procedures with mice were approved by the University of Florida Instituti onal Animal Care and Use Committee. RNA Extraction and Quantitative Real Time PCR Real Time PCR was performed by either onestep or twostep method. The onestep RTPCR was directly from the total RNA samples, while the twostep RTPCR was from reverse tra nscribed cDNA samples. Tissues were excised, and immediately homogenized (Polytron) with TRI reagent ( Ambion ). Total mRNA was isolated by TRIzol, and stored in DEPC water. The total RNA was treated with DNase f or 30min

PAGE 29

29 (Ambion) before RTPCR. TaqMan unive rsal PCR master mix was used as the reaction solution. 18S rRNA primers and the probe were from the TaqMan ribosomal RNA reagent kit (Applied Biosystems) were used for normalization. The RNA was reverse transcribed by reverse transcriptase using iScript reagents (Bio Rad) for 30 min at 42C, followed by 95 C for 10 min and then 95 C for 15 s and 60 C for 1 min for a total of 40 cycles. The reaction was performed and fluorescence detected with an iCycler instrument (Bio Rad). Standard curve and RNA sample reactions were run in triplicate with the standard curve over a 6 log range. In some experiments total RNA was isolated using TRI reagent (Applied Biosystems), and treated with TurboDNase (Applied Biosystems) for 30 min before RT PCR. Primer Express version 3.0 software (Applied Biosystems) was used to design the oligonucleotide primers and TaqMan probes. cDNA s were synthesized using high capacity cDNA reverse transcription (Applied Biosystems) with a thermal cycler (MJ Research). Quantitative Real Tim e PCR reactions were performed and fluorescence was detected with the StepOnePlus system (Applied Biosystems). Values were normalized to 18S rRNA. Isolation and Culture of Pancreatic Acinar Cells The murine pancreatic acini was prepared by limited collagenase digestion using a well established procedure (55) The pancreata were removed from mice minced and was incubated with a buffer containing 140 mM NaCl, 5 mMKCl, 1.8 mM CaCl2, 4.2 mM NaHCO3, 0.8 mM MgSO4, 10 mM HEPES, 10 mM g lucose and 0.1% bovine serum albumin (BSA). The buffer was supplemented with 10mM glucose and 0.02% soybean trypsin inhibitor and 2.5mg of collagenase. T he pancreatic tissue was incubated in the solution for 60 min at 37 C. After digestion, the acinar cells were separated by gradient centrifugation. Then the acinar cells were transfer red to the collagen precoated slide

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30 wells, and cultured in RPMI 1640medium. The cells were counted, and viability was determined. Zymogen Granules Isolation The zymogen granules were isolated from acinar cells on a Percoll gradient by the method described previously (56, 57) Briefly, pancreata were finely minced and homogenized in buffer containing, 0.3M sucrose 2nM MOPS, pH 6.8 and protease inhibitors (S igma). Nuclei and cellular debris were removed by centrifugation at 750 g for 10 min, and the supernatant was centrifuged at 1750 g for 20 min. The resulting pellet was resuspended in the same buffer mixed with Percoll and zymogen granules were further purified by separation with Percoll gradients at 60,000 g for 30 min. Mitochondrial and zymogen granule fractions were collected. FluoZin3 AM Labile Zinc Quantification The cellular labile Zn2+ was detected using fluorescence microplate reader by incubating the cells wi th the fluorescence Zn2+ indicator probe, FluoZin3 AM (Invitrogen) The experimental procedure was modified from the previous described method (58) Briefly, cells were loaded with FluoZin3 AM for 30 min at 37 C, and washed with PBS, and resuspended in DMEM supplemented with 0.3% BSA. Aliquots of the cell suspensions were incubated with TPEN, or zinc/pyrithione. The concentration of intracellular labile zinc was calculated from the mean fluorescence with the formula [Zn] = K d [(F (59) The dissociation constant of the FluoZin3/zinc complex is 15 nM. Fmin was determined by the addition of the zinc specific, membranepermeant chelator TPEN, and Fmax was determined by the addition of ZnSO4 and the ionophore pyrithione.

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31 Immunoblotting and Immunohistochemistry Protein concentrations were measured spectrophotometrically with Rc Dc reagents (BioRad) Equal amount s of protein were resolved on a 10% SDS PAGE gel and transferred to nitrocellulose membranes (Whatman). Blots were stained with Ponceau S and destained. For western detection, blots were blocked in Tris buffered saline (TBST) containing 0.1% Tween 20, 5% nonfat dry milk for 1 h (60) Affinity purified antibody then was applied at 2.0 g/ml for 1 h. After washing in TBS T, diluted (1:10,000) secondary ant i rabbit IgG horseradish peroxidase conjugated antibody (GE Healthcare) was applied. The blots were visualized by enhanced chemiluminescence by West Pico Chemiluminescent substrate (Pierce) and exposed to x ray film for detection. The tissue was fixed in 10% formalin in PBS buffer, and then embedded in were mounted on poly L ysine coated cover slips. Paraformaldehyde was used to treat the cells and was followed by permeabilization with Triton X 100. The tiss ue sections and permeable cells were incubated with primary antibodies and Alexa conjugate secondary antibody. Fluorescence images were obtained on an Axiovert 100 microscope. 65Zn Uptake and Efflux Assay Zinc uptake and efflux were measured using 65Zn. The cells were incubated with 65Zn2+ 30nCi/500ul, and then were washed and solubilized in 1%SDS, 0.2M NaOH solution. 65Zn was ray spectrometer and the total protein was measured using the BioRad RC DC protein assay reagent. s iRNA Mediated Knockdown The recent ly established RNAi technology for gene knock down gives us an alternative method for sequence specific post transcriptional gene silencing. Four s mall

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32 interfering RNA duplexes targeting the coding sequence s of ZnT1 and ZnT2 were synthesized targeting a distinct region of each gene. The mixture of these four siRNAs were transfected into AR42J cells. After transfection and a 48 h incubation, the mRNA and protein were measured, and zinc uptake and excretion assay s were done. Culture of Rat AR42J Acinar Cells AR42J cells (rat pancreatoma, ATCC CRL 1492) was purchased from American Type Culture Collection and were maintained at 37 C in Hams F12K medium (Mediatech) with 0.1 mg/ml L Glutamine, 15% fetal bovine serum (Mediatech) and 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 humidified atmosphere containing 5% CO2 (61, 62) C ells at 0.5 106 /well in 6 well plates were cultured for at least 48 h before treatments. C ells were treated with 100 nM DEX phosphate (Sigma) in culture medi um for 48 h for differentiationinduction. Some cultures also contained 1 M RU486 (63) or 1 M CpdA (64) as GR antagonist and selective agonist, respectively Zinc chelators : DPA (dipicolinic acid), DTPA (Diethylene triamine pentaace tic acid), and TPEN (N,N,N t etrakis(2 pyridylmethyl)ethylenediamine) were used separately to induce zinc deficiency in the cells (60) The cells were cultured in m edium supplemented with 15% DPA dialyzed fetal bovine serum, which was tested and confirmed to have a low concentration of zinc. The 4 added as a control medium. Alternatively, DTPA ( 50 ) or TPEN ( 3.5 ) were added to the cell culture medium, dissolved in PBS and DMSO respectively. C ontro l cultures contained PBS and DMSO, respectively at comparable concentrations. For siRNA experiments SMARTpool siRNA, and nontargeting pool negative control siRNA resuspended in siRNA buffer were purchased from Dharmacon (Thermo Scientific ). The cells were

PAGE 33

33 seeded at 1.0 106 cells/ well in 12 well plate. Immediately after seeding, the cells were transfected with 300ng siRNA using HyperFect transfection reagent (Qiagen). Scrambled negative control siRNA (Dharmacon, Thermo Scientific ) transfected cells were considered as negative control. Atomic Absorption Spectrophotometry Tissue samples were weighted and digested in concentrated HNO3 at 90 C T he zinc concentration absorption spectrophotometry was measured using air acetylene. Zinc concentrations of subcellular fractions were measured similarly after aliquots were removed for measurement of protein (as above) for normalization. Transfection and Luciferase Assay HeLa and COS 7 cells were seeded at 1x105 cells/ well in 48 well plates. Transfection began 12 h after seeding with 15 nM (final concentration) of siRNA for mMTF 1 (Smart Pool, Dharmacon) using HiPerFect transfection reagent (Qiagen), and was carried out for 48 h. For the luciferase assays, HeL a cells were seeded on 12well plates and transfected with 1 g pGL3 or pGL4 plasmid and 0.001 g pRLSV40 plasmid (Promega), as an internal control, using Effectene reagent (Qiagen) or FuGene HD (Roche) After a 48 h incubation, the cell medium was replaced by medium with or without 100 nM DEX or 40uM ZnSO4. After a 24 h incubation, ce lls were washed with PBS and lys ed by 100 l Passive Lysis Buffer (Promega) per well. Luciferease activities were measured with the Dual GloTM Luciferase Assay System (Pro mega) in a SpectraMax M5 microplate reader by following the manufacturers protocol. The raw values of firefly luciferase were normalized to renilla luciferase that transfected concurrently in all the assays to correct for differences in transfection effi ciency. The promoter activity assays were measured in triplicate in each experiment and shown as

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34 fol d change relative to control At least three sets of independent experiments were performed for each set of constructs. Statistical Analysis Results are expressed as mean SD from representative one of three independent experiments. The significance of variability was determined by an unpaired 2tailed Students t test or by twoway ANOVA. P 0.05 was accepted as statistically significant.

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35 CHAPTE R 3 ZINC REGULATES EXPRESSION OF ZINC TRANSPORTERS ZNT1 AND ZNT2 IN PANCREATIC ACINAR CELLS Introduction Over 90% of the pancreas is co mprised of exocrine cells (65, 66) Localization studies have shown that pancreatic zinc is concentrated in the granules of acinar cells, where digestive proenzymes are also stored prior to exocytosis through the apical membrane for entry into the intestine. Abnormal zinc metabolism has been r eported after pancreactomy (21, 66, 67) Zinc deficiency is believed to caus e pancreatic acinar cells to become depleted of zinc (22, 68) Radiotracer studies have shown that the pancreas exhibits high rates of Zn turnover (6972) The exocrine pancreas is a target organ of zinc toxicosis because of the highrate zinc flux Excessive dietary zinc alters acinar cell structure, and produces necrosis, causing depletion of zymogen granules and reduces digestive enzyme secretion (42, 73, 74) This sensitivity to zinc and high rate of zinc flux support the need for tight regulation of pancreatic acinar cell zinc transport. Identify ing the A bundance and L ocalization of Zip5, ZnT1 and ZnT2 in Acinar Cells The transport process for zinc release is proposed to involve three different cellular events. The transport of zinc into the acinar cell take s place at the basolateral surface. This bloodto exocrine flow of zinc is facili tated by a zinc importer, probably ZIP5, which has been shown to be expressed in pancreas acinar cells and localized to the basolateral membrane when dietary zinc is adequate. When zinc status is low, the basolateral localized ZIP5 is internalized in response to zinc deficiency (75) The intracellular compartmentalization of Zn2+ may occur concurrently with the formation of zymogen granules, where the zinc transporter ZnT2 was suggested to be localized and

PAGE 36

36 to mediate Zinc compartmentalization (51) Eventually, stimulation of acinar cells by secretagogues triggers fusion of zymogen granules with the apical membrane and the subsequent release of the content of zymogen granules (76) The ZnT1 is the primary cellular zinc efflux transporter and it appears to be the predominant ZnT family member at the plasma membrane. Therefore, ZnT1 may facilitate zinc efflux across the apical membrane. However, ZnT1 localizes not only to the plasma membrane, but also to the intracellular vesicles (46, 51) Determining the Effect of Exogenous Zinc Concentration on ZnT1, ZnT2 and MT Gene Expression Levels in AR42J Cells T he exocrine pancreas is the major route of human endogenous zinc loss, and the aim of this study is to understand how zinc is released and the role of zi nc transporters in this process. Therefore, it is important to fully understand the interaction between body zinc status and intracellular zinc and their regulation of ZnT1, ZnT2, and MT gene expression. Briefly, AR42J cells were cultured in F12K medium with 15% fetal bovine serum for 2 days. The zinc content in this standard medium was determined by a tomic a bsorption spectrophotomet ry. The cells were pretreated with 100 nM dexamethasone for 48 h prior to being placed in medium containing 5 1 0 20 40 80 120 and 160 Zinc as ZnSO4. The cells were harvested at 6 h post treatment. Relative amount s of ZnT1, ZnT2, and MT mRNA s were quantified by the TaqMan q PCR method. Cells were also cultured in medium with 40 ZnSO4 for a 16hour study. The cells were harvested, as above at 0, 2 4 6 8 and 16 hour time point s post zinc treatment. ZnT1, ZnT2 and MT mRNA levels were measured as above

PAGE 37

37 Zinc Transporters Experiment in Murine Pancreatic Zymogen Granules. The zymogen granules in pancreatic acinar cells ar e specialized organelles, where digestive enzymes are stored. The pancreatic secretion of zinc markedly increased over baseline when CCK infusion stimulates the exocytosis of zymogen granules in human subjects (35) Thus we speculate that, with an adequate dietary zinc intake, endogenous zinc in acinar cells is packaged via ZnT2 into a form that allows elimination with stimulated pancreatic secretions. Zinc deficiency causes a reduction in pancreatic zinc secretion and a reduction of the need for zinc packaging for export. Thus ZnT2 and perhaps other zinc transporters are related to zymogen granule formation for sequestering and providing zinc to the metalloenzymes and other constituents inside these granules. H erein, mouse pancreatic acinar zymogen granules were isolated from the animals fed with high zinc and zinc deficient diets Zinc content s were measured by atomic absorption spectrophotometr y. Granule membrane fractions were used to detect ZnT/Zip proteins associated with the membrane by immunoblotting. As described above, amylase is a loading control for zymogen granules. Amylase is a zymogen granule content protein, which is used as a marker for organelle enrichment using a n antibody against mouse amylase (77) The zinc concentrations in the zymogen granules were normalized to zymogen granule protein content Determining the E ffect of Low Exogenous Zinc on ZnT1, ZnT2 and MT gene E xpression Levels in AR42J cells. Similar to the rationale of the zinc treatment experiment s, zinc deficient status causes the reduction of zinc output from pancreas secretions. Zinc deficiency also decreases the pancreatic secretory response. D ipicolinic acid (Sigma) was used to chelate zinc from fetal bovine serum by over night

PAGE 38

38 dialysis (78) Picolinic acid is a metabolite of tryptophan and is considered a zinc chelator (79) Dipicolinic acid is a membraneimpermeable zinc selective chelator. It appears to be relatively well tolerated in patients with pancreatic insufficiency (80) It has been demonstrated that adding pic olinic acid to standard diets increased the amount of zinc actually absorbed So it is a more physiological zinc binding ligand than TPEN, which is a cellpermeable zinc chelator. TPEN causes apoptosis at low concentrations. In this study, the F12K medium with 15% dipicolinic acid dialysed FBS produced a medium with a zinc concentration as low as 0.45uM ZnT1, ZnT2, and MT mRNA levels were measured by the TaqMan q PCR method. The protein level changes were measured through immunoblotting and the localization of the transporters was also shown by immunocytochemistry Results M ice fed a zinc deficient diet developed signs of zinc restriction in 3 weeks, as shown by dramatically decreased ZnT1, ZnT2 and MT expr ession (Fig.31) as well as a depressed serum zinc concentration (Fig. 3 2 ). However, high zinc diet did not alter the expression of ZnT1, ZnT2, or MT. In order to understand how zinc affects the pancr eas, the zinc concentrations in subcellular fractions were measured. Zinc restriction resulted in pancreatic cytoplasm and zymogen granules having less than half the amount of zinc found in mice fed the zinc adequate diet (p < 0 .0 1 ). Interestingly, zinc concentrations of the mitochondria (data not shown) and c rude nuclear fractions were not reduced by zinc restriction (Fig. 3 2 ). By contrast, t he cytoplasmic and nuclear fractions from the liver did not change significantly (Fig. 32) Previously we showed that o f the 1 5 zinc transporter gene transcripts surveyed, only two, ZnT1 and ZnT2 mRNAs were differentially regulated by zinc restriction (51) The relative transcript levels of MT ZnT1, and ZnT2 in

PAGE 39

39 pancreatic RNA from the mice used in the present studies are shown in Fig. 3 1 These responses show the sensitivity of these zinc transporter genes to the dietary zinc intake level. Zip5 had previously been found in the pancreas, and its protein levels at the plasma membrane are regulated by zinc status (75, 81, 82) The immunoblotting results confirmed the disappearance of Zip5 during zinc deficiency (Fig. 33). Western blotting clearly showed the decrease of ZnT1 in the plasma membraneenriched fract ion during zinc restriction (Fig. 34, Fig. 35 ) The greater abundance of ZnT1 in the plasma membrane is consist e nt with the zinc efflux function of this transporter (46) Of particular interest is that ZnT2 was exclusively detected in the isolated zymogen granule fraction and showed a reduction in response to dietary zinc restriction (Fig. 34, Fig. 3 5 ) This novel finding of ZnT2 localized to isolated zymogen granules was confirmed by immunofluorescence confocal microscopy (Fig. 37 ). Consist e nt with secretory vesicle trafficking the presence of some ZnT1 in the zymogen fraction is to be expected. Also of note is that the two marker proteins for plasma membranes and zymogen granules, Na+/K+ ATPase and amylase respectively, were unaffected by zinc restriction. g body weight was given orally pancreatic zinc content increased (Fig. 38 Fig. 39 ) and there were transient elevations in pancreatic MT, ZnT1, and ZnT2 mRNA s ( Fig. 310). These results show ed both ZnT1 and ZnT2 were very se nsitive to elevated zinc intake, and suggested crucial roles in endogenous zinc excretion and homeostasis No ne from the Zip family was found to have changed at the level of mRNA expression (Fig. 31 1 ). Rat AR42J pancreatic acinar cells were used as a model to further understand the regulation of MT, ZnT1 and ZnT2 n pancreatic acinar cells. Various concentrations

PAGE 40

40 of zinc were treated to the cells, causing a dose dependent upregulation of ZnT1 mRNA expression (Fig 31 2 ) The highest ZnT1 mRNA level was fo und at 160 M zinc, a much higher concentration than the concentration causing zinc toxicity to other types of cells. In contrast to ZnT1, the expression of ZnT2 did not response to supplemental zinc in the medium in the same cells (Fig 31 2 ). The MT mRNA was significantly upregulated by supplemental zinc and the responsiveness was shown to be dose dependent (Fig. 31 3 ) This result as well as previous published findings, indica ted a high MTsynthesizing capacity in the pancreas when there is an excess o f zinc. By expressing a high level of MT, the pancreatic acinar cells can retain and tolerate a great amount of zinc This transient accumulated zinc in the pancreas can be secreted gradually through pancreatic secretions. High zinc secretion and low retention were observed in MT knock out mice. Because the metal responsive transcription factor MTF1 regulates both MT and ZnT1 genes, it was not surprising to see a similar response with the ZnT1 gene (Fig 3 1 2 Fig. 31 3 ) However, this is not the case of ZnT2 expression in AR42J cells treated with zinc. No zinc induced mRNA expression increase was found (Fig. 31 2 Fig. 31 3 ). The mechanism is not clear here. It is speculated that hypermethylation of the ZnT2 promoter might cause the gene silencing. This will be discussed more in detail later. To better understand the temporal regulation of MT and ZnT1 by zinc through MTF1 activat ion, MT and ZnT1 mRNA abundance were measured in a 48 h time course with 40 zinc Both MT and ZnT1 mRNA levels were regulated in a time dependent manner (Fig. 31 4 ). MT mRNA reached the peak level around 60fold at 18 h and gradually decreased (Fig. 3 1 4 ) In contrast, ZnT1 showed a rapid increase at 6 h

PAGE 41

41 for a 3fold elevation, and the mRNA abundance kept at 2to 3fold higher than control throughout the rest of the 16 h (Fig. 31 4 ) ZnT1 protein level changes were confirmed by wes tern blotting results (Fig. 31 5 Fig. 31 6 ). Although both genes are transcriptionally regulated through zinc coupled MTF1 activation, the expression patterns were found to be different. Again, we did not observe a transcriptional activation of the ZnT2 gene in this experiment (Fig. 31 4 ). AR42J cells were also used in a cell culture model of zinc deficiency. Cells were cultured in a low zinc medium Three different types of zinc chelators were tested in this series of experiments: DPA, DTPA, and TPEN. All three zinc chelators successfully induced zinc deficiency in AR42J cells in the culture medium, as confirmed by MT mRNA level as a sensitive cellula r zinc ind icator gene (Fig. 31 7 Fig. 31 8 ). B oth ZnT1 and ZnT2 gene expression were found to be decreased after 24 h in low zinc culture medium at mRNA level (Fig. 3 1 7 3 1 8 ) and protein level (Fig. 31 9, 320). A mZnT1 expression plasmid was transfected into HEK 293 cells for an over expression study twenty four, thirty six and forty eight hours after the transfection, cells were harvest ed and mZnT1 production was measured by western blotting with a polyclonal antibody raised against a ZnT1 peptide in a rabbit. A 60kDa band was detected at 36 h and 48 h after transfection, but not 24 h (Fig. 32 1 ) This 60kDa band match ed the expected molecular weight of mouse ZnT1. Immunofluorescence microscopy im ages showed that under membranepermeable condition s, high fluorescence signals were observed to be located not only to the plasma membrane, but also intracellular compartments (Fig. 3 2 2 )

PAGE 42

42 There are many studies that have shown a difference regarding the size of ZnT1 on western blotting, and the reason is not clear. To validate and confirm the correct bands for ZnT1, total IgG and affinity purified antibodies were compared in western blotting. Soluble protein fractions and membrane protein fractions of AR42J cells were revolved on PAGE gel s. The back ground was much less intense, and two clear bands of ZnT1 at about 36kDa and 20kDa were shown on western blotting by using the affinity purified antibody (Fig. 32 4 ) B oth of these two bands could be competed out by preincubating primary antibody with the peptide, to which the polyclonal antibodies were raised against. Another weak band around 60 kDa was also observed in ZnT1 membrane fractions, but not seen in soluble fraction. None of these three bands (60 36, and 20kDa) were changed by peptide N glyc osidase digestion, suggesting that there is no N glycosylation present in ZnT1 protein. Suprisingly, Nglycosyl ation modification was found in human ZnT1 protein with the total membrane protein sample prepared from HEK 293 cells as shown in Fig. 324. The same glycosylation was also found in other zinc transporter members (38, 83, 84) However t he significance and functionality of this post translati onal modification is not clear. The multiple band sizes of ZnT1 on western blot make it difficult to interpret the result s. The calculated molecular weight of ZnT1 is about 60kDa. However, a n all w estern analysis of ZnT1 from the lab, 36kDa band is a predominant one, present in the total membrane fraction. In some experiments, when the protein samples of plasma membrane and ZG membrane were prepared through a gradient centrifugation, this 36kDa ZnT1 band could still be found in both factions, even more intense band in ZG membrane. I suspect this 36kDa band is a short form of ZnT1 present in secretory

PAGE 43

43 vesicles in pancreatic acinar cells. When secretory vesicles are fusing to the plasma membrane to release their content the membrane of these vesicles becomes a part of plasma membrane. Thus, presence of some 36kDa ZnT1 in the plasma membrane is to be expected. However, the 60kDa band in ZnT1 western blot might have a distinct localization. Based on the calculated molecular weight, the 60kDa band would likely be the full length ZnT1 protein. The calculated molecular weight of ZnT2 protein is 48kDa; H owever band size of ZnT2 in western blotting has not been well documented. Herein, we tested our ZnT2 polyclonal antibody on total soluble and membrane protein samples from AR42J cells. By comparing total IgG and affinity purified antibody, a distinct 42 kDa band was found with much less background signal when probing with affinity purified antibody than total IgG antibody (Fig. 32 5 ) A peptide adsorption experiment showed a successful elimination of the 42kDa band, suggesting this band is a specific target of our polyclonal antibody binding (Fig. 32 5 ). This 42 kDa band could not be altered by peptide N glycosidase treatment to the total membrane protein samples (Fig. 32 4 ). Discussion Z inc transporter expression in the pancreas is of interest because pancreatic secretions constitute an important component of mammalian zinc homeostasis (22, 68, 72, 8589) ZnT1 and ZnT2 are expressed in the pancreas (51) and they are associated wit h isolated plasma membrane and zymogen granules, respectively. In this study we focus on regulation of the zinc transporter ZnT2 and have identified the role it may play in an endogenous zinc secretory pathway within pancreatic acinar cells. We propose th at zinc output from acinar cells follows two distinct pathways: cell to ductal zinc efflux via the apical membrane, which is zinc dependent and involves primarily ZnT1 for

PAGE 44

44 cellular efflux ; and zinc that is released along with digestive proenzymes from zymogen granules, where zinc is transported into the granules by ZnT2. These respective functions for ZnT1 and ZnT2 are in agreement with current proposed roles in zinc transport in other cell types (90) Zinc in stimulated pancreatic sections has been shown to be associated with highmolecular weight proteins, and is closely associated with the enzyme activities of zinc containing enzymes, especially carboxypeptidase A and carboxypeptidase B (22, 68, 85) Since zymogen granules are the storage sites of digestive enzyme precursors (91) our finding that ZnT2 influences zinc incorporation into zymogen granules suggests this transporter may provide zinc for inc orporation into digestive prometalloenzymes or maintain a zinc rich environment for the holo metalloenzymes for full activity upon release. The reduction in activity observed during dietary zinc restriction is in agreement with our finding that transcript abundance for nine pancreatic digestive enzymes is not influenced by the dietary zinc restriction model used in the present experiments (data not shown). Acute zinc toxicity has been shown in avian species, mice, and the pig to be detrimental to normal pancreatic exocrine function and produces pancreatic atrophy (42, 73, 74) This sensitivity suggests that secretory pathways of zinc loss, are essential for preventing pancreatic enzyme release, necrosis a nd atrophy. These signs of zinc toxicity are similar to the autodigestion of pancreatitis which is traced to abnormal calcium signaling within zymogen granules (92) The secretory zymogen granules hav e an acidic intragranule pH (93) w hich is in line with the notion that ZnT2 favors acidic vesicles for its maximal transport activity (47) It is relevant that ZnT8, expressed almost exclusively in the pancreatic Beta cell,

PAGE 45

45 facilitates zinc transport required for proinsulin aggregation, using a process t hat involves proton exchange (94, 95) The zinc and MT content of the pancreas are among the highest among tissues under normal conditions of dietary zinc intake Such high expression suggests MT has an important role in regulating zinc metabolism and function in the pancreatic acinar cells. MT declines almost completely with a zinc deficient diet, whereas in other organs it decreases only moderately (96) Pancreatic zinc secretion i n MT/mice is much higher than in wild type mice and they are more vulnerable to damage caused by zinc deficiency (33, 97) MT is also found in pancreatic secretions, however, through a route that does not involve granule secretory pathways (32) In that regard, MT bound zinc in pancreatic secretions could provide an endogenous zinc source for zinc reabsorption. Studies that could reflect on the glucocorticoid responsive expression of ZnT2 and a role in pancreatic zinc secretion are limited. It has been reported that adrenal insufficiency increases serum zinc concentrations while administration of glucocorticoids and ACTH and the excess control production in Cushing s Syndrome decrease these concentrations (reviewed in (98) ) Hypozincemia associated with glucocorticoid action has been related to induced synthesis of MT in rodents (99) Radiotracer kinetic studies with Zinc 69m given intravenously to humans revealed that carbohydrateactive steroids (glucocorticoids) may alter rate constants of the fecal excretion of zinc (98) The pancreatic acinar AR42J cell model has been widely used to characterize effects of gluc oc orticoid hormones on secretory activity of the exocrine pancreas (62, 100) Dexamethasone treatment of AR42J cells induces a highly differentiated

PAGE 46

46 phenotype. It has been reported that RU486 can reduce GR concentrations in AR42J cells by 50% with concomitant changes in GR regulated gene expression (101) That ZnT2 regulation is controlled by GR was established by demonstrating inhibition through RU486. This strongly suggests that the signaling pathway involv es GR dimerization, rather than the anti inflammatory pathway involving assembly of the NFkB GR dimer complex. GR involvement in pancreatic tissue organization and the differentiation of acinar cells, as well as enzyme and zymogen granule production, is compatible with a role in ZnT2 regulation. Furthermore, the control zinc provides via MTF1 responsive ZnT2 expression is consistent with a role in endogenous zinc secretion. That control, combined with the DEX responsiveness, makes ZnT2 analogous to MT i n terms of a teleological basis for dual regulation. To our knowledge ZnT2 may represent the first member of either the ZnT or Zip gene families to be glucocorticoidregulated. The next chapter will focus on this topic.

PAGE 47

47 Figure 31 ZnT1, ZnT2 and MT mRNA expression in mouse pancreas after 3 wks of dietary zinc restriction (ZnD) or zinc supplementation (ZnS) qPCR was used to measure the relative mRNA abundance change to zinc restriction and supplementation diet and the results were expressed as the percentage of ZnA control Y axis is in log scale. ZnA is zinc adequate control (n = 34 ). Percentage of ZnA controls

PAGE 48

48 Figure 3 2 Zinc concentrations in serum pancreas and liver from mice fed with zinc adequate or deficient diet. CD 1 mice were fed zinc ade quate or deficient diets for 3 wks Blood was drawn and serum was isolated. The pancreas and liver were collected and homogenized for cell fractionation. Cytoplasm, nucleus, and zymogen granule compartments were isolated and the zinc concentrations were measured by atomic absorption spectrophotometry and normalized against the protein concentration. The values are expressed as the mean of mg Zn /g protein standard deviation (n=3). ZnA ZnD

PAGE 49

49 Figure 33 Zip5 expression in mouse pancreas after 3 wks dietary zinc restriction (ZnD ) or a zinc adequate diet Mouse pancreas was removed and fractionated, plasma membrane (PM) protein and zymogen granule (ZG) were obtained. Western analysis showed Zip5 protein abundance in plasma membrane fraction. N o zip5 was detected in ZG fractions. Na+/K+ATPase is the loadi ng control D, is for zinc deficiency; A, is for zinc adequate. A nonspecific band (slightly lower than the Zip5 band) is detected in the plasma membrane (PM) and resistant to peptide competition.

PAGE 50

50 Figure 34 ZnT1 and ZnT2 western blotting in mouse pancreas after 3 wks dietary zinc restriction ( ZnD). Mouse pancreas was removed and fractionated, pl asma membrane enriched fraction (PM) and zymogen granule (ZG) were purified. Western blots here showed the protein abundance of ZnT1 and ZnT2 in plasma membrane, and zymogen granule. Na+/K+ ATPase is the plasma membrane loading control. Amylase is the zymogen granule loading control. ZnA is dietary zinc adequate control

PAGE 51

51 Figure 35 ZnT1 western blotting in mouse pancreas after 3 wks dietary zinc res triction (ZnD). Mouse pancreas was removed and fractionated, plasma membrane enriched fraction (PM) and zymogen granule (ZG) were purified. Western blots here showed the protein abundance of ZnT1 in plasma membrane, and zymogen granule. Na+/K+ ATPase is th e plasma membrane loading control. Amylase is the zymogen granule loading control. ZnA is dietary zinc adequate control

PAGE 52

52 Figure 36 ZnT1 western blotting in mouse pancreas after 3 wks dietary zinc restriction (ZnD). Mouse pancreas was removed and frac tionated, crude nuclear fraction (Nuc) with rough ER (rER), mitochondria (Mito), plasma membrane enriched fraction (PM) and zymogen granule (ZG) were purified. Western blots here showed the protein abundance of ZnT2 in each fraction. Amylase is the zymogen granule loading control. VDAC1 is the loading control for mitochondria. A, is dietary zinc adequate control.

PAGE 53

53 Figure 37 Confocal immunofluorescence analysis of ZnT2 in purified zymogen granules isolated from mouse pancreas.

PAGE 54

54 Figure 38 Zinc concentrations in the brain, pancreas and liver of mouse 3 h after a zinc gavage (ZnG). Saline was given as control (con). Tissue zinc concentrations were measured by at omic absorption spectrophotomet r y and normalized against wet wight (n = 3).

PAGE 55

55 Figure 3 9 Zinc concentrations in the pancreas, liver and serum in mouse 3h and 8h after zinc gavage. Saline was given as control. The pancreas, liver and serum were collected, and tissue samples were digested in nitric acid Zinc concentrations were measured by atomic absorption spectrophotomet r y, normalized against tissue wet weight The values from mice given zinc by gavage (Zn) are expressed as bars, and the saline controls are expressed as lines (n=34).

PAGE 56

56 Figure 310 ZnT zinc transport er family mRNA expression in the pancreas from mice 3 h and 8 h after zinc gavage of 35 mg zinc / g bwt Saline was given as control (n = 34 ).

PAGE 57

57 Figure 311 Zip zinc transporter family mRNA expression in the pancreas from mice 3 h and 8 h after zinc gavage of 35 mg zinc / g bwt. Saline was given as control (n = 34 ).

PAGE 58

58 Figure 3 1 2 Zinc influences ZnT1 mRNA levels in a dose dependant manner in AR42J cells. The cells were pretreated with dexamethasone (100 nM) for 48 h. Different concentrations of ZnSO4 the culture medium for 6 h q PCR was used to measure ZnT1 and ZnT2 mRNA levels after zinc treatment. A f itted correlation line is shown (n = 3).

PAGE 59

59 Figure 3 1 3 Zinc influences MT 1, and ZnT1 mRNA levels in a dose dependant manner in AR42J cells. The cells were pretreated with dex amethasone (100 nM) for 48 h. Different concentrations of ZnSO4 the culture medium for 6 h q PCR was used to measure MT (A), ZnT1 (B), and ZnT2 (C) mRNA levels after zinc treatment. Fitted linear correlation and Pearsons correlation coefficient (R) are shown (n = 3).

PAGE 60

60 Figure 3 1 4 Zinc influences MT and ZnT1 mRNA levels in a time dependant manner AR42J cells. The cells were pretreated with dex amethasone (100 nM) for 48 h ZnSO4 16 h. The c ells were collected at various time point s post treatment. The q PCR was used to measure MT (A), ZnT1 (B), and ZnT2 (C) mRNA levels after zinc treatment (n = 3).

PAGE 61

61 Figure 31 5 Influence of zinc on ZnT1 in AR42J cells. The cells were incubated with 40 zinc for 24 h. Total cell lysate was prepared, and the ZnT1 abundance was measured by western analysis Ponceau S

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62 Figure 31 6 Influence of zinc and ZnT1 siRNA on ZnT1 expression. AR42J cells were transfected with ZnT1 siRNA for 48 h and treated with zinc for 24 h and then harvested. Total membrane proteins were purified and ZnT1 was measured by western analysis Na+/K+ATPase was used as a loading control.

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63 Figure 31 7 MT, ZnT1 and ZnT2 mRNA abundance in AR42J cells incubated with low zinc medi um. 15% DPA dialyzed FBS was used in low zinc medium The DEX pretreated cells were incubated in the low zinc medium for 4 h, 6 h, 12 h, and 24 h qPCR was used to measure the mRNA levels, the values are expressed as t he percentages of relative changes to control (n=3) R elative MT mRNA Change Relative ZnT1 mRNA Change Relative ZnT 2 mRNA Change

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64 Figur e 3 1 8 Zinc chelators influences ZnT1, ZnT2 and MT mRNA levels and their protein expression. (A) AR42J cells were pretreated with dexamethasone (100 nM) for 48 h Three different zinc chelating reagents were used, by 15% DPAdialyzed FBS Cells were treated for 6 h q PCR was used to measure the mRNA levels of ZnT1, ZnT2, and MT after treatment (n = 3). p<0.05. (B) Immu nocytochemistry analysis of changes in ZnT1 (red) in AR42J cells responsiveness to DTPA, TPEN and DPA. Amylase (green) is also shown as a zymogen granule marker; DAPI staining shows the nuclei (blue).

PAGE 65

65 Figure 31 9 Low zinc medium influences ZnT1 and ZnT2 expression in AR42J cells. The low zinc medium contained 10% DPA dialyzed FBS, and normal medium was used as control. The cells were incubated with this low zinc medium for 24 h and protein abundance was measured by western analysis 42kDa

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66 Figure 320 Zinc chelating reagents influence ZnT1 expression in AR42J cells Zinc chelating reagents DPA, DTPA and TPENwere used to obtain zinc deficiency in AR42J cells. The cells were incubated with low zinc medium or control medium for 24 h and the protein abundance were measured by western analysis Na+/K+ATPase is the loading control.

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67 Figure 32 1 Western analysis of mZnT1 over expression in HEK 293 cells. pCMV SPORT6.1 mZnT1 was transfected into HEK 293 cells pCMV SPORT6.1 was the vector control. C ells were harvest at 24 h, 36 h, and 48 h after the transfection and mZnT1 protein abundance were measured by western analysis. A) Ponceau S red staining of the membrane. B) Western blotting of mZnT1.

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68 Figure 32 2 IF analysis of mZnT1 over expression in HEK 293 cells. 48 h after the transfection, as described in Fig. 321, cells were washed and treated for immunefluorescnece staining. DAPI was used to stain nuclei. A) Cells were transfected with control vector and stained without permeabilization B) Cells were tranfected with control vector and stained after permeabilization. C) Cells were transfected with mZnT1 and stained without permeabilization. D) Cells were tranfected with ZnT1 and stained after permeabilization.

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69 Figure 32 3 Western analysis of ZnT2. AR42J cell cytoplasm soluble protein (S) and total membrane protein (M) or total membrane protein digested with PNG F (PNG +) were probed with total IgG, affinity purified IgG antibody against ZnT2 or affinity purifi ed IgG antibody prein cubated with peptide (C) Ponceau red staining of the membrane (D) 42kDa 42kDa 42kDa

PAGE 70

70 Figure 32 4 Western analysis of N glycosation in rat ZnT1, ZnT2, ZnT4 in AR42J cell membrane fraction, and human ZnT1 in HEK 293 cell fraction by PNGase F diges tion AR42J and HEK 293 cell total membrane protein were prepared and digested with PNG ase F. Western analysis were performed to check the protein migration on SDS PAGE.

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71 Figure 32 5 Western analysis of ZnT2 in m ouse and rat pancreas total lysate (TL), plasma membrane (PM) protein samples by incubating with total IgG, affinity purified IgG, or affinity purified IgG antibody preincubated with peptide. Antibodies are polyc lonal antibodies raised in rabbit (UF609, UF610) or raised in donkey (Santa Cruz) against ZnT2 (A) B ) Western blotting of HEK cells transfected with pCMV 3tagZnT2myc or pCMV 3tagZnT2flag and measured by western analysis using anti myc or anti flag mo noclonal antibodies. A B WB: anti myc ZnT2 Con WB: anti flag Con ZnT2

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72 CHAPTER 4 GLUCOCORTICOID S REGULATE ZNT2 EXPRESSION IN PANCREATIC ACINAR CELLS Introduction Glucocorticoids and the Physiology of the Exocrine Pancreas. Numerous earlier studies have related the glucocorticoids with the physiology of the exocrine pancreas. I t has been clearly established that adrenalectomy (ADx) causes a marked depletion in the number of the zymogen granules in acinar cells (102, 103) and a profound decrease in protein secretion both in the basal state(104) and under CCK stimulation (105) This situation can be re versed by hyd rocortisone administration (104106) Additionally, it has been reported that the administration of glucoco rticoids to suckling rats caused hypertrophy of the pancreas (107111) and led to early maturation of the pancreatic acini in neonatal rats (112) Glucoc orticoid hormones have been thought to play a modulating role in the development of the pancreas (110, 111, 113, 114) M odulation of the balance in the exocrine/ endocrine differentiation has been suggested by in vitro studies of rat pancreatic explants treated with corticosterone, showing decreased insulin secretion and islet mass whi le exocrine enzyme contents and acinar mass were enhanced (114, 115) A ction s of glucocorticoids on secretory activity of the exocrine pancreas have not led to uniform conclusions In this sense, in vitro studies with AR42J cells have demonstrated an increase in CCK receptors and differentiation in the presence of dexamethasone (62, 100, 116) By contrast, in vivo studies have pointed to a dual effect ( both stimulatory and inhibitory) on secretagoguestimulated pancreatic secretion that depends on the dose of glucocorticoids employed (104, 105, 117, 118) Morisset et al. and Otsuki et al. reported that administrati on of hydrocortisone to rats increased the

PAGE 73

73 enzyme content and the size of the pancreas (109, 119) Conversely, Beaudoin et al. have shown that ADx caused a marked depletion of zymogen granules in the rat pancreas seven days after the surgery (120) S imilar results have been reported by several other investigators. However, in a sh ort term study of endogenous glucocorticoid depletion, zymogen granules of ADx rats were preserved based on ultrastructure (121) S hort term effects may be related to the susceptibility of acinar cells to apoptosis during acute pancreatitis whereas longterm effects may be related to the acinar cell differentiation and enzyme synthesis. G lucocorticoids promote acinar cell differentiation, showing that the increase in acinar cell area is a direct consequence of glucocorticoidmediated stimulation of differentiation and not acinar cell proliferation. Gesina et al. showed that in vitro treatment of the embryonic rat pancreas with DEX did not affect the number of precursor cells but decreased the number of differentiated beta cells and increased the differentiated acinar cell area(122) Their conclusion was further supported by the finding of decreased proliferation of amylase expressing cells upon DEX treatment, a result suggesting that glucocorticoids could also control the proliferation of already differentiated acinar cells and thereby prevent their overgrowth. The differentiation process from precursor to differentiated endocrine or exocrine cell s is altered, suggesting that just the precursor cells, not the differentiated beta cells are potential targets for glucocorticoids. Whether this in vitro situation also applies in vivo has yet to be fully investigated. In support of this idea, rats undernourished during their prenatal life and thereby exposed to increased corticosterone levels in utero show increased pancreatic weight at adult age.

PAGE 74

74 Determining the Effect of Dexamethasone Induced Acinar Secretory Maturation on ZnT1, ZnT2 and MT Gene Expression in AR42J Cells Since there a re no lines of murine pancreatic acinar cells, the rat pancreatic acinar cell line, AR42J (ATCC CRL 1492) in place of a murine model was used. AR42J cells were derived from a carcinoma of an azaserinetreated rat and have features of pluripotency of the common precursor cells of the pancreas. Dexamethasone converts pluripotent pancreatic acinar AR42J cells into the exocrine cell phenotype. I ntracellular and secreted amylase contents are markedly increased by dexamethasone (62) D examethasone stimulates secretory granules formation along with extensive reorganization of the ER. Expression of cholecystokinin (CCK) receptors and responsiveness of AR42J cells to CCK stimulation are also found to be upregulated by dexamethasone (100) The advantage of using cultured AR42J cells is that they are the only pancreatic acinar cell type studied in the culture. Also we could manipulate the concentration of exogenous zinc in the culture medium and eliminate the effects of other factors that can influence the zinc transport in vivo i.e. cytokines, hormones, cholecystokinin and growth factors etc. It is unclear if processing of zinc in imm ortalized culture cells will accurately reflect in vivo acinar cells responses. Presumably AR42J cells maintain the same responsiveness and regulation as acinar cells in vivo Therefore, to understand whether ZnT1 and ZnT2 are involved in the exocrine secretory granules maturation, AR42J cells were treated with 100 n M dexamethasone for 48 h and harvested at 0, 3, 6, 12, 24, and 48 h post treatment. ZnT1, ZnT2 and MT mRNA were measured by the TaqMan q PCR method.

PAGE 75

75 Potential Responsiveness of pGL3 and pGL4 Luciferase Reporter V ector to Steroid Hormones. The pGL3basic luciferase reporter vector has been widely used in the study of GRE responsiveness to glucocorticoid hormones. A recent article in BioTechniques reported both the pGL3Basic and pGL4 were induced by glucocorticoid in transient transfection of primary oviduct tubular gland cells, which c ontain GRs (123) M y results agreed with their findings on the effects of the pGL3Basic control vector but not the pGL4 vector, on dexamethasone treatment. Therefore, the pGL4 vector should be a better plasmid when considering the reporter system for glucocorticoid r egulation. Results ZnT2 Expression is Upregulated d uring Glucocorticoidstimulated Pancreatic Acinar Cell Differentiation. To further understand the regulation of ZnT1 and ZnT2 expression in pancreatic acinar cells, rat AR42J pancreatic acinar cells were used as a model. Dexamethasone (DEX) was added to the AR42J cell cultures for 48 h our to stimulate cell differentiation. An increase in amylase mRNA a signature of acinar differentiation and secretory enzyme production, was observed following the addition of DEX (Fig 4 1 ). A strong upregulation of ZnT2 mRNA was found, upon stimulation with DEX (Fig. 41, 42 4 4 ). Because ZnT2 was known to be localized to intracellular vesicles and may transport cellular zinc into these vesicles, increased expression of the ZnT2 gene during dexamethasoneinduced zymogen granule formation may indicate ZnT2 localization to zymogen granules and sequestration of cytoplasmic Zinc into the these granules. Surprisingly however, no change of ZnT1 expression was observed in response to DEX suggestive of selective GC regulation of these two zinc tra nsporter genes (Fig. 41, 42 ), and no interaction between zinc and DEX in regulating ZnT1 and ZnT2 expression (Fig 42 ). The synergic effects on the MT

PAGE 76

76 gene, which were documented before (99) were also confirmed in this experiment (Fig. 4 3 ). To examine if ZnT2 is regulated by GC hormones in vivo mice were given DEX by injection. As expected from previous work (99) serum zinc level decreased ( by 20% ) and liver zinc content increased ( by 30% ) Surprisingly pancreatic zinc content decreased significantly (Fig. 4 5 4 6 ). ZnT2 mRNA expression w as significantly upregulated nearly 2fold by 8 h after the injection and a high expression level was maintained at 16 h ( Fig. 47 ). In contrast, MT mRNA as a positive control was incre ased 7.0fold at 8 h and decreased to the normal level by 16 h a fter the DEX injection (Fig 4 7 ) These results are similar to what the data suggested in DEX treated AR42J cells (Fig. 41). Differences in half lives of these two mRNAs ar e apparent Pancreatic ZnT1 mRNA levels were not changed in r esponse to DEX (data not shown) ZnT2 mRNA was effectively knocked down in AR42J cells by using ZnT2 siRNA. With either the presence or absence of DEX ZnT2 mRNA was knocked down by about 70% at 48 h after ZnT2 siRNA transfection (Fig. 4 8 ). These results show that there was no interaction between the two independent events i.e. transcription initiation by GR and ZnT2 mRNA degradation by siRNA. W estern blotting confirmed the knock down of ZnT2 at the protein level (Fig. 48 ). E ffects of ZnT2 siRNA knockdown on intracellular zinc concentrations were accessed following transfection. MT is regulated by MTF1, therefore, knocking down ZnT2 in acinar c ells should produce a transient zinc accumulation in the cytoplasm and activat ion of MT gene expression via zinc induced MTF1 translocation to the nucleus. Significant elevation of MT mRNA was found to reach a peak around 24 h post transfection(Fig. 4 1 1 ) ZnT1 expression exhibited a

PAGE 77

77 comparable response (Fig. 41 0 ). In accord with the induction of MT, an increase in cytoplasmic (65Zn) zinc accumulation from the medium was observed through ZnT2 knockdown with siRNA (Fig. 4 9 ). C ytoplasmic 65Zn was increased by 36% with ZnT2 siRNA, which supports the hypothesis that ZnT2 transports cytoplasmic zinc into zymogen granules. Further support for this role of ZnT2 is that 65Zn in the zymogen granules was decreased by 15% (Fig. 4 9 ). However, no change in 65Zn content was found in the crude nuclear fraction suggesting specificity in cellular accumulation. It was hypothesized that overexpression of ZnT2 would enhance zinc loss via the zymogen granules. Since this could not be measured directly in AR42J cells, HeLa cells were transfected with a ZnT2 cDNA vector or empty vector (Fig. 3 25) and the cells were allowed to accumulate 65Zn. E fflux of 65Zn from preloaded cells was greater in t he overexpressing cells (Fig. 41 2 ). This finding is congruent with the ZnT2 transport function in zymogen granules. To understand the mechanism of ZnT2 regulation by DEX, the GC antagonist RU486 and a newly discovered GR modulator CpdA were exploited to study the association of ZnT2 gene transcription with signaling via the GR CpdA having a higher binding affinity competes with DEX for binding to GR, and induces GR release from chaperones and nuclear translocation and transrepression of NFdriven gene expression. CpdA exhibits no transactivat ion potential on GRE driven gene transcription (64) and was not able to activate ZnT2 gene expres sion However, when AR42J cells were treated with DEX and CpdA at the same time, the hormonal analogy could still initiate expression of MT and ZnT2 (Fig. 4 1 3 ) presumably via transactivation of GR In contrast, presence of the GR antagonis t RU486, prevented CpdA and DEX from

PAGE 78

78 stimulating the upregulation of MT and ZnT2 (Fig. 41 3 ). These differing results with the two antagonists indicate DEX stimulated ZnT2 expression via transactivation of GREdriven gene expression, but associated with N F B activation MT, a well characterized glucocorticoidregulated gene was used as positive control in these experiments (99, 124) The responsiveness of ZnT2 expression to DEX in both AR42J cells and pancreas of the intact mouse led to an analysis of GRE in the upstream promo ter region. Two half GRE site were found but no full GRE. While realizing that the noncanonical half sites may impart GC regulation for some genes (125, 126) t he involvement of STAT5 and GR in transductive pathways for regulation of other GC co ntrolled genes (127 1 29) was examined. Two STAT5REs were identified in the ZnT2 promoter (130) (Fig. 41 4 ) Consequently, we used a chromobased nicotinoyl hydrazone (CNH), a STAT5 specific inhibitor (131) and the Janus kinase 2 inhibitor (AG490) to examine STAT5 involvement in ZnT2 activation by DEX (Fig. 415) As shown in Fig. 41 6 4 1 7, 418, the inhibition of STAT5, particularly in combination with Ja k2 inhibition, completely blocked DEX induction of ZnT2. Notably, the DEXinduced increase in MT expression was not inhibited by either AG490 or the STAT5 inhibitor (Fig. 41 6, 417, 418) A n MRE sequence was identified in the downstream of TSS in mouse ZnT2 promoter, and it is highly co nserved across species (Fig. 419). A role for the transcription factor MTF 1 was shown through transfection with its vector cont aining human MTF1 cDNA (Fig. 4 2 0 ). Markedly enhanced luciferase was obser ved with hMTF1 transfection (Fig. 42 0 ). Zinc doubled promoter activity under these conditions.

PAGE 79

79 The contribution of the MRE sequence t o ZnT2 regulation was supported through mutation of this sequence in the ZnT2 promoter (Fig. 4 2 0 ). Cells transfected wit h the mutated ZnT2 promoter responded to DEX, but did not respond to zinc (Fig. 42 0 4 2 1 ). The pGL3basic and pGL4 vectors were test ed for dexamethasone responsiveness Agreeing with another groups finding (123) there is a 1.5to 2.0 fold increase in luciferase activities upon dexamethasone treatment (Fig. 42 2 ). But the increase was not seen in pGL4 vector This reengineered vector eliminated many potential transcription factor binding sites in its backbone sequences (Fig. 42 2 ). Discussion Z inc transporter expression in the pancreas is important because pancreatic secretions constitute an important component of mammalian zinc homeostasis (22, 68, 72, 8589) ZnT1 and ZnT2 are expressed in the pancreas (51) and ZnT1 and ZnT2 are associated with isolated plasma membrane and zymogen granules, respectively. In this chapter, special focus is given to the regulation of the zinc transporter ZnT2 and the role it may play in an endogenous zinc secretory pathway from pancreatic acinar cells. I t is propose d that zinc output from acinar cells follows two distinct pathways One of these is cellto ductal zinc efflux via the apical membrane, which is zinc dependent and primarily involves ZnT1 for cellular efflux The other is that zi nc is released along with digestive proenzymes from zymogen granules, where zinc is transported into the granules by ZnT2. These respective functions for ZnT1 and ZnT2 agree with current proposed roles for zinc transport in other cell types (90) Zinc in stimulated pancreatic sections has been shown to be associated with h igh molecular weight proteins, and is closely linked to th e activities of zinc containing enzymes, especially carboxypeptidase A and carboxypeptidase B (22, 68, 85) Since

PAGE 80

80 zymo gen granules are the storage sites of digestive enzyme precursors (91) our finding that ZnT2 influences zinc incorporation into zymogen granules suggests that this t ransporter may provide zinc to maintain full activity of digestive pro metalloenzymes. T he reduction in activity observed during dietary zinc restriction is in agreement with our finding that transcript abundance for nine pancreatic digestive enzymes is not influenced by the dietary zinc restriction model used in the present experiments (data not shown). High zinc consumption has been shown in avian species, mice, and pigs to be detrimental to normal pancreatic exocrine function and to produce organ damage (42, 73, 74) This sensitivity suggests secretory pathways of zinc loss are essential for preventing pancreatic enzyme release, cell necrosis and organ atrophy. These signs of zinc toxicity are similar to the autodigestion of pancreatitis which are due to abnormal calcium signaling within zymogen granules (92) In the current studies we have identified ZnT2 as a component for zinc transport into the secretory zymogen granules. T hese granules have an acidic intra granule pH (93) which is in line with the notion that ZnT2 favors acidic vesicles for its maximal transport activity. Also, ZnT8, which is expressed almost exclusively in the pancreatic cells, facilitates zinc transport required for proinsulin aggregation using a process that involv es proton exchange (94, 95) The zinc and MT content s of the pancreas are the highest among tissues under normal conditions of dietary zinc intake. Such high expression suggests MT has an important role in regulating zinc metabolism and function in the pancreatic acinar cells. MT declines almost completely with a zinc deficient diet, whereas in other organs it decreases only moderately (33, 97) Pancreatic zinc secretion i n MT/mice is much

PAGE 81

81 higher than in wild type mice and they are more vulnerable to damage caused by zinc deficiency (33) MT is also found in pancreatic secretions, although it is through a route that does not involve granule secretory pathways (32) In that regard, MT bound zinc in pancreatic secretions could provide a source of endogenous zinc for reabsorption. Studies that could reflect on the glucocorticoid res ponsive expression of ZnT2 and its role in pancreatic zinc secretion are limited. It has been reported that adrenal insufficiency increases serum zinc concentrations wh ile administration of gluco co rticoids and ACTH and the excess coris ol production in Cushing s Syndrome decrease these concentrations (reviewed in (98) ) Hypozincem ia associated with glucocorticoid action has been related to induced synthesis of MT in rodents (99) Radiotracer kinetic studies with Zinc 69m given intravenously to humans reveal that carbohydrateactive steroids (glucocorticoids) may alter rate constants of the fecal excretion of zinc (98) The pancreatic acinar AR42J cell model has been widely used to characterize effects of glucocorticoid hormones on secretory activity of the exocrine pancreas (62, 100) Dexamethasone treatment of AR42J cells induces a highly differentiated phenotype. It has been reported that RU486 can reduce GR concentrations in AR42J cells by 50% with concomitant changes in GR regulated gene expression (101) It was established t hat ZnT2 regulation is controlled by GR by demonstrating inhibition through RU486. This strongly suggests that the signaling pathway involves GR dimerization rather than the anti inflammatory pathway i nvolving assembly of the NFkB/ GR dimer complex. GR involvement in pancreatic tissue organization, the differentiation of acinar cells, and enzyme and zymogen granule production is compatible with its role in ZnT2

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82 regulation. Furthermore, the control by zinc via M TF1 responsive ZnT2 expression, is consistent with a role in endogenous zinc secretion. That control, combined with the DEX responsiveness, makes ZnT2 analogous to MT in terms of a teleological basis for dual regulation. To our knowledge ZnT2 m ay represent the first member of either the ZnT or Zip gene families to be glucocorticoid regulated. T he MRE mutation and deletion results confirm the importance of the functional MRE element in the ZnT2promoter. The crucial base level transcription is dependent on this MRE sequence in ZnT2 promoter, suggesting that the active MRE in the ZnT2 promoter is the dominant transcription element. As shown in Fig. 4 1 4 there is another potential transcription start site in the downstream of this MRE sequence. Thi s is based on the cDNA library sequences in the database. 4 different transcript variants of mouse ZnT2 were found in the database. In spite of the presence of inaccurate sequences in this database, another potential transcription start site needs to be co nsidered for the future study. Amylase and ZnT2 induction by DEX seem to be regulated through distinctly different mechanisms. The kinetics of dexamethasone induction of amylase gene expression are slow. In the case of the amylase gene, significant effect s were not observed in the first 6 h of treatment, and maximal effects were not observed until 2448 h. H owever, it does not seem to be the case for ZnT2. ZnT2 mRNA level s increased within 3 h and reached the maximal level at 12 h, gradually decreasing to the baseline level 48 h after the treatment. It has some similarities to MT induction by dexamethasone, in spite of the short half life of MT mRNA. The inhibition of protein synthesis blocked the ability of dexamethasone to increase amylase gene expres sion

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83 (132) Both cycloheximide and puromycin, agents with different mechanisms of action, blocked the induction of dexamethasone on the amylase gene (132) There are unknown newly synth esized factors by dexamethasone, which are involved in amylase transcription activation. The possibility of an unknown secondary regulatory factor involved in ZnT2 transcription should be tested by cycloheximide, puromycin, and/or actinomycin D treatments in the future. Epigenetic modification has been proposed in regulating zinc transporter expression. CpG island have be identified in the promoter region of ZnT2 gene (Fig. 423). Whether this CpG is functional in regulating gene expression needs further investigation. Computer based modeling (133, 134) of human ZnT2 structure showed tightly arranged six transmembrane domains along with a C terminal cytosolic domain (Fig. 42 4 ). There are four potential zinc binding sites in the struct ure of ZnT2, and one of these is in that C terminal intracellular domain. The other three sites are closed to the tunnel space embedded in six transmembrane helix, suggesting important function of these zinc binding sites in transport of zinc ion.

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84 Figure 4 1. Dexamethasone regulates MT, and ZnT2 mRNA levels in AR42J cells. The cells were treated with Dexamethasone (100 nM) for 48 h Cells were collected at various time point s post treatment. The q PCR was used to measure MT (A), ZnT1 (B), amylase (C), and ZnT2 ( D ) mRNA levels a fter the treatment. (n = 3). A C B D

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85 Figure 42 The responsiveness of ZnT1 and ZnT2 expression to Zinc and/or dexamethasone in AR42J cells. ZnSO4 nM) were added to AR42J cell culture medium for 6 h The q PCR measurement of ZnT1 and ZnT2 demonstrates mRNA levels change after treatment with dexamethasone and/or ZnSO4. The values are expressed as mean standard deviati on (n = 3).

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86 Figure 4 3 The responsiveness of MT1, and ZnT1 expression to zinc and/or dexamethasone in AR42J cells. The cells were treated with zinc and/or dexamethasone (100 nM) for 6 h q PCR was used to m easure mRNA levels of MT (A), and ZnT1 (B ) after treatments Each bar value is the mean + SD (n = 3).

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87 A B C Figure 4 4 Dexamethasone influences ZnT2 expression in pancreatic acinar cells. (A) AR42J cells were treated with 100nM dexamethasone for 24 h ZnT2 protein abundance was measured by western analysis. (B,C) Mice were given dexam e thasone via i.p. injection and ZG were purified from the pancreas 24 h after injection. ZnT2 were shown by IF in purified. (B) saline control; (C) dexamethasone injection. 42kDa

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88 Figure 45 Zinc concentration in the pancreas and serum in mouse 3 h after dexamethasone (DEX) i.p. injection. PBS was given as control. Serum (A) and pancreas (B) were collected and pancrea tic cytoplasmic soluble fraction was isolated. Zinc concentrations were measured by atomic absorption spectrophotometry Serum Zinc Concentration Pancreatic Zinc Concentration A B

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89 Figure 46 Dexamethasone influences z inc concentration in the pancreas, liver, and serum in mice 8 h af ter dexamethasone (DEX) i.p. injection. PBS was given as control. The pancreas liver, and serum were collected and the pancreas and liver were weighted and digested with nitric acid Zinc concentrations were measured by at omic absorption spectrophotomet r y and normalized against the weight.

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90 Figure 47 CD1 mice were injected i.p. with either dexamethasone or PBS and killed 3, 8, or 16 h thereafter. MT (A) and ZnT2 (B) mRNA were measured by qPCR. ( n = 34 ) A B

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91 Figure 4 8. ZnT2 knock down in AR42J cells by ZnT2 siRNA. AR42J cells were transfected with ZnT2 siRNA for 48 h and/or treated with DEX for 48 h. Scramble d siRNA was used as control siRNA The cells were harvested at 48 h after the transfection and ZnT2 mRNA expression was measured by qPCR (A) ZG were isolated and ZnT2 protein levels were detected by western blotting (B) Amylase was ZG loading control. A B Control siRNA ZnT2 siRNA

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92 Figure 49 Zinc concentrations in cytoplasm and ZG fractions after ZnT2 knock down by ZnT2 siRNA in AR42J cells. The cells were transfected with ZnT2 siRNA for 48 h Scramble siRNA was used as control siRNA. The cells were preloaded with 65Zn over night. Then cells were homogenized, cytoplasm and ZG fractions were purified. 65Zn were counted with ray spectrometer and normalized to total protein content Zinc concentration was calculated based on the specific activity.

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93 Figure 4 1 0 ZnT2 siRNA knockdown and dexamethasone influences ZnT1 and ZnT2 expression in AR42J cells The cells were transfected with ZnT2 siRNA or treated with dexamethaso ne for 48 h and total membrane protein was purified. ZnT1 and ZnT2 protein abunda nce were measured by western anal ysis Na+/K+ ATPase was shown as total membrane loading control.

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94 Figure 4 1 1 ZnT2 siRNA knockdown influences MT mRNA in AR42J cells. The cells were transfected with ZnT2 siRNA for 48 h and were harvested at various tim e points post transfection. Total RNA were isolated and MT mRNA was measured by q PCR. S crambled siRNA transfection was the control

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95 Figure 4 1 2 ZnT2 overexpression increases zinc efflux in HeLa cells. The cells were transfe cted with pCMV3tagZnT2 vector or a pCMV 3tag control vector for 48 h and loaded with 65Zn. Zinc efflux into the medium was calculated from the specific activity of 65Zn in the fresh medium and expressed on a cell protei n basis. Values are means SD ( n = 3)

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96 Figure 41 3 The effects of dexamethasone and glucocorticoid modulators, RU486 and CpdA, on the MT and ZnT2 mRNA expression in AR42J cells The cells were treated for 12 h. Total RNA was isolated, MT and ZnT2 mRNA expression levels were measured by qPCR (n=3).

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97 Figure 4 1 4 Transcription factor binding site analysis in the proximal region of m ouse ZnT2 promoter The sequences of activator protein 1 (AP 1) signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), estrogen receptor (ER ), Nuclear factor kappaB (NF kB) and Metal responsive transcription factor 1 (MTF 1) are shown in the box. Two transcription start site (TSS) are shown as arrows. The ZnT2 translated sequence is shown in green.

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98 Figure 41 5 Possible GR and Stat5 synergistic signaling pathway. Upon the glucocorticoids (GC) binding to glucocorticoid receptors (GR), GR form active homodimmer and translocate into nucleus. Upon activation, Stat5 SH2 domain is phosphorylated by Jak2, and the phosphorylated Stat5 also form homoor heterodimmer in nucleaus. GR and Stat5 interact and bind to the GAS/Stat5 RE elements in the promoter with other transcription activators for transcription activation.GR Antagonist RU486, Jak2 inhibitor AG490, and Stat5 inhibitor CNH are shown in the diagram

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99 Figure 41 6 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the MT mRNA expressions in AR42J cells. The cells were treated with 100nM DEX and/or 50uM AG490, 400uM CNH, and 1uM RU486 for 12 h, and cells were harvested. Total RNA were isolated, MT and ZnT2 mRNA expression levels were measured by qPCR (n=3).

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100 Figure 41 7 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the ZnT2 mRNA expressions in AR42J cells. The cells were treated with 100nM DEX and/or 50uM AG490, 400uM CNH, and 1uM RU486 for 12 h, and cells were harvested. Total RNA were isolated, MT and ZnT2 mRNA expression levels were measured by qPCR (n=3).

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101 Figure 41 8 The effects of dexamethasone and glucocorticoid antagonist RU486, Stat5 inhibitor CNH, and Jak2 inhibitor AG490, on the MT and ZnT2 mRNA expressions in AR42J cells. The cells were treated with 100nM DEX and/or 50uM AG490, 400uM CNH, and 1uM RU486 for 12 h, and cells were harvested. Total RNA were isolated, MT and ZnT2 mRNA expression levels were measured by qPCR (n=3).

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102 Figure 419 Conserved MRE sequences in the promoter sequence alignment of multiple sp ecies. The red dots marked the MRE consensus sequence.

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103 Figure 42 0 ZnT2 promoter activity in transfected HEK 293 and HeLa cells in response to zinc. HeLa cells were transfected with murine ZnT2 promoter constructs over the range ( +93) ligated into pGL3Basic vector. (A) HEK 293 cells were co transfected with an human MTF1 expression vector and the +93 ZnT2 promoter construct. (B) The Site Directed Mutagenesis was used to change the MRE consensus sequence into a restriction s ite. HeLa cells were transfected with mutant +93 ZnT2 promoter construct. The cells were treated with 100nM DEX or 100uM zinc for 24 h. Luciferase activity was measured 48 h after transfection. A B

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104 Figure 42 1 ZnT2 promoter activity in transfected HeLa cells in response to zinc. HeLa cells were transfected with mutant ZnT2 promoter constructs over the range p52( +93) or p22 ( +93) ligated into pGL3Basic vector. The Site Directed Mutagenesis was used to change the MRE consensus sequence to a XbaI, DraI, BstBI or PsiI site The cells were treated with 100nM DEX or 100uM zinc for 24 h. Luciferase activity was measured 48 h after transfection.

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105 Figure 42 2 pGL3 Basic and pGL4 vector luciferase activities under DEX treatment in HeLa cells. SV40 promoter construct was used as a positive control. p52 construct is the +93 ZnT2 promoter construct in pGL3Basic vector. pZip10 is the Zip10 promoter construct in pGL3Basic vector.

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106 Figure 42 3 CpG island analysis in ZnT2 gene.

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107 Figure 42 4 Predicted human ZnT2 protein structure (A) and zinc binding sites (B).

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108 CHAPTER 5 Z INC T RANSPORTERS IN PANCREATIC ACINAR CEL LS UNDER STRESS C ONDITION S Introduction The primary disease of the pancreas is pancreatitis a condition in which there is an inflammation localized to the pancreas (92) A disturbance in z inc metabolism has been documented in patients with p ancreatitis. Both urinary and serum zinc levels are increased in chronic pancreatitis along with a deterioration of exocrine pancreatic function (20) Stimulation with cholecystokinin and secretin increased secretion of zinc in healthy persons but not in p ancreatitis patients (135) Serum zinc level s in advanced chronic pancreatitis patients were higher when compared to patients with normal exocrine pancreatic function (20) H owever, dia betes, a complication due to chronic pancreatitis has generally been associated with decreased serum zinc concentration (136) By con trast, a cute pancreatitis presents a different scenario due to the acute p har se response and inflammation, where lower serum zinc level s and an increased liver zinc content are observed (137, 138) which could be explained by Zip14 activated through IL 6/Stat3 signaling in the liver (139) In alcoholic pancreatitis, the patients have a higher risk of acute zinc deficiency (140, 141) Altered mineral metabolism in the pancreas may contribute to the pathophysiology of m ice with acute pancreatitis (142) suggesting that z inc supplementation could be therapeutic in pancreatitis (142) In the early stages of acute pancreatitis, the pancreatic concentration of zinc is significantly decreased. Zinc could increase both MT and glutathione levels in the pancreas and combat oxidative damage in cerulein induced acute pancreatitis (143) MT is present in the exocrine and endocrine cells of patients with chron ic pancreatitis and chronic pancreatitis with concomitant diabetes. Increased expression of MT particularly in

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109 acinar cells, protects against ceruleininduced acute pancreatitis in MT overexpressing transgenic mice (144) It is believe d that MT can reduce the oxidative damage that occurs during acute pancreatitis. MT induction in pancreatic islets and specifically cells is regulated by cytok ines and DEX, and PKC activation might play a role (145) Because of the relationship of zinc to pathophysiologic events in the pancreas, i n this chapter of the dissertation, the zinc transporter expression was studied in relation to the supramaximal cerulein model of pancreatitis, as well as the in vitro alcohol related acinar cell damage. Methods and Results Supramaximal cerulein decreased ZnT1 and ZnT2 expression in AR42J pancreatic acinar cells. A s upraphysiological dose of cerulein, a synthetic analogue of CCK, was added to AR42J cells cultured at low (0.5uM), normal (5uM), and high (50uM) zinc levels to induce acute inflammatio n I found that at these various zinc conditions, both ZnT1 and ZnT2 mRNA expression levels were reduced during cerulein induced pancreatiti c damage (Fig. 51) However, MT mRNA were greatly induced by cerulein. These results might indicate that MT, as cellular stress response, was induced due to ceruleininduced acinar cell damage. Cellular zinc also mobilizes and interacts with MT, which at high cellular levels could lead to lower zinc MTF coupled transcription activation in the promoter r egions of ZnT1 gene. The question whether CCK could r e gulate MT and zinc transporters. To pursue this issue AR42J cells were treated with different concentrations of CCK. MT mRNA expression was not altered by CCK simulation (data not shown) However, both ZnT1 and ZnT2 mRNA were decreased in a dose dependent manner by CCK in AR42J cells. Most notablely ZnT1 mRNA decreased to only half the level s of the control samples

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110 (Fig. 52 ZnT2 mRNA is not sh own here) Previously, we found that CCK mRNA in the small intestine were up regulated during zinc deficiency in rat s (1 46) T hese present results suggest a repression of ZnT1 and ZnT2 in pancreatic acinar cells by CCK signaling pathways and suggest a dynamic regulation of zinc homeostasis in the pancreas through CCK signaling and crosstalk between the small intestine and pancreas. Longterm excess alcohol c onsumption could induce alcohol related pancreatic damage, chronic pancreatitis and zinc deficiency. However, the mechanism is unknown. In this study, AR42J cells were treated with 300mM ethanol for 12h and 24h. Cells were harvested and relative mRNA abundance of MT was found to be elevated during ethanol stimulation. W e found a progressive decrease in ZnT2 mRNA abundance, which was inversely correlated with MT level s (Fig. 54) These results indicat e a lowered zinc secretion from secretory pathway s and zinc accumulation could occur during the first 24h of alcohol related cell damage in pancreatic acinar cells. However, the longterm effects of excess alcohol consumption on the pancreatic zinc homeostasis need to be further investigated. Discussion Supramaximal cerulein is widely used to induce pancreatic acinar cell damage and pancreatitis. In this series of experiments, AR42J cells were treated with cerulein, and ZnT1 and ZnT2 expressions were found to be decreased. These results might indicate that MT as a ce llular stress response gene is induced during ceruleininduced acinar cell damage. H igh levels of metallothion ein, which could lead to a lower zinc MTF coupled transcription activity in the promoter regions of ZnT1 and ZnT2 genes. This could potentially result in a higher cellular zinc level as a defense response to oxidative

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111 stress induced by the supramaximal dose of cerulein. Further in vivo evidence is needed to fully understand how zinc transporter s and intracellular zinc homeostasis are changed during pancreatitis and the relationship between this interaction and disease progression and prognosis. This chapter provided a preliminary result of the zinc transporter regulation in acinar cells under stress conditions. Inconsistent results made it difficult to interpret the data. Further investigation is needed to fully understand the role of zinc homeos tasis and function of zinc transporter in the progression of pancreatitis. Especially when these experiments were designed, the role of STAT5 signaling in ZnT2 regulation was not known. Future experiments will need to consider the STAT5 signaling pathway i n study design.

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112 Figure 5 1 MT and ZnT1/2 mRNA expression in AR42J cells stimulated with cerulein. AR42J cells were cultured in low (0.5uM), normal (5.0uM), and high (80uM) zinc medium and stimulated with cerulein for 6 h. After stimulation, cells wer e harvested, and total RNAs were isolated. Relative mRNA abundance was measure by qPCR.

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113 Figure 5 2 Supreamaximal CCK8 stimulation decreased the expression of ZnT1 in AR42J cells. AR42J cells were stimulated with CCK8 at various concentration. After s timulation, cells were harvested, and total RNAs were isolated. Relative mRNA abundance was measure by qPCR

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114 Figure 5 3 MT, and ZnT1/2 mRNA expressions in AR42J cells stimulated with supreamaximal CCK8. AR42J cells were stimulated with CCK8 or together with PKC inhibitor (CalC) NF KB activation inhibitor (NFkB AI) MEK inhibitor (U0126) Relative mRNA abundance was measure by qPCR. A) MT, and ZnT1/2 mRNA expression level. B) Pancreatitis associated protein (PAP) mRNA expression A B

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115 Figure 5 4 MT and ZnT1/2 mRNA expressions in AR42J cells stimulated with ethanol. AR42J cells were stimulated with ethanol for 12 h and 24 h. After stimulation, cells were harvested, and total RNAs were isolated. Relative mRNA abundance was measure by qP CR.

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116 CHAPTER 6 ZINC TRANSPORTERS IN HUMAN PANCREATIC CANCER Introduction Pancreatic duct al adenocarcinoma is the fourthleading cause of cancer deaths in the United States with an average expected survival of less than 6 moths (147) Recently the study by Dr. Min Li and his group from Baylor College of Medicine found aberrant expression of Zip4 was involved in pancreatic cancer progression (148, 149) To further understand the cellular zinc homeostasis changes in pancreatic cancer progression, q PCR was utilized to create an mRNA expression profile of human ZnT and Zip genes from a normal human pancreas. Methods Adult human pancreas total RNA isolated by a modified guanidinium thiocyanate method, was obtained from a commercial source (Cell Application, San Diego, CA). Total RNA was. q PCR primers and the TaqMan probes were designed by using PRIMER EXPRESS V.3 .0 (Applied Biosystems). The 18S rRNA level was used for nor malization. ZnT and Zip transcript level s in a human reference RNA (Stratagene) were measured and considered as reference average level s. ZnT1 and ZnT2 were found to have higher transcript level amount s among ZnT transporters in the adult human pancreas on a relative basis where as Zip5 is the most abundant transcript of the SLC39A Zip family. Results Aberrant expression of zinc transporter in human pancreatic cancer. Recent findings suggest the aberrant ex pression of zinc transporter 4 Zip4, contributes to human pancreatic cancer pathogenesis and progression (149) To further understand

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117 how zinc transporter genes are regulated in pancreatic cancer, we compared the zinc transporter mRNA expression panel in the normal human and carcinoma pancreas. First, qPCR results confirmed the elevated pancreatic adenocarcinoma marker genes CD56 and Palladin expression. However, we noted that acinar cell marker s of digestive enzyme gene expression, such as amylase, elastase, chymotrypsinogen, and trypsinogen, were nearly completely lost These results indicate that pancreatic adenocarcinoma progression transform s the normal acinar cells into a duct cell like cell type. ZIP4 expression level s were found to be increased more than 100fold as we expected (Fig. 61 ) Surprisingly, as one of the major and most abundant zinc transporter in the normal pancreas, Zip5 expression decrease d more than 100fold in RNA from panc reatic cancer. The most abundant ZnT transporters in normal pancreas are ZnT2 and ZnT8, and are predominantly found i n acinar cells and islets, respectively. Both ZnT2 and ZnT8 expressions were found to decrease dramatically in pa ncreatic adenocarcinoma (Fig. 62) Also of note is the 50% reduction in MT transcript (MT1) in RNA from pancreatic carcinoma (Fig. 63) The caution of interpreting these result s is that total RNA from whole human pancreas tissue includes 85% from the exo crine pancreas. Another limitation is the use of the commercial human reference RNA These RNAs are derived from a combination of different types of human cultured cell lines. It is comprised of total RNA isolated from various cultures of human cell lines representing different human tissues. The cell lines were chosen to ensure a broad coverage of human genes on human arrays and other type of assays. The cell lines include adenocarcinoma, mammary gland; hepatoblastoma, liver; adenocarcinoma, cervix; embryo nal carcinoma, testis;

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118 glioblastoma, brain; melanoma; liposarcoma; histiocytic lymphoma; macrophase; histocyte; lymphoblastic leukemia and plasmacytoma; myoloma and B lymphocyte. This is a good representation of most genes, but these cell lines may not be representative of certain highly tissue specific or exclusively expressed genes, e.g. ZnT2, which has very low mRNA levels in human reference RNA. Discussion The initial aim of this experiment was to investigate the zinc transporter expression in acinar ce ll carcinoma. However, according to our cancer mar ker gene expression results, the pancreatic ductal adenocarcinoma marker genes CD56 and palladin are positive and all the acinar cell marker genes are negative, which indicated that the human pancreatic cancer sample was pancreatic ductal adenocarcinoma. Nonetheless, the full panel of zinc transporter gene expression results demonstrated a dramatic potential change of cellular zinc transport capability in cancer progression. Zip4 expression is dramatically i ncreased in pancreatic cancer. This result is in agreement with the previous fi nding of aberrant expression of Zip4 in pancreatic cancer progression (149) The turnoff of Zip5 expression observed in pancreatic cancer might suggest a complete change in zin c uptake behavior and kinetics and the possibility of a ce ll type change is not excluded in this experiment. The abnormal ly high expression of Zip4 may be important in providing zinc for cancer cell metastasis. shRNA mediated silencing of ZIP4 resulted in a dramatic decrease in tumorgenesis, including decreased volume, weight, and differentiation grade, and significantly increased the survival rate of the subjects (148) This molecular mechanism has been proposed to be associated with cyclin D1 activity (148)

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119 The only decreased ZnT transporters in pancreatic cancer tissue were ZnT2 and ZnT8. These zinc transporters are cell typespecific and are expressed at very high levels in acinar and beta cells respectively, in the pancreas However, this qPCR panel screening result showed a significant decrease in these two transcripts. Hence, it is curious that the transporter genes, which appear to be the major component of zinc metabolism in the pancreas, are down regulated in pancreatic cancer. Future studies are needed to fully understand the role of zinc transporters in pancreatic cancer progression.

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120 Figure 6 1 mRNA abundance panel of Zip family in human pancreatic cancer.

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121 Figure 6 2 mRNA abundance panel of ZnT family in human pancreatic cancer.

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122 Figure 6 3 MT mRNA abundance in normal human pancreas and pancreatic cancer.

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123 CHAPTER 7 CONCLUSIONS AND F UTURE D IRECTIONS Conclusions M ice fed a zinc restriction diet developed covert signs of zinc deficiency in 3 weeks, as shown by dramatically decreased ZnT1, ZnT2 and MT expression in pancreas as well as a depressed serum zinc concentration. Zinc deficiency could develop in a similar manner in human. Zinc restriction resulted in pancreatic cytoplasm and zymogen granules having less than half the amount of zinc found in mice fed the zinc adequate diet These responses show the sensitivity of these zinc transporter genes to the dietary zinc intake level. Western blotting clearly showed the decrease of ZnT1 in the plasma membraneenriched fract ion during zinc restriction. Of particular interest is that ZnT2 was exclusively detected in the isolated zymogen granule fraction and showed a reduction in response to dietary zinc restriction. This novel finding of ZnT2 localized to isolated zymogen granules was confirmed by immunofluorescence confocal microscopy g body weight was given orally pancreatic zinc content increased, and there were transient elevations in pancreatic MT, ZnT1, and ZnT2 mRNA s. Various concentrations of zinc were treated to the AR42J cells, causing a dose dependent upregulation of ZnT1 mRNA expression. The MT mRNA was significantly upregulated by supplemental zinc, and the responsiveness was shown to be dose dependent. Both MT and ZnT1 mRNA levels were regulated in a timedependent manner. All three zinc chelators successfully induced zinc deficiency in AR42J cells in the culture medium, as confirmed by MT mRNA level as a sensitive cellular zinc ind icator gene. Both ZnT1 and ZnT2 gene expression were found to be decreased after

PAGE 124

124 24 h in low zinc culture medium at mRNA level and protein level. N glycosylation modification was found in human ZnT1 protein with the total membrane protein sample prepared from HEK 293 cells. Dexamethasone (DEX) was added to the AR42J cell cultures for 48 hour to stimulate cell differentiation. An increase in amylase mRNA a signature of acinar differentiation and secretory enzyme production, was obse rved following the addition of DEX. A strong upregulation of ZnT2 mRNA was found, upon stimulation with DEX. To examine whether ZnT2 is regulated by GC hormones in vivo mice were given DEX by injection. The pancreatic zinc content decreased significantly ZnT2 mRNA expression w as s ignificantly upregulated nearly 2fold by 8 h after the injection and a high expression level was maintained at 16 h. ZnT2 mRNA was effectively knocked down in AR42J cells by using ZnT2 siRNA, w ith either the presence or absence of DEX. K nocking down ZnT2 in acinar cells should produce a transient zinc accumulation in the cytoplasm and activat ion of MT gene expression via zinc induced MTF1 translocation to the nucleus. Significant elevation of MT mRNA was found to reach a peak around 24 h post transfection. In accord with the induction of MT, an increase in cytoplasmic (65Zn) zinc accumulation from the medium was observed through ZnT2 knockdown with siRNA. C ytoplasmic 65Zn was increased by 36% with ZnT2 siRNA, which supports the hypothesis that ZnT2 transports cytoplasmic zinc into zymogen granules. Further support for this role of ZnT2 is that 65Zn in the zymogen granules was decreased by 15% HeLa cells were transfected with a ZnT2 cDNA vector or empty vector and the cells were allowed to accumulate 65Zn.

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125 E fflux of 65Zn from preloaded cells was greater in t he overexpressing cells This finding is congruent with the ZnT2 transport function in zymogen granules. To understand the mechanism of ZnT2 regulation by DEX, the GC antagonist RU486 and a newly discovered GR modulator CpdA were exploited to study the association of ZnT2 gene transcription with signaling via the GR. CpdA exhibits no transactivat ion potential on GRE driven gene transcription (64) and was not able to activate ZnT2 gene expression. However, when AR42J cells were treated with DEX and CpdA at the same time, the hormonal analogy could still initiate expression of MT and ZnT2 presumably via transactivation of GR In contrast, presence of the GR antagonist RU486, prevented CpdA a nd DEX from stimulating the upregulation of MT and ZnT2. These differing results with the two antagonists indicate DEX stimulated ZnT2 expression via transactivation of GREdriven gene expression, but associated with NFB activation Two half GRE site we re found but no full GRE. While realizing that the noncanonical half sites may impart GC regulation for some genes two STAT5 REs were identified in the ZnT2 promoter (130) Consequently, we used a chromobased nicotinoyl hydrazone (CNH), a STAT5 specific inhibitor (131) and the Janus kinase 2 inhibitor (AG 490) to examine STAT5 involvement in ZnT2 activa tion by DEX. T he inhibition of STAT5, particularly in combination with Ja k2 inhibition, completely blocked DEX induction of ZnT2. Notably, the DEXinduced increase in MT expression was not inhibited by either AG490 or the STAT5 inhibitor. An MRE sequence was identified in the downstream of TSS in mouse ZnT2 promoter, and it is highly conserved across species. Markedly enhanced luciferase was observed with hMTF1 transfection. Zinc doubled promoter activity under these

PAGE 126

126 conditions. The contribution of the MRE sequence to ZnT2 regulation was supported through mutation of this sequence in the ZnT2 promoter. A supraphysiological dose of cerulein was added to AR42J cells cultured at low (0.5uM), normal (5uM), and high (50uM) zinc levels to induce acute inflammation, at these various zinc conditions, both ZnT1 and ZnT2 mRNA expression levels were reduced during ceruleininduced pancreatitic damage. AR42J cells were treated with different concentrations of CCK. MT mRNA expression was not altered by CCK simulation. However, b oth ZnT1 and ZnT2 mRNA were decreased in a dose dependent manner by CCK in AR42J cells. AR42J cells were treated with 300mM ethanol for 12h and 24h. Cells were harvested and relative mRNA abundance of MT was found to be elevated during ethanol stimulation. We found a progressive decrease in ZnT2 mRNA abundance, which was inversely correlated with MT levels Zinc transporter expression in pancreatic cancer was studied. Recent findings suggest the aberrant expression of zinc transporter 4 Zip4, contributes to human pancreatic cancer pathogenesis and progression (149) As one of the major and most abundant zinc transporter in the normal pancreas, Zip5 expression decreased more than 100fold in RNA from pancreatic cancer. The most abundant ZnT transporters in normal pancreas are ZnT2 and ZnT8, and are predominantly found in acinar cells and islets, respectively. Both ZnT2 and ZnT8 expressions were found to decrease dramatically in pancreatic adenocarcinoma. Also of note is the 50% r eduction in MT transcript (MT1) in RNA from pancreatic carcinoma. Future Directions Zinc and glucocorticoidregulated ZnT2 expression in the pancreas is a key finding of this study. The transcription factors, MTF1, glucocorticoid receptor, and

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127 Stat5, have been found to be associated with this regulation. As a predominant zinc transporter involved in secretory pathways, ZnT2 is highly expressed in many of the other secretory glands and tissues, primarily the mammary glands, prostate, test e s, kidney s, adipose, placenta, small intestine, and adrenal glands. The functions and regulation of each transporter influence zinc secretion in these exocrine organs Many new technologies and methods are emerging in the nutritional sciences field. Gene knock out and tissue specific conditional knock out techniques increasingly provide powerful model s to study the functionality of genes including the zinc transporters Further study would be beneficial concerning nutrient gene interactions in specific genedeficient s ettings. Epigenetic modification has been proposed in regulating zinc transporter expression. CpG islands can be identified in many of the zinc transporter gene promoter s, including ZnT2 and ZnT5. A r ecent report by Dr. Coney worth suggested that the methy lation of the human ZnT5 pr o moter resulted in reduced expression, which could be associated with agerelated decline in zinc status. The epigenetic effects of aging and cancer are being studied, and more and more findings are suggesting the interrelationsh ip between epigenetic effects on zinc status and altered gene expression related to zinc metabolism F or example, zinc deficiency can decrease DNA methylation status, as in global DNA hypomethylation in rat liver. DNA hyper methylation status can alter the zinc metabolism. H ypermethylation in the promoter of the MT gene alters the MTF1 binding affinity to the multiple MREs in the promoter, thus changing MT gene transcription. There are other MTF1 regulated zinc transporter genes that have not been investigated concerning whether the methylation status can change the promoter

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128 activity. The consensus sequence of MRE is TGCRCNC, and all of the three Cs in this sequence can be a potential methylated mCpG site as follow: TGmCGCNC TGCRmCGC TGCRCNmCG Methylation status changes dramatically in aging and cancer cells consequently. It is important to investigate how the methylation status changes in the course of aging and cancer progression and alter s the zinc transporter gene expression. This in turn might affect cellular zinc metabolism, and consequently either promotes or inhibits aging and cancer progression. Cellular zinc homeostasis dysregulation has been associated with several types of cancer. For example, in pancreatic cancer, an aberrant expression of Zip4 increases cellular zinc concentration, which is associated with cancer progression (149) Another example is found in the LIV 1 subfamily which is involved in invasive behavior of breast cancer (150) The future question for investigation is how intracellular zinc influence s cancer progression and what therapeutic strateg ies can be developed to target those involved zinc transporter s in cancer treatment. MT plays an important role in zinc homeostasis, particularly in the pancreas, where it not only protects against zinc deficiency, but it also prevents any toxic effects of zinc on the pancreas (32) MT 1 is a wellstudied metallothionein protein. H owever there ar e other forms of metallothionein that are not so well studied. For example, it has been suggested that MT2 is found in the pancreatic secretions (32) however the molecular mechanism is still not well understood. Another interes ting observation is that

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129 MT 3 overexpressing transgenic mice die from pancreatic necrosis. It would be of interest to study t he function of MT 3 in the pancreas MT 4 is another novel metallothionein protein, which is not well studied either Genome wide t ranscript analysis from the Genomics Institute of Novartis Research Foundation suggests a highly tissue specific expression is found only in the epiderm and stomach, and MT4s function and regulation in these tissues need further investigation. microRNA s function in gene regulation has been unknown until recent. Plant microRNAs have been found to be important in sensing and responding to the mineral content of the environment (151, 152) In humans, miRNA584 was found to mediat e post transcriptional regulation of the lactoferrin receptor and was involv ed in regulation of nutrient metabolism in newborns (153) Several genes in mineral metabolism have been found to be translational ly regulated. Therefore, the ro les of microRNAs in micro nutrient gene regulation need to be further explored. Another future direction of study could be the development of new molecular sensors that can reveal the amount of zinc in cells (154) Zinc dysregulation is becoming more and more important in the progression of a number of diseases Zinc imaging methods will continue to emerge as a powerful tool s, not only in disease prevention, but al so in the diagnosis, treatment of many diseases including diarrhea, cancer, lower immunity, diabetes, Alzheimers disease, acrodermatitis enteropathica, fume fever, and agerelated macular degeneration.

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130 Fi gure 7 1 Hypothetical schematic model of zinc transport and secretion in pancreatic acinar cells. Zinc influx is influenced by ZIP5 located at the basolateral plasma membrane. Zinc secretion at the apical plasma membrane is regulated through two different pathways. Zinc is transported into lumen through ZnT1 localized on apical plasma membrane. Cytosolic zinc is sequestrated into zymogen granules by ZnT2 and is released during regulated exocytosis. Expression of ZnT2 is mediated by MTF1, depending on the intracellular zinc level. MT and ZnT2 expression is regulated through the glucocorticoid receptor (GR) through an essential interaction of the GR with STAT5. ZnT2 expression is more likely to be associated with secretory stimulation of digestive enzymes.

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143 BIOGRAPHICAL SKETCH Liang was born in 1981 in Hami, Xinjiang province, China. He received his Bachelor of Science in b iological sciences at Fudan University (Shanghai, China) in 2004. He joined the graduate program in the Food Science and Human Nutrition Department at University of Florida in 2005. In 2009, the N utritional Sciences d octoral p rogram was developed and im plemented by D r. Robert J. Cousins. Liang com pleted his dissertation under Dr. Robert J. Cousins supervision, and became the first Ph.D. graduate in the Nutritional Sciences d octoral p rogram at the University of Florida. Liang won the first place in Vitamin and Mineral Research Interest Section Poster Competition in Experimental Biology 2009. During his study, he also received several awards, including the Checkers Scholarship from Checkers Inc., the Outstanding International Student Award, the Outstanding Achievement f rom the International Center, and numerous travel awards ( Travel Funds from the Office of Vice President for Research and Graduate Program, the Food Science and Human Nutrition Graduate Travel Award twice, the Graduate Student Council Travel Grant twice, and the Institute of Food and Agricultural Sciences Travel Grant three times )