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1 ROLE OF ZINC AND ZINC TRANSPORTERS IN LIVER REGENERATION By TOLUNAY BEKER AYDEMIR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF D OCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011
2 2011 Tolunay Beker Aydemir
3 For peace
4 ACKNOWLEDGMENTS Writing an acknowledgment became the most difficult part of my dissertation since I am having a hard time finding suitable words to fully describe my gratitude. My first and most sincere acknowledgement must go to my mentor Dr. Cousins for providing the greatest training that any graduate student could ever imagine (in my opinion). He was always approachable and accessible (24/7) and always willing to go the extra mile to help me. His endless energy, enthusiasm, and dedication inspired me and many people around me throughout my doctoral training. He set the perfect example in my life, as a scientist and person, and for that I would like to g ive him my heartfelt thanks. Next, I would like to thank my committee members, Drs. Michael S. Kilberg, Jorg Bunger t, Brian K. Law, and Harry S. Sitren for their invaluable inputs, advice and critiques. I would also like to thank past lab member, Dr. Juan P. Liuzzi, and present lab members Dr. Shou Mei Chang, Moon Suhn Ryu, Greg Guthrie and in particular Alyssa Maki for her help in correcting the English spelling and grammar in my dissertation. Virginia Mauldin deserves the much appreciation for her mother ly friendship and support. Lastly, I would like to thank my family and friends for their unconditional love and support that they provided me through my entire life. The most special thanks goes to my love, Fikret, for sleeping on the very uncomfortable c ouch for many nights just to be right next to me, while I was writing this dissertation and making the beautiful handmade notebooks for me.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ 4 TABLE OF CONTENTS ................................ ................................ ................................ 5 LIST OF TABLES ................................ ................................ ................................ ........... 7 LIST OF FIGURES ................................ ................................ ................................ ........ 8 LIST OF ABBREVIATIONS ................................ ................................ .......................... 10 ABSTRACT ................................ ................................ ................................ .................. 12 1 INTRODUCTION ................................ ................................ ................................ ... 14 Liver Regeneration ................................ ................................ ................................ 14 Cytokine Dependent Pathway ................................ ................................ ......... 16 Growth Factor Dependent Pathway ................................ ................................ 17 Cell Cycle and Gene Expression after Partial Hepatectomy ............................ 20 Termination of Liver Regeneration ................................ ................................ .. 24 Zinc and Zinc Transporters ................................ ................................ .................... 26 Zinc, Zinc Transporters and Liver Regeneration ................................ .................... 28 2 MATERIAL AND METHODS ................................ ................................ ................. 33 Animals ................................ ................................ ................................ ................. 33 Genotyping ................................ ................................ ................................ ............ 34 Partial Hepatectomy ................................ ................................ .............................. 34 Cell Culture and Treatments ................................ ................................ .................. 35 Macrophage Conditioned Medium ................................ ................................ ......... 35 Interleukin 6 Enzyme Linked Immunos orbent Assay ................................ ............. 36 Measurement of Serum and Cell/Tissue Zinc Concentration ................................ 36 Alanine Amino Transferase Assay ................................ ................................ ......... 37 RNA Isolation and Quantitative Real Time Polymerase Chain Reaction ................ 37 Zinc Transporter Antibodies ................................ ................................ ................... 38 Immunoblotting ................................ ................................ ................................ ...... 38 Bromodeoxyuridine Assay ................................ ................................ ..................... 40 Tyrosine Protein Phosphatase 1B Assay ................................ ............................... 40 Zip14 Overexpression ................................ ................................ ........................... 41 Deoxyribonucleic Acid Pull Down Assay ................................ ................................ 42 3 ZINC ENHANCES LIVER REGENERATION ................................ ......................... 46 Introductory Remarks ................................ ................................ ............................ 46
6 Results ................................ ................................ ................................ .................. 50 Hepatic Zinc Increases in Response to Partial Hepatectomy .......................... 50 Zinc Transporters are Differentially Expressed in Response to Partial Hepatectomy ................................ ................................ ................................ 51 Zinc Supplementation Enhances Liver Regeneration ................................ ...... 52 Discussion ................................ ................................ ................................ ............. 55 4 ZINC ENHANCES C MET PHOSPHORYLATION BY INHIBITION OF PR OTEIN TYROSINE PHOSPHATASE 1B ................................ ........................... 76 Introductory Remarks ................................ ................................ ............................ 76 Results ................................ ................................ ................................ .................. 81 Zinc Inhibits Protein Tyrosine Phosphatase 1B Activity ................................ ... 81 Zinc Enhances c Met Phosphorylation ................................ ............................ 82 Discussion ................................ ................................ ................................ ............. 83 5 ZIP14 FACILITATES HEPATIC ZINC UPTAKE DURING LIVER REGENERATION ................................ ................................ ................................ .. 89 Introductory Remarks ................................ ................................ ............................ 89 Results ................................ ................................ ................................ .................. 93 Overexpressed Zip14 Enhances Hepatocyte Proliferation .............................. 93 ZIP KO Caused Decrease in Hepatocyte Prolife ration ................................ .... 94 Transcriptional Regulation of Zip14 during liver regeneration .......................... 96 Discussion ................................ ................................ ................................ ............. 96 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 107 LIST OF REFERENCES ................................ ................................ ............................ 112 BIOGRAPHICAL SKETCH ................................ ................................ ......................... 129
7 LIST OF TABLES Table page 2 1 ZIP transporter primer/probe sequences ................................ ........................... 43 2 2 ZnT transporter primer/p robe sequences ................................ .......................... 44 2 3 Other primer/probe sequences ................................ ................................ .......... 45 2 4 ZIP and ZnT transporter antibody sequences ................................ .................... 45 2 5 List of Antibodies ................................ ................................ ............................... 45
8 LIST OF FIGURE S Figure page 3 1. Proliferation marker change in response to part ial hepatectomy. ...................... 59 3 2. Changes in hepatic zinc in response to partial hepatectomy. ............................ 60 3 3. Differential expression of zinc tra nsporters in response to partial hepatectomy.. ................................ ................................ ................................ .... 61 3 4. Changes in Zip6 and Zip14 expression in response to partial hepatectomy. ..... 62 3 5. Changes in ZnT7 and ZnT8 expression in response to partial hepatectomy. ..... 63 3 6. Changes in hepatic zinc in response t o both dietary zinc and partial hepatectomy. ................................ ................................ ................................ ..... 64 3 7. Effect of dietary zinc on liver function after partial hepatectomy.. ...................... 65 3 8. Effect of dietary zinc on liver regeneration. ................................ ........................ 66 3 9. Dose and time dependent effect of zinc treatment on AML12 proliferation in response to hepatocyte growth factor stimulation. ................................ ............. 67 3 10. Effec t of in vitro zinc treatment on AML12 proliferation in response to hepatocyte growth factor stimulation.. ................................ ............................... 68 3 11. Effect of in vitro zinc treatment on deoxyribonucleic acid replication in res ponse to hepatocyte growth factor stimulation. ................................ ............. 69 3 12. In vitro simulation of initiation of liver regeneration in vivo. ................................ 70 3 1 3 Effect of lipopolysaccaride on interleukin 6 production by RAW macrophages and the influence of conditioned medium on Zip14 expression. ........................ 71 3 14. Effect of macrophage conditioned medium on Zi p14 expression. ...................... 72 3 15. Validation of zinc transporter expression pattern in the in vitro model of liver regeneration initiation. ................................ ................................ ....................... 73 3 16. Effect of Zip14 expression on the intracellular zinc concentration of AML12 hepatocytes. ................................ ................................ ................................ ...... 74 3 17. Effect of macrophage conditioned media on AML12 hepatocyte proliferation. ... 75 4 1. Validation of phosphatase assay. ................................ ................................ ...... 86 4 2. Inhibitory effect of zinc on protein tyrosine phosphatase 1b activity. .................. 87
9 4 3. Effect of zinc on c Met signaling pathway. ................................ ......................... 88 5 1. Effect of Zip14 overexpression on AML12 hepatocyte proliferation. ................ 100 5 2. Effect of Zip14 overexpression on c Met signaling pathway. ........................... 101 5 3. Genotyping of mouse tail samples.. ................................ ................................ 102 5 4. Validation of Zip14 knock out.. ................................ ................................ ........ 103 5 5. Effect of Zip14 k nock out on hepatic zinc in response to partial hepatectomy. 104 5 6. Effect of Zip14 knock out in liver regeneration. ................................ ................ 105 5 7. Transcriptional regulation of Zip14 during liver regeneration. .......................... 106 6 1. Shematic representation of invo lvement of zinc and Zip14 in liver regeneration ................................ ................................ ................................ .... 111
10 LIST OF ABBREVIATION S AAS Atomic Absorbtion S pectrophotometry ALT Alanine Amino T ransferase AE Acrodermatits E nteropathica BrDU 5 bromo 2' deoxyuridine cAMP Cyclic AMP CCl4 Carbon Tetra C hloride CD1 Cyclin D1 CDF C ation Diffusion F acilitator Cdk Cyclin Dependent K inase DTT Dithiothreitol EGF Epidermal Growth F actor elF4E Eukaryotic Initiati on F actor 4E HB EGF Heparin Binding Epidermal Growth F actor HGF Hepatocyte Growth F actor IGFR Insulin L ike Growth Factor R eceptor IR Insulin R eceptor IL6 Interleukin 6 LDLT Live Donor Liver Transplantation LPS Lipopolysaccharide LR Liver R egenerat ion Met Mesenchy mal epithelial Transition F actor MMP Matrix M etalloprotease MRE Metal response element mRNA Messenger Ribonucleic A cid
11 MT Metallothionein MTF 1 Metal Response Element Binding Transcription F actor 1 PCNA Proliferating Cell N uclear A ntigen PHx Partial H epatectomy PMSF P henylmethanesulfonylfluoride PI3K Phosphatidylinositol 3 kinase PTP1B Protein Tyrosine P hosphatase 1B RTK Receptor T yrosine K inase qPCR Quantitaive Polymerase Chain R eaction SLT Split Liver Transplantation S NP Single Nucleotide P olymorphism TBP TATA B ox binding P rotein TGF Transforming Growth F actor TM Transmembrane Tumor Necrosis F actor alpha TPEN Tetrakis (2 pyridylmethyl) Ethylenediamine ZIP Zrt Irt like Zinc Transporter S uper family ZnA Zinc A dequate ZnD Zinc D eficient ZnH Zinc H igh ZnT Zinc T ransporter
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Doctor of Philosophy ROLE OF ZINC AND ZINC TRANSPORTERS IN LIVER REGENERATION By Tolunay Beker Aydemir August 2 011 Chair: Robert J. Cousins Major: Medical Science Biochemistry and Molecular Biology Chronic liver disease and cirrhosis rank twelfth and fifth among the leading causes of death in the America and Europe respectively. Zinc deficiency has been observed in acute and chronic liver disease, thus z inc supplementation has been used in a few clinical trials for treatment of chronic liv er injury. Zinc supplementation esponsiveness to treatment as determined by measuring the level of liver enzymes in serum However, with this measurement method it has not been possible to delineate a role for zinc/zinc transporter s in the regeneration process. Hepatocyte proliferation is the main event in liver regeneration. Zinc is involved in cell proliferation both as a structural and as a regulatory factor in many mitogenic signaling pathways, and its homeostasis in cells is mai ntained by zinc transporters. Zinc transporter, Zip14 expression is inducible by proinflammatory cytokines and the priming step of liver regeneration is cytokine dependent. Using 70% partial hepatectomy as an in vivo model of l iver regeneration, Zip14 up regulation and increased hepatic zinc have been shown. R esults reveal that increased hepatic zinc caused inhibition of PTP1B, enhancement of c Met phosphorylation and consequently enhanced hepatocyte proliferation. Identical results were obtained with Zip 14 overexpression. The
13 hepatic zinc increase and subsequent hepatocyte proliferation in response to partial hepatectomy were diminished in ZIP14 knockout mice. I n this study, Zip14 was identified as a main transporter for the hepatic zinc uptake during liv e r regeneration. Therefore, zinc and Zip14 could be considered as new therapeutic target s for new approaches that take advantage of the regenerative capacity of the liver for the treatment of chronic liver diseases.
14 CHAPTER1 INTRODUCTION Liver Regeneratio n Liver Regeneration (LR) is compensatory hyperplasia after cell/tissue loss to restore original mass and architecture. Loss of liver mass can be induced by s urgical removal of tissue or administering hepatotoxic chemicals (e.g., carbon tetrachloride) Par tial hepatectomy (PHx) was used in this dissertation project, as this procedure is the model that most clearly demonstrates the regenerative capacity of the liver. In the model, 2/3 (~70%) of the rodent liver is removed and the remnant liver enlarges until the mass of the liver is restored. T he reproducibility of PHx and precision of timing of the sequence of ensuing events has made PHx the preferred approach for the experimental study of liver regeneration. The PHx technique is also important because it re sembles regeneration following resection of the liver in a clinical setting. In adult liver hepatocytes are in G 0 phase of the cell cycle and rarely divide (1 ) However after PHx approximately 95% of hepatocytes that are normally quiescent enter the S phase of ce ll cycle. PHx triggers a sequence of events that proceed in an orderly fashion and can be observed from 5 min to 5 7 days after surger y Hepatocytes are the first cells to enter into deoxyribonucleic acid ( DNA ) synthesis. After 70% PHx hepatocytes of the remnant liver undergo one round of DNA synthesis, which peaks at approximately 36 h for the mouse. A second smaller percent of cells enter into a second round of DNA synthesis and establish the original number of hepatocytes. The proliferation of hepatocyt es advance from periportal to pericentral areas ( 2 ) To totally appreciate the regeneration process, knowledge about structural unit of liver, its cells and their localization in the structure is helpful. The structural unit of the
15 l iver i s called the liver lobule The blood supply for this structure comes from portal vein and hepatic artery, which becomes mixed at the edge of portal tract (bile duct and branches of the hepatic artery and hepatic portal vein) where the liver sinusoids are found. Blood travels through sinusoids from portal tract to central vein. Liver sinusoids between hepatocytes and endothelial cells ( 3 ) There are four different cell types found lining the sinusoid. (1) Endothelial cells, the majority of the lining cells of the sinusoids, do not have a basement membrane. They are specialized fenestrated endothelial cells. This structure allows direct contact between hepatocytes and plasma for metabolic processes. (2) Hepatic stellate cells reside in the subendothelial space, between the basolateral surface of hepatocytes and si nusoidal endothelial cells. In normal liver, stellate cells store vitamin A droplets in their cytoplasm and play an important role in liver inflammation because of their ability to recruit inflammatory cells through expression of adhesion molecules and sec retion of chemokines and cytokines. (3) Kupffer cells are liver macrophages. They are mainly located in the lumen of hepatic sinus oids, where they particulate foreign materials from the portal circulation ( 4 ) (4) He patocytes, paranchimal liver cells, extend along the sinusoids to the centriolobular region as polarized epithelial cells. Roughly 80% of the mass of the liver is composed of hepatocytes. These cell s are the main cell type in the liver and are where all metabolic events are carried out. Liver regeneration signaling process can be divided into three main pathways: (a) a cyto kine dependent pathway that is largely responsible for entry of quiescent hepa tocytes into the cell cycle (transition from G 0 G 1 ), (b) a growth factor dependent
16 pathway that is responsible for cell cycle progression (progression through the G 1 phase to S phase), and (c) a still poorly described pathway that links metabolic signals w ith ribosome synthesis and DNA replication ( 5 ) Each of these pathways is required but not sufficient alone for regene ration. Interactions of these pathways are also essential for regenerative growth. Cytokine Dependent P athway Cytokines are involved in the initiation of liver regeneration. They prime hepatocytes to enter the cell cycle and to respond to the mitogenic eff ect of growth factors. The main participants in the cytokine network are t umor necrosis factor alpha (TNF nterleukin 6 (IL and IL 6 increase very rapidly after PHx, and are components o tumor necrosis factor receptor 1 ( TNFR1 ) nuclear factor kappa light chain enhancer of activated B cells ( ) IL 6 Signal transducer and activator of transcription 3 ( STAT3 ) pathway. Each component appears sequentially in the regenerating rodent liver during the first 12 h after PHx. The propose d pathway is that initially TNF plasma membrane of Kupffer cells causing an increase in IL 6 expression through the ( 6 ) IL 6 causes activation of ST AT3 and translocation to the nucleus in Kupffer cells and also in hepatocytes ( 7 ) PHx in mice lacking the TNFR1 is associated DNA synthesis ( 8 ) however, Injection of IL 6 prior the PHx restore d the DNA synthesis. IL6 k nock out ( KO ) mice also showed an impaired regenerative resp onse that is corrected by an IL 6 injection ( 9 ) Neit her TNF 6 is a direct mitogen for hepatocytes. They do not induce DNA synthesis in primary cultures in serum free medium nor do these mediators induce
17 hepatocyte DNA synthesis when injected in the intact animals ( 10 ) H owever, mice over expressing IL 6 and its soluble receptor (IL 6 binds to a soluble receptor and the complex binds to the receptor gp130) have areas of periportal hepatocyte hyperplasia ( 11 ) Thus, even though these mediators are not direc t mitogens for hepatocytes, they are important contributors to the initiation of signaling pathways leading to proliferation of hepatocytes at the very early stage of regeneration. It is particula rly intere 6 increase after PHx since they are pro inlammatory cytokines and important mediators of the acute phase response. In PHx part of the liver is removed surgically thus there is no tissue damage or inflammation produced by the procedur e. However, there is increased portal blood pressure to remnant liver that may cause stress. The recovery from these stress conditions can resemble wound healing conditions following PHx ( 10 ) This process will be explained in below in the section describing very earl y events in liver regeneration following PHx. Growth Factor Dependent P athway In the wound healing scenario, the injury to the tissue results in disruption of capillary vascular networks and extravasation of blood, accompanied by local release of coagulati on factors, platelets and growth factors ( 12 ) This is cl early not the case following 70% PHx. In PHx 70% of the liver is surgically removed without damage to the residual tissue However, portal blood flow per unit of liver tissue is tripled. It has been shown that if the pressure of the portal vein is kept constant, there is a deficient activation of hepatocyte growth factor (HGF) and increased apoptosis. These changes after PHx closely resemble the wound healing process and suggest they are necessary for the initiation of regeneration. As seen in early stages of wound healing one of the
18 first observed biochemical changes is increase in activity of urokinase plasminogen activator (uPA) ( 13 ) Even though the exact relationship between increased uPA and portal blood pressure in remnant liver is not clear in the PHx model, it has been shown in several cell types, including endotheli al cells, that there is an increase in uPA after mechanical stress associated with increased turbulent blood flow ( 14 ) Thus, alteration of vascular flow patterns alone can trigger some of the early events in regeneration process. uPA is present in extracellular matrix and its primary physi ological substrate is plasminogen. After PHx the increase in uPA activity is accompanied by activation of plasminogen to plasmin and as a result appearance of fibrinogen (a main part of the extra cellular matrix) degradation products ( 15 ) Other evidence that supports extra cellular matrix (ECM) remodeling at the very early stages of liver regenerati on is activation of metalloproteinase 9 (MMP9) and metalloproteinase 2 (MMP2) after PHx ( 16 ) Studies from wound healing and tumor biology have shown that matrix remodel ing causes signaling through in t egrins and is associated with release of locally bound growth factors and peptides that have signaling capacities ( 17 ) Hepatic ECM binds many growth factors. One of the main matrix binding growth factors in liver is HGF ( 18 ) Inactive, single chain HGF bound to hepatic biomatrix is locally released during matrix remodeling and is activated to its active for m by uPA (HGF and plasminogen have the same consensus sequence at their activation site) ( 19 ) Activated HGF is available locally, but it enters the circulation. HGF in liver is produced predominantly by the stellate cells, but also hepatic endothelial cells ( 20 ) HGF messenger ribonucleic acid ( mRNA ) increases not only in liver it also increases in lung and spleen ( 21 ) The signal
19 triggering this extrahepatic participation is not clear. How ever, it has been shown that IL 6 stimulates production of HGF in responding cells ( 21 ) Thus IL 6 might be the candidate for this effect. Release and activation of HGF result in activation of c Met ( 22 ) MET (mesenchymal epithelial transition factor) is a proto oncogene that encodes a protein called c Met o r hepatocyte growth factor receptor (HGFR). c Met is a membrane receptor that is essential for embryonic development and wound healing and liver regeneration. HGF is the only known ligand of the c Met receptor. The epidermal growth factor receptor (EGFR) is also activated with the same kinetics as c Met ( 22 ) HGF and all the EGFR ligands (epidermal growth factor EGF, transforming growth factor alpha ( TG F ) heparin binding EGF like growth factor ( HB EGF) are direct mitogens for hepatocytes in chemically defined serum free medium ( 23 ) when injected alone into intact normal mice and rats ( 24 ) EGF are produced by hepatocytes and functions through an autocrine mechanism, as hepatocytes both produce the ligand and appropriate receptor binding ( 25 ) Mice with and develop tumors ( 26 ) On th affect liver regeneration ( 27 ) HB EGF is produced by endothelial cells and kupffer cells ( 28 ) Expression of HB EGF is greatly increased in regenerating liver after PHx. HB EGF transgenic mice with liver targeted over production have enhanced regeneratio n ( 29 ) whereas HB EGF KO mice h av e deficient regeneration respon se ( 30 ) In summary all the EGFR ligands are direct mitogens for hepatocytes. Moreover there is a great
20 redundancy in the regeneration process, probably because of the overlap between various ligands of the EGF family. The only irreplaceable growth factor in the liver regeneration process is HGF because of the following reasons: (a) HGF levels in plasma increase 10 20 fold a fter PHx, (b) active HGF is consumed from intrahepatic stores and is followed by new HGF synthesis, (c) HGF causes a strong mitogenic response in vivo and in vitro, (d) the liver HGF receptor (c Met) becomes activated short after PHx and (e) targeted genet ic elimination of c Met expression in liver is associated with a very diminished or absent regenerative respond. The degrees of suppression of regeneration suggest that there are unique signaling pathways associated with c Met that are not compensated by E GFR or other mechanisms (the same dependency on c Met has also been found in wound healing ) ( 31 ) Cell Cycle and Gene Expression after Partial Hepatectomy There is evidence showing increases in cell cycle proteins after PHx. The cell cycle process is regulated by cyclin, cyclin d ependent kinases (cdk) and cell cycle inhibitors. Gene expression after PHx can be groupe d in two phases. (1) Activation of pre existing transcription factors (TF) to induce expression of immediate early and delayed early response genes during the priming phase of LR. (2) De novo synthesis of TFs to regulate mitosis, termination of proliferati on and size adjustment in the regenerating liver. Pre existing TFs are activated by post translational modification s without de novo protein synthesis. This allows rapid expression of genes that promote hepatocytes to leave their G 0 state and enter the ce ll cycle (G 0 G 1 transition). Thre e of the TFs that are first to be activa ted are NF B, STAT3 and activator protein 1 ( AP 1 ) (3 2, 33 ). These TFs stay activated for the first 5 hours after PHx. TNF l ipopolysaccharide ( LPS )
21 complement factors, complement component C3a ( C3a ) and complement component 5a ( C5a ) and inter cellular adhesion molecule 1 ( ICAM ) activate NF B mediated transcription in Kupffer cells. Kupffer cell produce d IL6 binds to the IL6 receptor on hepatocytes and activates STAT3 dependent tran scription in hepatocytes. Activation of STAT3 causes an increase in expression of Myc, Fos, Jun, Gadd45 and e arly response 1 (E gr ) (34 ) Liver specific deletion of STAT 3 causes lower expression of cyclin D1 and cyclin E required for G 1 S transition of the cell cycle c Jun and c Fos bind to the AP 1 DNA binding site of early response genes. Conditional inactivation of Jun in the perinatal liver causes high morbidity and severe impairment of LR because of higher level s of cell cycle inhibitor p21 and stres s kinase p38 (35, 36 ) Decreased c Fos expression and delayed hepatic proliferation were observed in the cAMP responsive promoter element modulator (Crem) KO mice. As a result of diminished c Fos expression, cell cycle regulators cyclin A, B, D, E and cyc lin dependent kinase 1 ( Cdk1 ) were down regulated after PHx (37 ) Beta catenin ( 3 8 ) and notch 1 ( 3 9 ) intracellular domain appear in hepatocyte nuclei within 15 30 min after PHx. Elimination of the expression of these proteins by RNA interference decreases the regenerative response. Ligation of the lef t lateral and median lobes dur ing PHx surgery leads to hypoxia in the remaining lobes of the liver (40, 41 ) Hypoxia induced factor 1 translationally modified and 48 hours following PHx ( 42, 43 ) Conditional hepatic KO mice caused a delay in DNA replication ( 44 ) Additionally, liver specific K O mice develop severe hypoglycemia and accumulate more glycogen in the regenerating liver when compared
22 to wild type ( WT ) mice Pepck and Pgk1 ( 45 ) At the same time, KO mice have a higher level of phosphorylated Akt and g lycogen synthase kinase 3 ( GSK ) kinases, leading to accelerated glycogen synthesis in regenerating liver. Thus the activity of HI meet ing the metabolic demand imposed by PHx. Ex pression of immediate early and delayed early response genes during the priming phase of LR leads to the do novo synthesis of TFs, thus starting the new cascade of transcriptional activity during the G 1 S and S G 2 transitions. The newly synthesized TFs inc lude Myc, CCAAT/en hancer binding proteins (C/EBP) and FoxM1 (46 ) Liver specific expression of c Myc transgene resulted in a 10 hour earlier hepatocyte proliferation during LR ( 47, 48 ) This result was correlated with early expression of cyclin A and Cdk1 genes. The C/EBP form a transcription factor family with key roles in the regulation of several hepatocyte specific genes. ( 36 ) Anti ( 1, 50 and 51 ) Additionally, K O mice displayed a 75% reduction in replicating hepatocytes and decreased expressions of Egr 1 TF, cyclin B and E following PHx ( 52 ) Increased hepatic expression of FoxM1 occurs during G1/S transition of the cell cycle and remained elevated throughout th e proliferation period, thus it displays the expression kinetics of delayed early TF. Liver specific expression of FoxM1 transgene revealed 8 hours acceleration of hepatocyte entry to S phase and mitosis ( 82 5 4 ) This accelerated proliferation was associ ated with an earlier expression of S phase phophatase and diminished induction of cdk inhibitor p21 in a regenerating liver.
23 Conditional liver FoxM1 KO mice displayed a reduct ion in hepatocyte DNA replication and mitosis ( 55 ) Up regulation of Egr 1, Ets2 and Atf3 genes after the first 4 hours after PHx were found by high density microarray analysis ( 5 6 ). The Egr1 transcription factor was implicated in expression of the cell division cycle 20 (cdc20) gene, a key regulator of the anaphase promoting complex during mitosis ( 5 7 ) Egr1 KO mice displayed impaired metaphase to anaphase transition during mitosis. The Ets2 transcription factor interacts with C/EBP stimulation in primary mouse hepatocyte s Atf3 is a member of ATF/cAMP resp onsive element binding protein (CREB) TF family, and is highly up regulated. Atf3 expression stimulates proli feration of mouse hepatoma cells by Atf3 dependent activation of cyclin D1 transcription ( 5 8 ). Late response TFs control mitotic entry and progression during LR. G2 M transitions occur during the second day after PHx, and require expression of cyclin A, cy clin B, cyclin mediated kinases Cdk1, Cdk2 and the cdc25 family of phosphatases. Some of these genes such as cdc25a and cdc25b are induced by FoxM1 ( 55, 59 ). Transcriptional activity of FoxM1 was associated with increase d cyclin F and p55cdc, which ar e in volved in completion of mitosis during regeneration ( 6 0 in the G 2 responsible for cell cycle progression ( 6 1 KO mouse, the regulatory role of C/EBP p eroxisome proliferator activated receptor ( ) coactivator 6 2 ). gluco neo genesis and ketogenesis in the regen erati ng liver. These findings show the
24 roliferative and metabolic functions during regeneration. In contrast with proliferative phase of liver regeneration ( 6 3 a multi protein complex with histone deacetylase 1 (HDAC1), cyclin D3 and Brm, silencing E2F dependent promoters to decrease hepatocyte proliferation ( 6 2 ). Termination of Liver R egeneration The regeneration process stops when the liver mass has been restored, however ; the exact stop signal has not known t o date. Because of its known anti mitotic properties, t ransforming growth factor ( ) has been the most a ttractive candidate. Despite its anti regeneration. At the same time however TGFRI and II expression is decreased markedly particularly during first 48 h. This decrease may explain why hepaocytes are ( 6 4 ) have normal termination of liver regeneration, unless there is a concurrent elimination of activin ( 6 4 ) Activin i s also a mito inhibitor and it is possible that termination of Regardless of its potential to terminate regeneration as a mito has a role in the assembly of hepatic tissue towards the end of r egen eration ,s timulat ing stellate cells to synthesize new ECM. It is possible that the reassembly of ECM and the sinusoidal capillary network provides matrix driven signaling through integrin to terminate regeneration.
25 Enhanced hepatocyte proliferation and hepa tomegaly were reported in a liver specific KO of an ECM integrin linked kinase (ILK) ( 6 5 ). Changes in ECM actively signal to ILK, which then modulated expression of hepatic genes via TFs such as C/EBPs ( 6 5 ). C/EBP antagonizing fashion during LR. Thus C/EBP between proliferation and termination phases of LR. leads to gluconeogenesis and growth suppression during termin ation phase regulates transcription of E2F target genes during proliferation phase of LR. increased in quiescent ILK KO hepatocytes, mimicking the proliferating phase of LR ( 6 6 ). A d efect in the termination of LR in l iver specific ILK KO mice was also reported ( 6 6 ). The ILK KO liver exhibited sus tained cellular proliferation following PHx, causing the ILK KO mice to have 158% of their original weight compared to the normal 100% liver mass restoration in WT mice. In t he same study they also observed decreased level s of the phosphorylated Yes activator protein (YAP) in ILK KO mice at 3 14 days following PHx when compared to WT mice (66) YAP is an important effector of Mst1/2 signaling and in the dephosphorylated form binds to the promoter of target genes to promote cellular proliferation and tissue growth ( 6 7 68 ). YAP becomes inactive when it is phosphorylated. Mst1/2 mediated phosphorylation/inactivation of YAP was shown to limit organ size in Drosophila and mice ( 68 ) These results from ILK KO studies collectively suggested a role for ILK for the termination of LR through either regulation of C/EBP TFs or the Mts1/2 YAP pathway.
26 Zinc and Zinc T ransporters Zinc is essential micronutrient that has been identified in mo re than 300 different enzymes including, alcohol dehydrogenase, alkaline phosphatase, matrix metalloproteinases, reverse transcriptase, RNA polymerases, and superoxide dismutase ( 69 70 ) Zinc finger proteins are a family of more than 2,000 transcription factors and accessory factor that bind specifically to DNA modify that binding and therefore regulate transcription. Zinc is also involved in intracellular sig naling and neurotransmission ( 71 72 ) Zinc transporter proteins maintain zinc homeostasis through tight regulation of zinc influx, efflux, and distribution to intracellular organelles. Zinc transporter proteins are essential for these metabolic and functional adjustments. Zinc transporters are found within two gene families, the ZIP (SLC39) and ZnT (SLC30) ( 73 74 ). ZIP transporters function in zinc influx into the cytosol, while ZnT family transporters function in zinc efflux from the cytosol. Zinc transporter contributions to cellular events can be illustrated through published data on dendric cell maturation ( 75 ) In this study it has been shown that dendric cell maturation required a decrease in intracellular zinc. This decrease was provided through a decrease in exp ression of the ZIP6. Increased ZIP8 expression during T cell activation was also s howed by our lab ( 76 ). The T cell activation process required increased cytoplasmic zinc while the zinc content of the lysosomal vesicles was decreased. ZIP8 localized on the lysosome functions in translocating zinc from the lysosome to cytoplasm and result in sustained cAMP response element binding protein (CREB) phosphorylation.
27 Expression of zinc transporters change s depending on the need for zinc in the cell. Abnormality i n their expression causes diseases. Best known example of this is Acrodermatitis Enteropathica (AE). AE is caused by impaired zinc uptake in small intestine and is produced through a mutation of the ZIP4 gene. More recently it has been reported that a sing le nucleotide polymorphism (SNP) of ZnT8 is associated with type 2 diabetes with reduced insulin secretion ( 77 ) Zinc availability is important for tumor growth and progression. Zinc transporter involvement to different types of cancers has been reported. High Zip4 expression in pancreatic adenocarcinoma was shown ( 78 ) Forced expression of ZIP4 increased cell proliferation and pancreatic tumor volume in the nude mice model. In another study, ZIP6 and ZIP7 have been found to be associated with estrogen positive breast cancer and metastasis to lymph nodes ( 79 ) ZIP10 invo lvement to invasive behavior of breast cancer cells has also been reported ( 80 ) These fi ndings are particularly important since cancer development and liver regeneration process share many pathways that regulate cell proliferation and metastasis. Changes in zinc transporter expressions detected by microarray during liver regeneration have bee n reported ( 81 ) ZIP3, ZIP6, ZIP7, ZIP10, ZIP14, ZnT3, ZnT4, ZnT5, ZnT7 were increased and ZIP4, ZIP8, ZnT1 were decreased suggesting zinc transport was potentially involved in liver regeneration. Among the se zinc transporters ZIP14 is of specific interest to us since we previously reported that IL 6 results in ZIP14 up regulation in WT mice liver after injection of either LPS or turpentine ( 82 ) However, in IL6 KO mice there was an impaired induction of liver ZIP14. Relevant to liver re generation, in a small pilot study mice were injected with CCl4 (2mg /kg) and zinc
28 transporter expression was measured. Result showed that ZIP14 had the highest induction among all others. Collectively these observations lead to the hypothesis that ZIP14 mi ght be the main player for zinc homeostasis and function in liver regeneration. Zinc, Zinc Transporters and Liver Regeneration As mentioned in earlier sections, liver regeneration is multi step process. In this section potential zinc and zinc transporter i nvolvement in specific steps will be discussed. One of the first steps in liver re generation is production of TNF As a result of binding of TNF pffer cells and hepatocytes, IL 6 is a zinc finger transcription factor that inhibits TNF ( 83 84 ) It has been shown in the HL 60 ( human pro myelocytic leukemia cell line that differentiates to the monocyte macrophage phenotype by PMA) cells that expression of A20 is zinc dependent ( 85 ) B iphasic effect of zinc on TNF was also shown in our lab (unpublished data) suggesting that zinc is an impo rtant component to regulate TNF Another early event in liver regeneration is elevated uPA activation. Activated uPA causes conversion of plasminogen to its active form plasmin, which is a proteolytic enzyme responsible for degrading ECM proteins. When plasmin detaches from the cell surface it becomes inactivated very quickly. A relatively new, histidine rich glycoprotein, has been found that tethers plasmin to the cell surface ( 86 ) The importance of this protein from a zinc perspective is that elevated levels of zinc are required to tether plasmin to the cell surface.
29 Increased activation of MMP2 and MMP9 is also observed in early liver regenera tion. These are members of zinc depen dent endopeptidases family enzymes that collectively degrade ECM constituents. All the members of these family enzymes possess zinc containing catalytic domain meaning that they require zinc to be activa t ed. Degradation of ECM causes HGF release from the m atrix and activation to its active heterodimeric form by uPA. The activated form of HGF binds to its receptor c M et on the hepatocyte membrane. c Met is a tyrosine kinase receptor. Its activation through HGF binding induces c Met kinase catalytic activity, which triggers transphosphorylation of the tyrosines (Tyr) Tyr 1234 and Tyr 1235 in the int r acellular kinase domain ( 87 ) Consequent phosphorylation of tyrosine 1349 and 1356 at the docking domain engage various signal transducers, thus initiating a whole spectrum of biological activities The rat s arcoma ( RAS ) pathway mediates HGF induced scattering and proliferation signals. Of note, HGF, compared to most mitogens induces sustained RAS activation, and thus prolonged m itogen activated protein kinase ( MAPK ) activity. The MAPK cascade activation is the key signaling pathway involved in G1 phase progression. MAPK cascade is also activated by EGF. It has been shown that EGF dependent hepatocyte proliferation through MAPK pathway is delayed by inhibition of the metal responsive transcription factor 1 (MTF 1) which requires zinc for activation and nuclear localization ( 88 ) Another consequence of MTF 1 inhibition is increased production and activation inhibitor suggesting that MTF 1 might function as a it functions on
30 ZIP10 expression ( 89 ) regeneration. Potential MTF effective liver regeneration. Another zinc transporter that its expression is regulated by MTF 1 is ZnT1. C. elegans cation diffusion facilitator (CDF) protein is a homologue of mammalian ZnT1, and responds to zinc in a mode consistent with activity as a zinc exporter ( 90 ) CDF 1 activity increases Ras mediated signaling. Similarly, CDF 1 and ZnT1 bind to Raf 1 and may be responsible for full activation of this downstream component of RAS pathway ( 91 ) This mechanism could be taking place in HG F induced RAS pathway in hepatocyte proliferation. The activation of c Met by HGF binding is also linked to cell growth and survival through activation of PI3 kinase/Akt. Interestingly, it has been found that zinc can also activate this pathway ( 92 95 ) Activation of Akt is via P13K, as it is prevented by P13K inhibitors. Furthermore, P13K activity was shown to be enhanced in cells exposed to zinc (92 95 ) Zinc is known to be a phosphatase inhibitor. The phosphatase and tensin homolog (PTEN) protein acts as a phosphatase in the Akt signaling resulting in inhibition of the p athway. One possibility for zinc to enhance the Akt activation might be through inhibition of PTEN since it has been shown that PTEN protein levels are lost in zinc treated airway epithelial cells ( 96 ) Akt the downstream target of PI3K exerts its anti apoptotic effects in a variety of ways, including phosphorylation and activation of IKKs (I KappaB Kinases). This results in I KappaB degr adation and allows to enter the nucleus and activate transcription of anti apoptotic genes.
31 Activation of Met ty rosine kinase also activates phospholipase C that results in the elevation of intracellular calcium and activation of protein kinase C (PKC) pathways. PKCs are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. Zinc is found in the regulatory domain of PKC isoforms, where it is thought to influence the activity and translocation of PKC. HGF is also known to be a scatter factor, which increases the motility of a variety of cell types. HGF is produced by several tissues, including neoplasms; it can therefore provide a stimulus for increased motility of malignant cells. It has been reported that ZIP6 expression is related to metastasis to lymph nodes in breast cancer ( 97 ) ZIP6 is a downstream target for STAT3. The mechanism of ZIP6 involvement to breast cancer metastasis through e cadherin regulation is also reported ( 98 ) It was shown that silencing of ZIP6 caused down regulation of e cadherin while overexpression caused up regulation HGF signaling may induce signaling f or hepatocytes to gain mobility to reestablish original liver architecture during regeneration. Therefore, ZIP6 may have role in liver regeneration through regulation of hepatocyte cell motility. In conclusion al l observations discussed above led to the hy pothesis that zinc and one or more zinc transporters may have an important role in liver regeneration. To investigate this hypothesis, three specific aims were proposed in this dissertation project: Determination of changes in zinc transporter expression a nd which of those are zinc responsive during liver regeneration Exploration a role for zinc in the HGF pathway during hepatocyte proliferation
32 Delineation of a specific role and regulation of ZIP14 during hepatocyte proliferation
33 CHAPTER 2 MATERIAL A ND METHODS Animals Young adult (8 12 weeks) male C57BL / 6 strain mice were purchased from Charles Riv er Breeding Laboratories. Zip14 /+ and Zip14 +/+ mice were purchased from Mutant Mouse Regional Resource Center (MMRRC) Un iversity of California, Davis. A t argeted mutation in Zip14 gene ( exons 3 through 5) was generated in strain 129/SvEvBrd derived embryonic stem cells. The chimeric mice were bred to C57BL/6J a lbino mice to generate F1 Zip14 /+ mice. Zip14 / mice were obtained through further breeding of f ounder Zip14 /+ mice in the Genetic and Cancer Research Complex ( GCRC ) animal facility, University of Florida. Mice were given free access to tap water and received commercial rodent diets [Harlan Teklad 7912 030811M ] with a 12 h our light dark cycle. Proto cols were approved by the University of Florida Institutional Animal Care and Use Committee. The design of mice experiments in this dissertation project were composed of different dietary treatments and PHx Dietary treatments were low zinc (ZnD, <0.5 mg Zn/kg diet ), zinc adequate (ZnA, 30 mg Zn/kg diet ) and high zinc (ZnH, 180 mg Zn/kg diet ). In the first set of experiments mice were fed with ZnA diet for 4 days then underwent either PHx or a sham operation under anesthesia. Mice were sacrificed over a 4 8 h ou r s time course post PHx to collect blood and tissue. In the second set of experiments, after 4 days of acclimation with ZnA diet, mice were randomly assigned to be fed ZnA, ZnD or ZnH diets for a week then unde rwent PHx or a sham operation. Mice wer e sacrificed at 24 h our post PHx to collect blood and tissue. Five mice for the surgery and three mice for sham operations were used in all experiments. Mice were anesthetized by isoflurane inhalation for surgery, postoperative
34 blood/tissue collection and euthanasia. The flow of 3 % isoflurane was at the rate of 2 L/min ( 99 ). Maintenance anesthesia dur ing surgery was achieved with 2 % isoflurane at the same flow rate. Genotyping Genomic DNA was extracted from mouse tail samples by using ZyGem PrepGEM kit. W ater (89 l), 10x Buffer GOLD (10 l) and prep GEM (1 l) were added to the tail sample. Mixture was incubated at 75C for 15 minutes and at 95C for 5 minutes. DNA containing supernatants were collected and used as a template in polymerase chain reaction ( PCR ) reactions. Two sets of primers were used F irst set was s pecifically design to amplify 164 bp wild type ( WT ) TCATGGACCGCTATGGAAAG sense primer GTGTCCAGCGGTATCAACAGAGAG A s econd set was specifically design ed to amplify 469 bp mutant DNA (sense primer TGCCTGGCACATAGAATGC GCAGCGCATCGCCTTCTATC for PCR amplifications and products were visualized in a 1.5% agarose gel. DNA that was only amplified by WT specific primers were WT mice, DNA that was only amplified by mutant specific primers were knockout ( KO ) and DNA that was amplified by both WT and mutant specific primers were heterozygous ( HET ) Partial Hepatectomy A rat partial hepatectomy method ( 100 ) was modified for the mouse PHx (99, 101 ) After appropriate steril ization of the surgery site, a 1.5 cm long, upper midline incision in the skin was made Once adequate exposure of the liver was obtained, partial resection of the liver was performed b y excisions of the left and medi an lobe by 5 0 vicryl sutures After excisions, the peritoneum was reapproximated with 5 0 vicryl
35 suture s and skin was closed with 4 0 nylon suture s B uprenorphine (0.1mg/kg) analgesic, in sterile saline was injected subcut aneously. According to the design of experiments blood and tissue were collected either in a 48 h ou r s time course or at 24 h ou r s post PHx. Cell Culture and T reatments AML12 hepatocyte cell line (American Type Culture Collection, CRL 2254) was established f rom hepatocyte regulates normal growth in epithelial tissues through binding to the EGFRs. Therefore at serum starvation and for subsequent steps media was s upplemented with 100 ng/ml EGFR inhibitor (C albiochem). Expressio n of liver specific proteins decreases with time in culture. Thus AML12 hepatocytes were used only in the first ten passages. AML12 hepatocytes were maintained at 37C in 5% CO2 in DMEM/F 12 containing 10% (v/v) FBS, 40 ng/mL dexamethasone, ITS (insulin, transferrin, selenium) supplement (BD Biosciences) and penicillin, streptomycin (Cellgro). Before the treatments, AML12 hepatocytes were always serum starved for 20 hours. After serum starvation, AML12 hepatocytes were either pretreated with a combination of pyrithione and zinc or conditioned media that was collected from RAW264.7 mouse macrophage supernatants then HGF (Imgenex) f or either 30 minutes or 48 hour time period RAW264.7 mouse macrophages (American Type Culture Collection, TIB 71) were maintain ed 37C in 5% CO2 in DMEM containing 10% (v/v) FBS. Macrophage Conditioned Medium RAW 264.7 cells were frozen from the same passage in multiple vials (3x10 6 cell/vial). For each experiment a vial of cells was thawed and treatments and media collection were always conducted at the second passages. RAW264.7 cells were
36 seeded at the density of 3 x10 6 cells /15 cm 2 plate and after 48 hours cells were incubated with either 20 g/ml LPS (Sigma) or without any supplement as a control for two hours. Supernatants wer e collected from both and mixed in 1:1 ratio with the medium usually used for hepatocytes. Supernatants that were collected from LPS treate d and control macrophages were denot ed as LPS CM and CM, respectively. AML12 hepatocytes were treated with either t hese mixtures or mixtures supplemented with 20 ng/ml IL6 (Prospec) for 2 to 4 hours. Interleukin 6 Enzyme Linked Immunosorbent Assay instructions for the enzyme linked immunosorbent a ss ay ( ELISA ) kit (eBioscience). Supernatants were obtained from LPS treated and control RAW264.7 cells. IL6 standards were provided with the kit and ranged between 0 250 pg/ml. Plates were coated with capturing antibody overnight at 4C. Blocking and detecti on antibo dy incubations with samples followed. Absorbances were read at 450 nm (Molecular Devices) after Avidin Horse Radish Peroxidase ( HRP ) and substrate incubations. Measurement of S erum and C ell/ Tissue Zinc C oncentration The blood was collected by ca rdiac puncture under anesthesia. Serum was obtained by a two stage centrifugation at 2000 x g for 10 min utes These serums were used for serum zinc and ALT measurements. Liver tissue was collected (~50mg) and digested in 2 ml HNO 3 for 3 h ours at 90C. AML1 2 hepatocytes were washed with wash buffer [ 0.9% sodium chloride ( NaCl ) 10 mM ethylenediaminetetraacetic a cid ( EDTA ) and 10 mM N 2 Hydroxyethylpiperazine N' 2 Ethanesulfonic Acid ( HEPES )] twice and scraped into the iced cold phosphate buffered s aline ( PBS ) Cells were centrifuged at 3000 rpm for 5 minutes then cell pellets were digested in 200 l HNO 3 for 2 h ours at
37 80C. Serum, liver tissue and AML12 hepatocyte cellular zinc concentrations were measured by flame atomic absorption spectro photo metry (AAS). Absorbance values for tissue and cellular zinc were normalized to the wet tissue weight and total protein concentration measured by RC / DC assay (BioRad ), respectively. This method allowed us to obtain g zinc in gram protein/wet tissue. Alanine Amino Tran sferase Assay Serum alanine amino t ransferase ( ALT ) measurements were used for the assessment of liver health status of the mi ce following PHx or sham operations. S erum ALT levels were measured by a colorimetric end poin t method that is modified from World Health O rganization standard operating procedures for clinical chemistry. ALT is responsible for transfer of an amino group from alanine to an keto glutaric acid. The products that are produced by this reaction are glutamate and pyruvate. Therefore pyru vate standards were prepared in phosphate buffer (50 mM Na 2 HPO 4 2H 2 O, 16 mM KH 2 PO 4 at PH: 7.4) and used for formation of the standard curve. An a lanine substrate solution [ (1M DL alanine, 10 mM keto glutarate and 5 mM sodium hydroxide ( NaOH) ] was prepare d in phosphate buffer. The a lanine substrate and serum samples were incubated at 37C for 30 minutes. 2, 4 Dinitrophenylhydrazine ( DNPH ) [ (1 mM 2, 4 DNPH in hot 1 M hydrochloric a cid ( HCl) ] was added and incubated in room temperature for 20 minutes for the formation of hydrazone from the ketoacids. The r eaction was stopped with 0.4 M NaOH and absorbance was read at 510 nm (Molecular Devices) RNA Isolation and Quantitative Real Time Polymerase Chain Reaction At certain time points during post PHx or sha m operations, liver tissues were collected in RN ase Later (Ambion) and homogenized (Polytron Brinckman Instruments)
38 in TriZol reagent (Ambion). Cells were harvested after indicated treatment conditions (above) in TriZol reagent as well. After total RNA iso lation, to protect against residual DNA contamination, all RNA samples were treated with Turbo DNA free reagents (Ambion) as described by the manufacturer. The total RNA concentrations were measured by optical density at 260 nm (Nanodrop) and then samples were diluted to a 2 l of the diluted sample was used. The primer/probe sequ ences are provided in Table 2 1, 2 2, 2 3. All the primers and probes were used at 900 and 250 nM, respectively. RNA reaction mixtures were incubated at 48C for 30 min utes followed by 95C for 15 min utes and amplification of 40 cycles at 95C for 15 s, and then 60C for 60 s. All assays were conducted as one step reverse transcriptase reactions (Applied Biosystems), with fluoresc ence measured with Applied Biosystems instruments. Relative quantitation for all assays used tata binding protein ( TBP ) mRNA as the normalizer. Zinc Transporter Antibodies Polyclonal rab bit antibodies against ZIP6, ZIP14 and ZnT8 were raised to the peptide s listed in the Table 2 4 Each was synthesized (Biosynthesi s) with an additional N terminal cysteine to facilitate conjugation to maleimide activated keyhole limpet hemocyanin (Pierce). The conjugated peptides were used to produce polyclonal antibodies in rabbits and IgG fractions were affinity purified (Pierce). Immunoblotting Liver tissue samples were flash frozen in liquid nitrogen at collection. Frozen liver tissues are homogenized in r adioimmunoprecipitation assay ( RIPA ) lysis buffer (Santa Cruz) con tain ing, protease inhibitor cocktail (Santa Cruz), phosphatase inhibitor (Santa Cruz) and p henylmethane sulfonyl f luoride ( PMSF ) (Santa Cruz) and incubated on ice
39 for 15 minutes. Following indicated treatments (above), AML12 hepatocytes were washed and har vested into iced cold PBS that contained protease inhibitors (Roche). Cells were centrifuged at 3000 rpm for 5 minutes at 4 C and cell pellets are resuspended with iced cold lysis buffer (20mM Tris HCl pH:8, 137 mM NaCl, 10% glycerol, 1% Trit on X 100 and 2mM EDTA) containing protease inhibitor, pho s phatase inhibitor and PMSF. To remove tissue and cell debris, lysates were centrifuged at 400 x g and 14,000 x g for 10 minutes at 4C respectively. Upper fractions were transferred to the new tubes and total p rotein concentrations were m easured by RC/DC assay kit (BioR ad). Depending on the protein abundance either 25 or 40 mg total protein was denaturated in a 6 x sample buffer (350 mM Tris, pH6.8, 600 mM dithiothreito l ( DTT ) 10% sodium dodecyl sulfate ( SDS ) 0.012% Bromophenol blue and 30 % Glycerol) for 5 minutes at 90 C. Denaturated proteins were separated by either 10% or 12% sodium dodecyl sulfate polyacrylamide gel e lectrophoresis ( SDS PAGE ) based on to be evaluated size. Proteins were transferred to the nitr ocellulose membrane (Whatman) overnight at 30 volt s Protein transfer was confirmed by Ponceau Red staining (0.25% Ponceau stain red, 40% Methanol and 15% Glacial acetic acid). Next, mem branes were blocked with 5% non fat dry milk in TBS T for an hour on an orbit al shaker at room temperature. Time, antibody diluents and wash buffers were different for the prima ry antibody incubati ons and are listed in the Table 2 5 Membranes were then incubated with either anti rabbit (GE Healtcare) or anti mouse IgG ( GE Healthcare) secondary antibody (1/2000 dilution of an antibody in 5% non fat dry milk in tris buffered saline with t ween ( TBS T ) ) conjugated to horseradish peroxidase for one hour at room
40 temperature. After three washes with TBS T for 5 minutes each, im munoreactivity was visualized by enhanced chemiluminesc ence (Thermo Scientific) and X ray film. Bromodeoxyuridine Assay A b romodeoxyuridine ( BrDU ) assay was conducted after AML12 hepatocytes were subjected to three d ifferent treatment conditions For the first two conditions, AML12 hepatocytes were seeded at the density of 2x 10 4 cell/ml in 96 well plate s (200l/well). After 24 hours of incubation cells were serum starved for 20 hours and pretreated with either 50 M p and 8 M ZnSO 4 for 30 minutes or macr ophage conditioned media for 2 hours. Cells were wa shed with wash buffer twice then 40 ng/ml HGF was added. For Zip14 overexpression AML12 hepatocytes were reverse transfected with pCMV SportZip14 vector by using Effectene transfection reagent (QIAGEN) (s ee Zip14 overexpression section). After 48 hours cells were serum starved for 20 hours then 40 ng/ml HGF was added. A B rDU cell proliferation assay (Calbiochem) protocol was followed thereafter. BrDU was added at the last 20 hours of incubation. Following fixation cells were incubated with anti BrDU primary antibody and peroxidase goat anti mouse secondary antibody. Color formation was obtained by addition of substrate and absorbance was measured at 450 nm (Molecular Devices) Tyrosine Protein P hosphatase 1B Assay AML12 hepatocytes were seeded at the density of 2x10 5 cell/ml and after a 24 hour incubation they were serum starved for 20 hours. Cells were either treated with 50 M pyrithione and Zn Ac etate at different concentrations for 30 minutes or macroph age conditioned medium for 2 hours. The nonspecific phosphatase inhibitor Sodium Orthovanadate (Na 3 VO 4 ) (Sigma) and p rotein tyrosine phosphatase 1B ( PTP1B ) specific inhibitors (Calbiochem, Cat# 539741), (Santa Cruz, Cat# sc 221378) were
41 incl uded as positiv e controls. Next cells were washed with the w ash buffer twice and harvested in to iced cold PBS that was supplemented with protease inhibitor cocktail (Roche). Cell pellets were obtained by centrifugation at 3000 rpm for 5 minutes. Membrane proteins were is protocol for membrane protein extraction (BioVision). Briefly, cells were homogenized with a D ounce hom ogenizer using iced cold homogenizing buffer supplemented with protease inhibitor cocktail (BioVisi on). Membrane protein pe llets were obtained through a two step centrifugation at 700 x g for 10 min utes and at 10000 x g for 30 minutes. Pellets were dissolved in 20 l of 20 mM HEPES buffer that wa s supplemented with protease inhibitor cocktail (Roche). A 10 l lysate was incubated with phosphopeptide substrate [ELEF pY MDYE NH 2 ] (AnaS pec) in the 50 l final r eaction volume for 30 minutes at 30 C. Inorganic phosphate rele ase was measured using a colorimetric phosphate assay kit (Biovision). Briefly, phosph ate solution was added and after 30 minutes absorbance was measured at 650 nm in a plate reader (Molecular Devices) Total protein concentrations were m easured by RC/DC assay kit (BioR ad) and used as a normalizer. Zip14 Overexpression AML12 hepatocytes wer e reverse transfected with pCMV SportZip14 vector by using Effectene transfection reagent (QIAGEN). The protocol wa s modified from a 293 cell reverse transfection. Cells were plated at the density of 9.4 x 10 5 cell/ml at the same time of tran s fection. DNA ( 1 g ) was used for the pCMV SportZip14 and the empty control vector transfection Total transfecti on time was 96 hours and all secondary treatments were conducted between 24 96 hours of transfection. In the serum free treat ments medium was supplemented with 4 M zinc.
42 Deoxyribonucleic A cid Pull Down Assay Hepatic n uclear extracts were prepared from both partially hepatectomized and sham operated m ice by using NE PER nuclear extraction kit (Thermo Scientific) Briefly, after washing with PBS supplemented with protease and phosphatase inhibitors, liver tissue was homogenized in CER I buffer and incub ated on ice for 10 minutes. After addition of CER II buffer a pellet was obtained by centrifugation at 16000xg for 5 minutes, and dissolved in NER buffer. Following multiple vortexing steps, lysates were centrifuged at 16000xg for 10 minutes Supernatants (nuclear extract) were collected and used in DNA binding experiments. The Zip14 promoter region (2 kb) was amplified biotinylated primers. Amplified DNA was further purified by using a PCR purification kit (Invitrogen). Purified PCR DNA was incubated with hepatic nuclear extracts and DNA bound proteins were isolated by using magnetically labeled streptavidin beads (Dyna beads from Invitrogen) DNA bound proteins were separated on SDS PAGE and bands were visualized by silver staining (Invitrogen)
43 Table 2 1 Z IP transporter primer/probe sequences Transporter Sequence Zip1 Forward TCCTCAAGGTCATTCTGCTCCTA Reverse CCCTTTCTCTTGAAGCACCTTAGA Probe FAM CTGCTCACTGGCCTTCTCTTTGTCCAA BHQ1 Zip2 Forward CTGCTTGCTCTTCTGGTTCTCA Reverse GACCTGTAGCTGCATCCATCTG Probe FAM ACTGGGCTGTGGCCTTACTCCCATCTAC BHQ1 Zip3 Forward CTGGGCTACGCCGTTCTG Re verse GGGACGTGCTCTGTGTCCTT Probe FAM CTTTCTCAAGTGGTGAGCCCTGAATCCC BHQ1 Zip4 Forward CTCTGCAGCTGGCACCAA Reverse CACCAAGTCTGAACGAGAGCTTT Probe FAM CAATCTCCGACAGTCCAAACAGACCCAT BHQ1 Zip5 Forward GGGCAGCCTCATGTTTACCA R everse CCACATCAGCCGTCAGGAA Probe FAM CCCTATTGGAGGAGCAGCTAGTGCCC BHQ1 Zip6 Forward GCCACAGCCAGCGCTACT Reverse Probe FAM CGGCGTCCTTCAGCTCCTCTCGA BHQ1 Zip7 Forward AGGCATCAAACACCACCTGG Reverse T GCGGAGATCAGCACTGTG Probe FAM CTGTCACCCTCTGGGCCTACGCACT BHQ1 Zip8 Forward CTAACGGACACATCCACTTCGA Reverse CCCTTCAGACAGGTACATGAGCTT Probe FAM ACTGTCAGCGTTGTATCCCTCCAGGATG BHQ1 Zip9 Forward AAATTCCCGTTTGCTTGGAA Reverse CAGTTTCGAAAGGCGCTTAGG Probe FAM ACCACGCGTTTAAACA BHQ1 Zip10 Forward CGGCAGTCGGTCAGTATGC Reverse AACATGCCGGCAGTGATTG Probe FAM AACAACATCACACTCTGGAT BHQ1 Zip11 Forward CACTGAGTGGAAGGCATCTTTCT Reverse TGAGGTGTTGAAG TTGAGTCTAGTGA Probe FAM TCGAGGCTAACCCCTACTTGTCCCACC BHQ1 Zip12 Forward GGTTGTAAATTTGTCCTGCATGAA Reverse TTGGGCTTGGGTTGTGTTG Probe FAM CCTCCCATTCACCC BHQ1 Zip13 Forward AGGAATGTGAACTGGAAGAATGC Reverse GGTGTCAGCCAAGG GAAATAGT Probe FAM AAGCCATAATCCCC BHQ1 Zip14 Forward GTAAACCTTGAGCTGCACATTAGC Reverse TGCAGCCGCTTCATGGT Probe FAM TGGCCTCACCATCCTGGTATCCGT BHQ1
44 Table 2 2 Z nT transporter primer/probe sequences Transporter Sequence ZnT1 Forward CACGACTTACCCATTGCTCAAG Reverse CTTTCACCAAGTGTTTGATATCGATT Probe FAM AGTCTGCTCTCATTCTTCTACAAACTGTCCCTAAGC BHQ1 ZnT2 Forward CCGACCAGCCACCAAGAC Reverse TGGAAAGCACGGACAACAAG Probe FAM CGGCTCGATGCCAGCCGAA BHQ1 Zn T3 Forward GGTGGTTGGTGGGTATTTAGCA Reverse CAAGTGGGCGGCATCAGT Probe FAM ACAGCTTGGCCATCAT BHQ1 ZnT4 Forward GCTGAAGCAGAGGAAGGTGAA Reverse TCTCCGATCATGAAAAGCAAGTAG Probe FAM CAGGCTGACCATCGCTGCCGT BHQ1 ZnT5 Forward CTGCTCGGCTTTGGTCATG Reverse CGGCCATACCCATAGGAGGA Probe TTTGCTGCCCTGATGAGCCGC BHQ1 ZnT6 Forward TCCCAGGACTCAGCAGTATCTTC Reverse GCCCCAGCAAGATCAATCAG Probe FAM TGCCCCGCATGAATCCGTTTG BHQ1 ZnT7 Forward CCTCTCTTTCGCT TTTGTGGAA Reverse GTGGAAGGAGTCGGAGATCAAG Probe FAM ACTCTACGGCATCTGGAGCAACTGCCT BHQ1 ZnT8 Forward TGGGTGGTATCGAGCAGAGAT Reverse ACACCAGTCACCACCCAGATG Probe FAM TCGGTGCCCTGCTGTCTGTCCTT BHQ1 ZnT9 Forward GCACTGGGCATCA GCAAAT Reverse GAAAAGCCGTACGGGTGAGA Probe FAM TGTTCAAACACCAGATCC BHQ1 ZnT10 Forward GCACTGGGCATCAGCAAAT Reverse GAAAAGCCGTACGGGTGAGA Probe FAM CTCTGAACTGGAGTGAGC BHQ1
45 Table 2 3 Other primer/probe sequences Transpo rter Sequence MT Forward GCTGTGCCTGATGTGACGAA Reverse AGGAAGACGCTGGGTTGGT Probe AGCGCTGCCACCACGTGTAAATAGTATCG BHQ1 CD1 Forward AGCCAGCTGCAGTGCTGTAG Reverse CTGGTGGTGCCCGTTTTG Probe FAM CCCAAGTTCCCTAGCAAGCTGCCA B HQ1 PCNAForward GCGCAGAGGGTTGGTAGTTG Reverse CCCGATTCACGATGCAGAA Probe FAM CGCTGTAGGCCTTCGCTGCCG BHQ1 TBP Forward TCTGCGGTCGCGTCATT Reverse GGGTTATCTTCACACACCATGAAA Probe FAM TCTCCGCAGTGCCCAGCATCA B HQ1 Table 2 4 ZIP and ZnT transporter a ntibody sequences Antibody Sequence ZIP6 MIAHAHPQEVYNEY ZIP14 NSELDGKAPGTD ZnT8 CQKPVNKDQCP Table 2 5 List of Antibodies Antibody Company 1Ab Diluent Wash B. Time/Temp. CD1 Santa Cruz 5% milk in TBS T TBS T 2hr/RT PCNA Santa Cruz 5% milk in TBS T TBS T 2hr/RT ZIP6 In house 5% milk in TBS T TBS T O/N/4C ZIP14 In house 5% milk in TBS T TBS T O/N/4C ZnT7 In house 5% milk in TBS T TBS T O/N/4C ZnT8 In house 5% milk in TBS T TBS T O/N/4C c Met Upstate 5% milk in TBS Water O/N/4C pc Met Cell S ignaling 5% BSA in TBS T TBS T O/N/4C ERK1/2 Cell Signaling 5% BSA in TBS T TBS T O/N/4C pERK1/2 Cell Signaling 5% BSA in TBS T TBS T O/N/4C e lf4E Cell Signaling 5% BSA in T BS T TBS T O/N/4C Actin Sigma 5% milk in TBS T TBS T 1hr/RT Tubulin Abcam 5% milk in TBS T TBS T 1hr/RT
46 CHAPTER 3 ZINC ENHANCES LIVER REGENERATION Introduct ory Remarks The liver is the largest internal organ in the human body, and it has many important functions in metabolic homeostasis. Among these one of the most important functions is metabolism and clearance of xenobiotics. The liver receives 80% of its blood directly from the small and most of the large intestine, spleen and pancreas thro ugh the portal vein while 20% of oxygenated blood is received through the hepatic artery. Because most of the liver blood supply is received via the portal vein, the liver is in the first line to be injured by ingested toxins. These toxins may cause acute or chronic liver injury depending on their persistence. In acute liver injury after elimination of the injurious agent the liver recovers lost mass without jeopardizing viability of the entire organism because of its unique ability to regenerate with hig h capacity ( 102 ) If acu te liver injury is continuous over an extended period of time it causes chronic liver injury. Progressive fibrosis is the hallmark of chronic liver injury; it can eventually result in cirrhosis, liver failure, or hepatic carcinoma. Fibrosis is pr edominantly viewed as a repercussion of the wound healing response that occurs after persistent liver injury. Recent studies suggest that early stage liver fibrosis is a reversible process ( 103 ) Thus new therapeutic approaches are mostly concentrated on drugs that can reverse fibrosis. Along with this new approach it is also recognized that enhancing regeneration capacity of the healthy part of the liver is an important component. By contrast cirrhosis or hepatic carcinoma is not a reversible process and is very difficult to treat. Until recent application of split liver transplantation and live related donor transplantation, whole liver transplantation was the only effective therapy
47 available for these conditions. Split liver transplantation and live related donor transplantation have a common approach that takes advantage of regenerative capacity of the liver. After removal of the certain part of the recipient liver, a graft will be transplan ted. While a healthy graft would support the maintaining of liver functions, the liver reaches the original mass by regeneration ( 104 106 ) Effective regeneration is also important for the donor in live related donor transplantation. Given the fact that recipient livers are generally severely damaged their capacity for regeneration is lessened. Thus therapeutic approaches for the enhancement of regene ration are required. To develop such therapies a better understanding of the regeneration cascade is required. Zinc may be a factor upon which to develop new therapies. Zinc deficiency is one of the parameters that has been observed in people who suffer fr om acute and chronic liver disease and hepatocellular carcinoma ( 107 ) Zinc has been used in few clinical trials during the treatment of patients with chronic liver in jury and hepatocellular carcinoma ( 108 113 ) Result s from these studies showed that zinc enhances patient responsiveness to treatment and also decreases mortality. However, since these data are mostly obtained by testing liver enzymes it is not possible to delineate the exact function of zinc in the therap eutic process. Consequently, further investigation of zinc metabolism and function during these pathophysiologic states are needed. Zinc participates in a wide variety of cellular processes as a cofactor for many enzymes and influences gene expression thr ough transcription factors. Zinc may also affect mitogenic hormonal signal pathways that specifically direct cell proliferation. Zinc is present in the cell nucleus, nucleolus and chromosomes, and zinc stabilizes the structure of DNA, RNA and ribosomes ( 114 ) Numerous enzymes associated with RNA
48 synthesis are also zinc metalloenzymes, including RNA polymerase. Proliferation of hepatocytes is the main event in liver regeneration. Since zinc is highly involved in proliferation at different steps it might be an important component of liver regeneration. Proliferative effect of zinc was reported in adult stem cells in the brain ( 115 ). Adult stem cells in the human brain have the capacity to undergo neurogenesis ( 116 ). When rats were fed with ZnD diet or NT2 cell line (human teratocarcinoma) was treated with Tetrakis (2 pyridylmethyl) Ethylenediamine ( TPEN ) a significant decrease in proliferation was detected by either Ki7 staining or BrDU incorporation, respectively. Zinc was shown to be stimula ting cell proliferation, differentiation and protein synthesis in pre osteoblas tic cells ( 117, 118 ). Differentiation of pre osteoblastic cells to osteoblastic cells consists of many steps. The first step is proliferation of pre osteoblastic cells until the y reach the cell number that expression of the osteogenic phenotype starts. In pre osteoblastic cell line, MC3T3 E1 proliferation was increased by time in a zinc concentration dependent manner as it was shown by a m ethylthiazol t etrazolium ( MTT ) prolifera tion assay ( 119 ). A m ore relevant study to zinc and LR was reported by Kang et. al. ( 120 ). In this study, LR in mice was induced by long term ethanol administration. During ethanol administration mice were further divided into two groups as control and zinc supplemented. Liver regeneration was assessed by immunohistochemical staining of p roliferating c ell n uclear a ntigen ( PCNA ) and BrDU incorparation. Results revealed much higher staining for both proliferation markers PCNA and BrDU, in the liver of the zin c supplemented group when compare to liver of the control group. They also showed
49 lower serum ALT detection in zinc supplemented group of mice. Lower ALT detection in the zinc supplemented group agrees with the clinical trials in which zinc supplementatio n was used for the treatment of liver diseases. Z inc may enhance proliferation by influencing cytokine/growth factor signaling pathways. For instance enhanced proliferation in the T cells in response to interleukin 2 ( IL2 ) stimulation was shown ( 121 ). Aft er stimulation with IL2, cytosolic zinc was increased as a result of mobilizatio n of labile zinc from lysosomes to cytoplasm of T cells of the CTLL 2 line measured by zinc selective fluorescent probe FluoZin 3 An increase in cytosolic zinc caused an enh anced phosphorylation of extracellular signal related kinases ( ERK1/2 ) Enhanced ERK1/2 phosphorylation was not present when TPEN treatment wa s applied along with IL2. Since ERK1/2 was involved in proliferation and cell survival in response to IL2 ( 122 ), t hey measured proliferation. Result s showed a dose dependent inhibitory effect of zinc chelater, TPEN on T cells. Besides showing a necessity for the presence of zinc for proliferation related cytokine pathway this study sugge sts zinc transporter involve s to the mobilization of zinc from lysosomes to cytoplasm where zinc is needed In fact Aydemir et. al. previously showed the mobilization of zinc from lysosomes to cytoplasm was facilitated by the zinc transporter Zip8 in primary human T cells, when T c ells were activated through T cell receptor ( 76 ) Zinc homeostasis in cells is maintained through a tight regulation of zinc influx, efflux, and distribution to intracellular organelles ( 73, 74 ) Zinc transporter proteins are essential for th ese metabolic and functional adjustments. It has been shown in many cell systems that these transporters function in a tissue specific manner. Their localization also may differ in different tissue/cell systems and in response to specific
50 stimuli Moreover for specific metabolic or cellular events different zinc transporter(s) might be needed to maintain zinc homeostasis. This need may be enhanced during pathophysiologic events. Thus determination of the chang es in zinc transporters during the liver regen eration process may provide a better understanding on the action of zinc within the context of cellular proliferation and organ growth. Therefore, I aimed in this chapter to examine if zinc enhances liver regeneration and if any transporter is differential ly expressed during the regeneration process using an in vivo PHx model and an in vitro AML12 hepatocyte cell line. Results Hepatic Zinc I ncrease s in R esponse to Partial Hepatectomy A 70% PHx causes remnant liver cells to proliferate until the original ti ssue mass is restored ( 10 123 ) Liv er hepatocytes are the first cell s that enter the G1 phase of the cell cycle ( 10 ). Hepatocyte proliferation peaks between 24 t o 48 hours depending on the mouse strain ( 124 ) There fore, I concentrated on first the 48 hour s post PHx. The e xpression of CD1 as a G1 phas e and PCNA as an S phase marker of two stages of cell cycle were measured Both CD1 and P CNA mRNA and protein expression were increased in a sequential manner after PHx (Figure 3 1B ). These result s confirmed th at the PHx s urgeries conducted in our lab are working properly to study liver regeneration. Liver is among those tissues that have a fast exchangeable zinc pool. A n increase in liver zinc concentration s in response to PHx have been report ed ( 125 ). These me asurements were analyzed at the earliest time point, a t 24 hours post PHx. However, I hypothesized that change s in liver zinc concentration might start earlier since zinc might be involve d in hepatocyte proliferation. Therefore, at the same time points o f CD1 and PCNA measurements, I also meas ured the total zinc concentration in
51 serum and liver tissue by AAS. There were significant decreases in serum and an increase in liver zinc concentrations peaking at 10 hours post PHx (Figure 3 2). For further confi rmation zinc regulated gene metallothionein ( MT ) mRNA in liver tissue samples was measured. My result showed that MT mRNA was significantly up regulated at 10 hours post PHx (Figure 3 2) These results suggest that zinc might be a requir ed factor for the LR process. Zinc T ransporters are D ifferentially E xpress ed in R esponse to P artial Hepatectomy Changes in liver zinc content in response to PHx suggest that zinc transporters might have a role in the LR process. Since major changes in serum and liver tissue zinc concentrations were observed a t 10h post PHx differential expression of ZIP and ZnT zinc transporters were measured at the same time point (Figure 3 3 ). Zip1, Zip3, Zip6, Zip7, Zip10, Zip14 ZnT7 and ZnT8 were up regulated while Zip8 was the only t ransporter that was down regulated Among all the ZIP transporters, Zip6 and Zip14 had the most significant increase (p<0.001) in comparison to the sham control. Therefore, Zip6, Zip14 ZnT7 and ZnT8 mRNA and protein expressions in the 0 48 hour time perio d were measured by quantitative real time PCR ( qPCR ) and western blot, respectively (Fig ure 3 4, 3 5). All of up regulated transporters had the highest expression at 10 hour post PHx. We have previously reported that Zip14 is an IL6 regulated gene ( 82 ) Si nce LR process also start s with a cytokine (IL6 and TNF ) dependent pathway I hypothesized that Zip14 may play a major role in facilitating zinc mobilization to the hepatocytes. Zip14 up regulation started as early as 2 hours and at 24 hour s expression ha d returned to the same level as sham control (Figure 3 4 ). This
52 result supported the hypothesis that zinc is required for proliferation at 24 hour post PHx and that is provided mainly by the zinc transporter Zip14. Zinc S upplementation E nhances L iver R ege neration Zinc supplementation has been used in a few clinical trials ( 108 113 ) during the treatment of liver diseases. In these studies, ALT measurements were used for the assessment of liver health status and r esults showed that the zinc supplemented grou p had lower serum ALT levels. A dietary zinc study was conducted t o test the effect of zinc supp lementation in mouse model (Figure 3 6A) At the end of feeding period PHx or sham operations were conducted. At 24 hours post PHx serum and liver tissue wer e collected. First, the changes in serum and hepatic zinc were measured by AAS (Figure 3 6). Serum and hepatic zinc concentration were lower in ZnD fed mice and were higher in ZnH fed mice, in comparison to ZnA fed mice. Next, serum ALT levels were measure d (Figure 3 7). The ZnH fed mice had significantly (p<0.001) lower serum ALT levels than ZnA fed mice suggesting that ZnH fed mice had improved liver function This finding did not specify the function of zinc in the process of proliferation, t hus, a prol iferation marker PCNA, was measure d in liver tissues (Figure 3 8 ). The livers of ZnH fed mice had significantly (p<0.001) higher PCNA mRNA and protein expression in comparison to the livers of ZnA fed mice To further investigate the mechanism in which z inc may play a role in LR, in vitro experiments were conducted with AML12 murine hepatocytes. First, AML12 hepatocytes were treated with 4 and 8 M zinc along with pyrithione After two washes with metal wash buffer (see Material and Methods) hepatocytes w ere treated with 40 ng/ml HGF. Hepatocytes were harvested at 24 and 48 hours, and PCNA mRNA was measured by qPCR (Figure 3 9). In both the 24 and 48 hour time points, a dose
53 responsive effect of zinc pretreatment was seen in PCNA expression. The effect of zinc on AML12 hepatocyte proliferation was evident; however, a change in intracellular zinc by the pyrithione and zinc treatment needed to be confirmed through subsequent experiments. Therefore, following zinc and pyrithione treatment, total intracellular zinc concentrations were measured by AAS (Figure 3 10A). There was a 3 fold increase (p<0.001) in intracellular zinc concentration assuring that intracellular zinc concentration was increased. Next, the effect of zinc on hepatocyte proliferation was tested by the measurement of PCNA at th e mRNA and protein level (Figure 3 10B). Zinc (8 M ) pretreated hepatocytes had significantly (p<0.001) higher PCNA expression than only HGF treated hepatocytes. Further confirmation of the zinc effect on proliferation was obtained from a BrDU incorporation assay (Figure 3 11A, B). BrDU is a thymidine nucleotide analog and its incorporation into newly synthesized DNA is direct e vidence of DNA replication First, the dose response effect of HGF on AML12 hepatocyte s was tested and shown in Fig ure 3 11A. Next, BrDU incorporation was measured in 8 M zinc, pyrithione pretreated and HGF stim ulated AML12 hepatocytes (Figure 3 11B). The results revealed almost identical patterns of PCN A expression presented in Figure 3 10B, strongly suggesting that hepatocyte proliferation was enhanced by zinc. For a better simulation of the i n vivo initiation step of LR, I constructed a model where conditioned medium obtained from RAW cells were used to treat AML12 hepatocytes (Figure 3 12) LPS tr eatment causes RAW cells to produce cytokines such as IL6 and TNF similar to K upffer cells and innate immune system cells in response to PHx. Production of IL6 by LPS treated RAW cells was co n firmed by qPCR and ELISA (Figure 3 13A, B). Since Zip14 is IL6 regulated gene ( 82 ), Zip14 mRNA was measured
54 in LPS CM treated AML12 hepato cytes (Figure 3 13C). Zip14 mRNA was 3 fold higher in LPS CM treated AML12 hepatocyte s. Even though this result suggested that RAW cell produced IL6 might induce Zip14 expression, further confirmation was necessary for the IL6 regulation of Zip14. Two approaches were used; IL6 supplementation a nd depletion from LPS CM (Figure 3 14A, B). Recombinant IL6 (20 ng/ml) was added to the LPS CM which enhanced Zip14 expression. For the deple tion of IL6 in the LPS CM, neutralizing IL6 antibody was used. When IL6 was depleted, the LPS CM induced Zip14 increase was diminished to the control level, confirming that IL6 regulated Zip14 up regulation was similar to LR initiation in vivo. This in vi tro model was further tested for expression levels of the MT and Zip1, Zip3, Zip6, Zip7, Zip10, Zip11 ( up regulated ZIP transporters during LR) (Figure 3 15). They were all significantly higher than the control in response to combination of LPS CM and IL6 treatment. To investigate physiological consequences of Zip14 up regulation total zinc and MT expression in AML12 hepatocytes were measured by AAS and qPCR, respectively. MT expression and t otal intr acellular zinc concentration were higher in both LPS CM and a combination of LPS CM and IL6 treated AML12 hepatocytes than CM treated control cells (Figure 3 16B, C) Next, the question was ask ed whether LPS CM pretreatment would affec t the proliferation of AML12 hepatocyte s. To test this possibility after ma crophage CM pretreatment, AML12 hepatocyte s were treated with HGF and PCNA expression was measured. Both mRNA and protein levels of PCNA were significantly higher in cells treated with the LPS CM alone and combined LPS CM and IL6 treated cells (Figure 3
55 1 7 ). These result s collectively suggest that zinc improved liver function through enhanced proliferation of hepatocytes. Discussion The main findings of this chapter were (i) hepatic zinc concentration increased while serum zinc concentration decreased duri ng LR; (ii) at 10 hours post PHx, among the 14 ZIP transporters Zip1, Zip3, Zip6, Zip7 and Zip14 were up regulated while Zip8 was down regulated ; (iii) at 10 hours post PHx, among the 10 ZnT transporters only ZnT7 and ZnT8 were up regulated ; (iv) the liver was functioning better when mice were fed a ZnH diet; (v) LR was enhanced in the ZnH fed mice; (vi) enhancement in AML12 hepatocyte proliferation in response to HGF treatment was observed when cells were pretreated with either zinc or macrophage conditione d medium. Following PHx all the quiescent hepatocytes enter the G1 phase of the cell cycle ( 10 ) For the compensation of tissue lost due to 70% PHx approximately 1.66 cell divisions are necessary. In the PHx mod el all these events happen in a very synchro nized manner, however ; the proliferation rate changes by species. For instance, the peak time for DNA synthesis is at 24 hours post PHx in the rat while it can change between 36 48 hour postPHx for the mouse (124 ) To detect the most appropriate time for the measurement of hepatocyte proliferation during LR in the mouse, e xpression of CD1 as a G1 phas e marker and PCNA as an S phase marker of two stages of the cell cycle were measured DNA synthesis seemed to start at 24 hours post PHx. Thus, 24 hours post PHx was chosen as the best time to measure changes in proliferation in the following experiments. This decision enabled me to detect even small differences of proliferation between treatment groups before the system reached the maximal rate of proliferati on.
56 The p riming step of LR resembles an acute phase res ponse during inflammation (10) Hypozincemia is one of the hallmarks of inflammation and redistribution of hepatic zinc during inflammation was reported. Taken together, these observations suggest th at hepa tic zinc may change during LR. A h epatic zinc increase was previously shown during LR at 24 hours postPHx ( 125 ) As it was emphasized above, proliferation of hepatocytes started at 24 hours after PHx suggesting that any change that could potential ly a ffect proliferation should happen earlier than 24 hours post PHx. This idea led to the novel finding that hepatic zinc started to increase as early as 10 hours post PHx and stayed elevated for up to 48 hours. Later time points were not the focus of thi s study. This novel finding is important in terms of the requirement of zinc in early steps of hep atocyte proliferation during LR in the mouse. Expression of zinc transporters was measured in response to PHx Finding an increase in ZIP transporters was exp ected because of the increased hepatic zinc accumulation observed. H igh up regulation in the relative expression of ZnT8 was a most interesting finding E xpression of ZnT8 is highly specific to the insulin producing cells of the pancreatic islets ( 126 ). Intracellular localization of ZnT8 was shown to be in granules that were in close proximity to the plasma membrane. Zinc is required for insulin crystallization and this required zinc transport to the granules is facilitated by ZnT8 ( 126 ) I nsulin is norm ally produced in pancreas and t h ymus However ; in a relatively new study, insulin mRNA and proinsulin positive cells were detected in liver, adipose tissue and bone marrow by RT PCR and immunohistochemistry, re s pectively ( 127 ) An i nsulin injection caused a reversal of liver atrophies and was associated with rapid hepatocyte proliferation ( 10 ) suggesting a role for insulin in the LR process.
57 Taken together, besides obtaining endocrine insulin liver may produce insulin during LR and required zinc for insu lin crystallization could be transported by ZnT8. The d ietary zinc study revealed that ZnH fed mice had higher PCNA mRNA and protein when compared to ZnA fed mice. Therefore, zinc supplementation became the main focus for the following experiments. All tho ugh not a specific objective of this dissertation project there was a decrease in PCNA expression only in protein level in ZnD fed mice when compared to ZnA fed mice This result could be explained by possible post transcriptional /translational regulatio n of PCNA expression. Even though, PCNA expression is increased in mRNA and protein level in cycling cells ( 128 ), regulation via post t ranslational modi fications such as sumoylation and ubiqutinylation has also been reported ( 129 ) Ubiqutinylation of PCNA is necessary for its degradation Zinc involved control for ubiqutiny lation dependent degradation of the proteins was reported ( 130 ) Collectively, post translational regulation for PCNA in zinc deficient conditions might have caused a decrease in PCNA pr otein level in ZnD fed mice. In an effort to generate an in vitro model for the simulation of the initiation step of LR, macrophage conditioned media was used to pre treat AML12 hepatocytes. Macrophage conditioned media was obtained by an LPS treatment of m urine macrophage cell line, RAW264.7. IL6 was the most important component of macrophage conditioned media because of both its role in priming step of LR and regulatory role on Zip14 expression. The reason for the preference of conditional media over recom binant IL6 treatment was the controversy over the consistency of IL6 effect on hepatocytes proliferation in vitro. Both inhibitory ( 131, 132 ) and enhancing ( 133, 134 and 135 ) effects of IL6 on hepatocyte proliferation were reported. T he reason for thes e
58 co ntroversies was shown by Sun et.al. ( 136 ) In their experiments, IL6 was added either to the hepatocytes alone or to hepatocytes that were c o cultured with non parenchymal cells. While the addition of IL6 inhibited proliferation in hepatocytes alone it en hanced proliferation in co cultured hepatocytes. These results suggested that the presence of non parenchymal cell s produced components were required for IL6 to have a proliferative effect on hepatocytes. They also showed that the IL6 effect was related to the HGF pathway. When co cultured hepatocytes were pretreated with c Met antibody then IL6 they detected a decrease in hepatocyte proliferation. Therefore, macrophage condition media was used along with HGF in the in vitro model used in my experiments.
59 Figure 3 1. Proliferation marker change in response to partial hepatectomy (A) Study design for the time course experiment with C57BL6 mice. (B) Liver tissue was collected at the indicated times from partially hepatectomized and sham operated mi ce. RNA and protein were extracted and used for qPCR and western blot analysis for the detection of CD1 and PCNA expression. Values shown are means SD (n=5 for partial hepatectomized group and n=3 for sham operated group).
60 Figure 3 2. Changes i n hepatic zinc in response to partial hepatectomy (A) Liver tissue was collected at the indicated times from partially hepatectomized and sham operated mice. Total zinc concentrations were measured by AAS. (B) Serum was collected at the indicated times from partially hepatectomized and sham operated mice, and total serum zinc concentrations were measured by AAS. (C) Liver RNA was extracted and used for qPCR to detect expression of MT. Values shown are means SD (n=5 for partial hepatectomized group and n=3 for sham operated group).
61 Figure 3 3. Differential expression of zinc transporters in response to partial hepatectomy Liver RNA was extracted and used for the measurement of ZIP and ZnT transporter mRNA at 10 hours post PHx. Values shown are mean s SD ( *= P < 0.05 ; **=0.01; ***= P< 0.001). (n=5 for partial hepatectomized group and n=3 for sham operated group).
62 Figure 3 4. Changes in Zip6 and Zip 14 expression in response to partial hepatectomy Liver tissue was collected at the indicated times f rom partially hepatectomized and sham operated mice. Total RNA and tissue lysates were prepared for qPCR and western blots, respectively. (A) A quantitative PCR. (B) Western blots. Values shown are means SD (n=5 for the partial hepatectomized group and n =3 for the sham operated group).
63 Figure 3 5. Changes in ZnT7 and ZnT8 express ion in response to partial hepatectomy Liver tissue was collected at the indicated times from partially hepatectomized and sham operated mice. Total RNA and tissue lys ates were prepared for qPCR and western blots, respectively. (A) Quantitative PCR. (B) Western blots. Values shown are means SD (n=5 for the partial hepatectomized group and n=3 for the sham operated group).
64 Figure 3 6. Changes in hepatic zin c in respo nse to both dietary zinc and partial hepatectomy (A) Experimental design of the dietary zinc study with C57BL/6 mice. (B) Tissue and serum were collected at 24 hours post PHx. After nitric acid digestion, the tissue zinc concentration was measu red by AAS. Serum zinc concentrations were measured by AAS as well. Total liver RNA was extracted and used for the qPCR and normalized to TBP mRNA. Values shown are means SD (n=5 for the partial hepatectomized group and n=3 for the sham operated group).
65 Figure 3 7. Effect of dietary zinc on liver function after partial hepatectomy (A) Blood was collected at the indicated times from partially hepatectomized and sham operated mice. (B) After 4 days of acclimation, mice were fed with either a ZnA, ZnD or ZnH diet for a week. Next, mice underwent either partial hepatectomy or a sham operation. At 24 hours post PHx blood was collected. Serum was separated by two step centrifugation and used for ALT measurements. Values show n are means SD (n=5 for the partial hepatectomized group and n=3 for the sham operated group).
66 Figure 3 8. Effect of dietary zinc on liver regeneration After 4 days of acclimation, mice were fed with either a ZnA, ZnD o r ZnH diet for one week. Next, mice underwent either partial hepatectomy or a sham operation. At 24 hours post PHx liver tissue was collected. Total RNA and tissue lysates were prepared to use for qPCR and western blots, respectively. Values shown are mean s SD (n=5 for the partial hepatectomized group and n=3 for the sham operated group).
67 Figure 3 9. Dose and time dependent effect of zinc treatment on AML12 proliferation in response to hepatocyte growth factor stimulation. Following the 20 hours o f serum starvation, AML12 hepatocytes were pretreated with a combination of pyrithione and different concentrations of zinc for 30 minutes. Next, cells were washed and incubated with 40 ng/ml HGF. At 24 and 48 hours cells were harvested and total RNA was i solated to measure PCNA mRNA by qPCR with normalization to TBP mRNA. Values shown are means SD (n=3)
68 Figure 3 10. Effect of in vitro zinc treatment on AML12 proliferation in response to hepatocyte growth factor stimulation. (A) Following 20 hours of serum starvation, AML12 hepatocytes were pretreated with a combination of pyrithione and 8 M zinc for 30 minutes. Next, cells were harvested and digested in nitric acid for 2 hours. Total zinc concentrations were measured by AAS. (B) After zinc pretreatment cells were incubated with HGF. At 48 hours cells were harvested, and total RNA and tissue lysates were isolated to measure PCNA mRNA and protein. Values shown are means SD (n=3 )
69 Figure 3 11. Effect o f in vitro zinc treatment on deoxyribo nucleic acid replication in response to hepatocyte growth factor stimulation. Following the 20 hours of serum starvation, AML12 hepatocytes were stimulated by HGF either immediately (A) or after pretreatment of pyrithione and 8 M zinc (B). At the last 20 hours of the 48 hours of HGF stimulation, BrDU was added. BrDU incorporation to newly synthesized DNA was measured by reading the absorbance at 450nm. Values shown are means SD (n=3 )
70 Figure 3 12. In vitro simulation of init iation of liver regener ation in vivo. RAW cells were treated by lipopolysaccharide to stimulate cytokine production. After 2 hours, supernatant was collected from control (CM) and LPS treated (LPS CM) cells and used for the treatment of AML12 hepatocytes.
71 Figure 3 13. Effect of lipopolysaccaride on interleukin 6 production by RAW macrophages and the influence of conditioned medium on Zip14 expression (A) Control or LPS treated RAW cells were harvested and RNA was isolated for the measurement of IL6 mRNA by qPCR with n ormalization to TBP mRNA (B) At the time of harvest, supernatan ts (macrophage conditioned medium; LPS CM ) were collected and used to measure IL6 by ELISA assay (C) AML12 hepatocytes were incubated with macrophage conditioned media for the indicated times. Next, AML12 hepatocytes were harvested, and total RNA was isolated to measure Zip14 mRNA by qPCR. Values shown are means SD (n=3)
72 Figure 3 14. Effect of macrophage conditioned medium on Zip14 expression. Macrophage conditioned media was obtained fro m either LPS treated or control RAW cells. (A) AML12 hepatocytes were incubated with macrophage conditioned medium for 2 hours. Cells were harvested and total RNA was isolated to measure Zip14 mRNA by qPCR, normalized to TBP mRNA. (B) LPS CM medium was pre incubated with either Rat IgG or IL6 specific antibody for an hour and used for the treatment of AML12 hepatocytes. After 2 hours of incubation, cells were harvested and total RNA was isolated to measure Zip14 mRNA by qPCR. Values shown are means SD (n=3 )
73 Figure 3 15. Validation of zinc transporter expression pat tern in the in vitro model of liver regeneration initiation. AML12 hepatocytes were treated with either CM/LPS CM alone or in combination with the recombinant IL6 for 2 hours. Next, cells w ere harvested and total RNA was isolated to measure MT and Values shown are means SD (n=3)
74 Figure 3 16. Effect of Zip14 expression on the intracellular zinc concentration of AML12 hepatocytes. Macrophage cond itioned media was obtained from either LPS treated or control RAW cells. (A) AML12 hepatocytes were incubated with macrophage conditioned media for 2 hours. Cells were harvested, and total RNA and cell lysates were prepared to measure Zip14 mRNA and protei n levels. (B) The same RNA samples were used to measure MT mRNA by qPCR. (C) After nitric acid digestion, total zinc concentrations of AML12 hepatocytes were measured by AAS. Values shown are means SD (n=3 9) Values with a different superscript are stat istically different (P < 0.05 0.001).
75 Figure 3 17. Effect of macrophage conditioned media on AML12 hepatocyte proliferation. Macrophage conditioned media was obtained from either LPS treated or control RAW cells. (A) AML12 hepatocytes were incub ated with macrophage conditioned media for 2 hours, then washed and stimulated by 40 ng/ml HGF for 48 hours. Cells were harvested, and total RNA and cell lysate were prepared to measure PCNA mRNA and protein levels. Values shown are means SD (n=3) Value s with a different superscript are statistically different (P < 0.05 0.001).
76 CHAPTER 4 ZINC ENHANCES C MET PHOSPHORYLATION BY INHIBITION OF P ROTEIN TYROSINE PHOSPHATASE 1B Introduct ory Remarks Liver regeneration is a process of compensatory hyperplasia in response to hepatic tissue loss, either from liver injury or from resection, until the original liver mass is restored. LR after PHx is carried out by proliferation of all the existing mature cellular populations, composing the intact organ ( 137 ). These ce ll populations include hepatocytes, biliary epithelial cells, endothelial cells, Kupffer cells and stellate cells. Among these, hepatocytes are the first to proliferate and are a major target for parenchymal regeneration. In normal adult liver, hepatocytes are usually quiescent and only 0.0012 to 0.01% of them undergo mitosis in any given time ( 138 ). However, after 70% PHx, 95% of the hepatocytes in the remnant liver participate in one or two proliferative events to restore the original number of hepatocyte s ( 137 ). To answer the question of what triggers hepatocytes to proliferate after PHx, extensive research h as been conducted in rodent models. In early studies, when isolated hepatocytes were transplanted into extrahepatic sites, these cells enter into DN A synthesis after PHx of the liver in the host ( 139 ). When rats were join ed in pairs through parabiotic circulation, hepatectomy in one member of the pair caused regeneration of the intact liver in the other member ( 140 ). These studies collectively provide d convincing evidence that a mitogenic signal for hepatocytes appeared in the blood during LR. An effort to identify blood borne hepatic mitogens arising during LR l ed to the discovery of the hepatocyte growth factor in 1984 ( 141 ).
77 HGF is essential for LR HGF or HGF receptor, c Met, KOs are associated with embryonic lethality due in part to arrested hepatic development ( 142 ). Liver specific deletion of c Met caused severe impairment in LR after PHx. (143). HGF is synthesized as a pro HGF consisting of 728 amino acids, and mature HGF is formed by proteolytic cleavage ( 144 ). In mature HGF, cysteine ( Cys ) chain form an interchain bridge. Both the paracrine and endocrine effects of HGF are involved in LR after 70% PHx. The hepatic biomatrix contains a large amount of scattered inactive, pro HGF. Shortly after PHx, the hepatic biomatrix is subject to proteolysis, thus pro HGF is released. Released pro HGF is activated through proteolytic cleavage by uPA. Therefore, the amount of active HGF increases in the liver. Expression of HGF mRNA also increases in hepatic stella te cells, 3 to 6 hours after PHx and lasts for 24 hours. Besides an increased level of HGF in the liver, the plasma concentration of HGF rises, both in humans ( 145 ) and rodents ( 137 ). Previous studies showed that the sources of plasma HGF after PHx were d istant organs such as lungs, kidneys and spleen ( 146 147 ). This endocrine effect of HGF supported the initial idea that hepatocyte proliferation during LR is triggered by blood born mitogen, HGF. However, injection of HGF in normal rats through the portal vein caused only a relatively small number of hepatocytes to show DNA synthesis. This result suggested that hepatocytes in normal liver were not ready to respond to mitogenic signals without priming events that switched them into a responsive state ( 148 ). Priming events include cytokine dependent pathways that are largely responsible for the entry of quiescent hepatocytes into the cell cycle (transition from G 0 to G 1 ) ( 149 ).
78 e xperiments with knockout mice suggested that TNFR1 and IL6 may be essential for full hepato cyte DNA synthesis during LR was suppressed ( 9 ). Additionally, STAT3, AP1, Myc, and cyclin D1 activation were markedly reduced. Changes, both in DNA synthesis and in cell cycle gene expression, were corrected by an injection of IL6 to the IL6 KO mice. Bes ides their importance on priming of hepatocytes for G 0 to G 1 conversion, regulation of HGF expression for the G 1 to S progression of hepatocytes ( 150 ). HGF acts via binding to the hepatocyte growth factor receptor, c Met, on hepatocytes during LR ( 151 ). The tyrosine kinase receptor c Met is a heterodimer composed of three port ions. The first portion is a jux tamembrane sequence that downregulates kinase activity following phosphorylation of Ser975. The second portion is a catalytic region that positively regulates kinase activity. The third portion is a mul tifunctional docking site at the carboxy terminal domain. HGF binding causes c Met receptor dimerization and trans phosphorylation of two catalytic residues at Tyr1234 and Tyr1235 in the kinase activation domain ( 152 ). The carboxy terminal tail has phospho rylation site at residues Tyr1349 and Tyr1356 ( 152, 153 155 ). These residues Tyr1349 and Tyr1356 when phosphorylated serve as a docking site for the recruitment of many adaptor proteins to
79 activate downstream signaling pathways. The c Met receptor is also a substrate for the protein tyrosine phosphatases (PTP) ( 156 158 ). Such phosphatases oppose c Met signals by triggering dephosphorylation of tyrosine residues. PTPs are a family of e nzymes all characterized by their sensitivity to vanadate, ability to hyd rolyze p nitrophenylbphosphate, insensitivity to okadaic acid, and lack of metal io n requirement for ca talysis (159 ). PTPs can exert both positive and negative effects on a signaling pathway and play crucial physiological roles in a variety of mammalian ti ssues and cells ( 160 ). A relatively recent evaluation of the human genome suggested that humans have 112 PTPs ( 161 ), which include both the tyrosine specific and dual specific phosphatases. The tyrosine specific PTPs can be further categorized into recepto r like and intracellular. The intracellular PTPs, exemplified by PTP1B, contain a single catalytic domain and various amino or carboxyl terminal extensions. PTP1B contains an endoplasmic reticulum (ER) targeting signal sequence at its carboxyl terminus. Th is targeting signal anchors PTP1B to the cytoplasmic face of the ER ( 162, 163 ). PTP1B biochemically interacts with multiple receptor tyrosine k k inase s ( RTK ) including the insulin, insulin like growth factor (IGF), EGF and platelet derived growth f actor ( ) receptors ( 164 ). Recent studies show that internalization of the EGF and PDGF receptors is required for their localization with PTP1B ( 165 ), supporting a role for PTP1B in the dephosphorylation of internalized RTKs. Research has also shown that the affinity of PTP1B for a substrate containing double tyrosines is 70 fold higher than for a substrate containing single tyrosine ( 166 ). The se obsevations collectively l ed to the idea that c Met double tyrosine residues in the activation domain could be dephosphorylated by
80 PTP1B. Research has shown in the PTP1B KO mouse model that the phosphorylation state of Tyr 1234/1235 is regulated by PTP1B ( 159 ). In response to HGF, injected via hepatic portal vein, phosphorylation of tyrosines 1234 and 1235 was ele vated in PTP1B KO mice when compared with WT mice. The HGF c Met pathway is essential for LR. Therefore the effect of partial hepatectomy was tested using PTP1B KO mice ( 167 ). BrDU incorporation studies revealed that there was increased liver cell prolife ration in PTP1B KO mice when compared with PTP1B WT mice. The PCNA levels increased after PHx in both genotypes and the magnitude of induction was higher in the absence of PTP1B. Moreover, c Met phosphorylation was enhanced in PTP1B KO mice liver after PHx These data collectively showed that PTP1B is involved in LR process after PHx, as a regulatory factor in the HGF c Met pathway. Phosphatases do not dephosphorylate their substrate continuously. Dephosphorylation is tightly controlled by mechanisms simila r to the ones that control phosphorylation ( 168 ). One of the controlling mechanisms for the regulation of PTP activity is oxidation of the sulfhydryl group of the catalytic cysteine of the enzyme. The PTPs employ a covalent catalysis, utilizing the thiol g roup of the active site cysteine (Cys215 for PTP1B) residue as the attacking nucleophile, to form a thiophosphoryl enzyme intermediate ( 169 ). This active site cysteine residue was particularly important for the discovery of new PTP1B inhibitors, since PTP1 B inhibition has a physiological importance. Liver and muscle from PTP1B KO mice show hyperphosphorylation of the Insulin receptor (IR) upon stimulation with insulin. These mice are resistant to weight
81 gain caused by a high fat diet ( 170, 171 ), emphasizing the importance of PTP1B as a therapeutic target for the diabetes. Zinc has long been shown to be a PTP inhibitor ( 172 173 ). I n glial C6 cells, increased cellular influx of zinc augments general tyrosine phosphorylation, in particular phosphorylation of th e activating tyrosine residues of the IR and IGF1R ( 174 ). The phosphatase assays that were conducted with specific phosphopeptide substrates showed that zinc specifically inhibited PTP1B activity. Combining the facts that the HGF c Met pathway is essential for LR; c Met kinase domain phosphotyrosils are substrates for PTP1B; and zinc inhibits PTP1B activity, lead to the hypothesis that zinc might be enhancing LR through inhibition of PTP1B resulting in enhanced c Met phosphorylation. Results Zinc I nhibits Protein T yrosine Phosphatase 1B A ctivity It has been shown in PTP1B knockout mice that c Met receptor kinase phosphorylation sites are dephosphorylated by PTP1B ( 158 ) It has also been shown that zinc inhibits PTP1B enzymatic activity in glioma cells resu lting in enhanced phosphorylation of insulin like growth factor receptor ( IGFR ) ( 174 ) IGFR and c Met share a common feature of having two consecutive phosphorylation site s in their kinase domain. Interestingly, PTP1B has a 70 times higher affinity for the double phosphate domains than single phosphate domain ( 158 ). Consequently, I hypothesized that the contribution of zinc to the hepatocyte proliferation might be through inhibition of PTP1B activity during LR. In order to test PTP1B enzymatic activity, an in vitro phosphatase assay was established in the lab. In this assay, after treatments, cell lysates were prepared and were incubated with a phosphopeptide substrate. The phosphate ion
82 release from the phosphopeptide was measured as an indicator of PTP1B e nzyme activity. The nature of the assay requires no usage of phosphatase inhibitors in the cell lysates, introducing the possibility of preexisting phosphate ion interference. To test whether zinc treatment caused any change in the amount of preexisting ph osphate ion in cell lysates, the first assay was done with the cell lysates containing phosphatase inhibitors to prevent any phosphatase enzyme activity. There was no difference between zinc pretreated and control cell lysates in the amount of phosphate io n (Figure 4 1). After validation of the assay, AML12 hepatocytes were incubated with different concentrations of zinc (2 32 M) and pyrithione, and then total cell lysates and membrane fractions were prepared for the measurement of the phosphate ion releas e from phosphopeptide substrate. Dose dependent inhibition of PTP1B activity by zinc was detected in both total and m embrane fractions (Figure 4 2A ). In the membrane fraction the effect was more pronounced possibly because PTP1B is a membrane bound prot ein. Thus further experiments were conducted with only membrane fractions. PTP1B enzymatic activity was diminished 60% in 8 M zinc pr etreated cells (Figure 4 2B). The a mount of inhibition by zinc was similar to the amount of inhibition by nonspecific NOV and PTP1B specific phosphatase inhibitors (Figure 4 2B ). A 40% decrease in PTP1B activity was detected in LPS CM and IL 6 treated cells, as well (Figure 4 2C ). These results collectiv ely showed that zinc inhibits PTP1B activity Zinc E nhances c Met Phospho rylation The inhibition of PTP1B may result in enhanced c Met phosphorylation. To test this hypothesis zinc/LPS CM and IL6 pretreatment, cells were treated with HGF and the changes in c Met kinase domain phosphorylation were measured by using phospho spec ific antibodies. The results revealed that there was a higher amount of
83 phosphorylation of c Met in the zinc /LPS CM and IL6 pretreatment conditions in comp arison to their control (Figure 4 3 ). Protein c Met was used as a loading control and there was no c hange in the band intensities between treatment groups. c Met phosphorylation activates many downstream signaling pathways. Among them ERK1/2 was shown to be the main target during LR, since there was no change in ERK1/2 phosphorylation in c Met conditi onal KO mice after PHx. Therefore phosphorylation of ERK1/2 was visualized by western blot s Similar to c Met, ERK1/2 phosphorylatio n was higher in the zinc/LPS CM and IL6 treated conditions (Figure 4 3 ). Protein ERK1/2 was used as a loading control and there was no change in the band intensities between treatment groups. Eukaryotic initiation f actor 4E ( e lf4E ) was used as a loading control, as well. These results further proved the role of zinc in c Met activated hepatocyte proliferation pathway during L R process. Discussion The m ain findings of this chapter ar e that (i) zinc inhibited PTP1B phosphatase activity; (ii) c Met phosphorylation at the active site residues Tyr 1234 and Tyr 1235 were enhanced as a result of PTP1B inhibition; (iii) enhanced c Met activation caused increased phosphorylation in ERK1/2. Phosphatase assays were conducted to evaluate the effect of zinc on PTP1B activity. Total cellular membrane fractions were used as a source of endogenous PTP1B since PTP1B is a membrane bound enzyme. However, this approach did not eliminate the possibility that there could be other tyrosine phosphatases in the lysate. One solution t o solve this issue was the use of a PTP1B specific substrate in phosphatase assays. I used the highly potent ( kcat/Km of 2 .20.05x10 7 M 1 s 1 ) consensus peptide, ELEFpYMDYE as a substrate. This consensus peptide sequence
84 was obtained from studies that used kinetic ( 175 ), structural ( 176 ) and alanine sc anning ( 177 ) approaches. The peptide corresponds to residues 988 998 in EGFR was used as a substrate in those initial studies These studies collectively showed that PTP1B could accommodate acidic (Glu/Asp), aromatic (Phe/Tyr), and hydrophobic (L eu) residues at the 1 position PTP1B preferred Leu at the 3 and Met at +1 position, and strongly preferred acidic residues (Glu/Asp) at +2 and aromatic residues (Phe/Tyr) at+3 positions. By using this specific peptide as a substrate, specificity of the assay was enhanced. PTP1B inhibition by zinc was further proven by the increased phos phorylation of physiological substrate of PTP1B, c Met kinase domain phosphotyrosils, as a result of zinc pretreatment (Figure 5 3) In this chapter, a dose dependent inhibition of PTP1B activity by zinc was shown. The possible mechanism for the zinc inhib ition of PTP1B could be related to the cysteine residue at in the active site of PTP1B. One of the controlling mechanisms of the PTP1B enzymatic activity is through reversible oxidation of this cysteine residue. Zinc interacts strongly with cysteines of pr otein such as metallothionein ( 178, 179 ). Therefore, zinc may possibly bind to the cysteine residue of PTP1B active site and cause the inactivation of enzymatic activity. This idea was supported by the fact that the inhibitory effect of zinc diminished whe n the reducing reagent, DTT, was included in the reaction (data not shown). DTT increases the redox potential, thus causing PTP1B to return to the active form. Such i nterplay between zinc and redox was shown previously with regard to sequestration and rele ase of zinc ions by cysteine rich metallothioneins ( 180 ).
85 The inhibition of PTP1B activity caused increased phosphorylation the of c Met receptor at t he kinase domain Downstream of c Met signaling produces activat ion of Ras/ERK/MAPK PI3K/Akt, Rac/Pak, a nd Crk/Rap1 pathways ( 181, 182 ). Among these pathways, phosphorylation of the ERK1/2 was not detected in the regenerating liver of the conditional Met mutant mice ( 143 ) This suggested that c Met signaling contributes dominantly to ERK1/2 activation in the regen erating liver. Therefore, I cho se to investigate ERK1/2 phosphorylation to show the effect of zinc on the downstream signaling of c Met activation. Zinc inhibition of MAPK phosphatases resulted in increased phosphorylation of ERK1/2 in neural cells ( 183 ), raising the possibility that the increase in ERK1/2 phosphory lation could be independent from upstream c Met activation. This possibility was eliminated by further investigation of ERK1/2 phosphorylation in cells treated with zinc (without HGF) In t hese cells, there was no significant change in ERK1/2 phosphorylation between control and zinc pretreated cell preparation (data not shown). Therefore, I concluded that the increase in ERK1/2 phosphorylation was a downstream effect of HGF c Met activation after zinc pretreatment. In summary, a role of zinc appears essential in the HGF c Met pathway in hepatocyte proliferation demonstrated in this dissertation project. Zinc re gulates the HGF c Met pathway during hepatocyte proliferation through inhibition o f PTP1B phosphatase activity.
86 Figure 4 1. Validation of phosphatase assay. (A) Representative standard curve of the phosphate assay. (B) Total cell lysates were prepared from AML12 hepatocytes after 30 minutes of pyrithione and zinc treatment. Phosphata se inhibitors were added to the lysis buffer, and free phosphate contaminations from the lysate were measured.
87 Figure 4 2. In hibitory effect of zinc on protein tyrosine phosphatase 1b activity. (A) AML12 hep atocytes were treated with pyrithione and different concentrations of zinc for 30 minutes. Next, cells were harvested and total cell lysate (grey bar) and membrane fractions (black bar) were prepared. Lysates were incubated with phospho substrate and inorg anic phosphate release was measured. AML12 hepatocytes were treated with either prythione and 8 M zinc, along with phosphatase inhibitors for 30 minutes (B) or macrophage conditioned media for 2 hours (C). Next, cells were harvested and the total membrane fraction was used for the phosphatase assay.
88 Figure 4 3. Effect of zinc on c Met signaling pathway. AML12 hepatocytes were treated with either two different concentrations of zinc or macrophage conditioned medium for 2 hours. Next, cells were harvested and total cell lysates were used for the detection of indicated proteins by western blot.
89 CHAP TER 5 ZIP14 FACILITATES HE PATIC ZINC UPTAKE DU RING LIVER REGENERATION Introduct ory Remarks The Zip14 gene was first identified in an effort to accumulate inf ormation on the coding sequences of unidentified human genes in 1994 ( 184 ). In the study cDNA clones that carry unreported sequences at the 5 ends were isolated from human cDNA libraries and sizes of their inserts were then compared with those correspond ing transcripts by Northern hybridization. After sequence determination of these inserts, Zip14 was named KIAA0062 a mong 40 newly identified coding sequences. Sequence alignment studies revealed that Zip14 belongs to the LZT (LIV 1 subfamily of zinc tra nsporters) subfamily of the ZIP (ZRT/IRT related protein) transporter family proteins ( 185 ). ZIP transporters are responsible for the control of zinc transport into the cell cytosol and can be divided into four separate subfamilies ( 186 ): subfamily I is ma inly fungal and plant sequences, subfamily II consist s of mammalian, nematode and insect genes, the gufA subfamily is related to the gufA gene of Myxococcus xanthus and LIV 1 subfamily is related to the oestrogen regulated gene, LIV 1 LIV 1 is implicat ed in metastatic breast cancer ( 187 ). Its detection was associated with oestrogen receptor positive breast cancer ( 188 ) and with the metastatic spread of these cancers to the regional lymph nodes ( 189 ). Computer analyses of LIV 1 for secondary structure p rediction revealed that LIV 1 has six to eight transmembrane domains, a long extracellular N terminus, a short extracellular C terminus and numerous histidine rich repeats, relating LIV 1 to the ZIP family of zinc transporters ( 185 ). Multiple alignment ana lyses with an effort to find other members of
90 LZT subfamily led to the discovery of Zip14, named LZT Hs4 at that time. The alignment analyses also revealed an important conserved motif (HEXPHE) among all the possible LZT proteins that were tested. This m otif localized in the TM domain V, similar to the HXXXE motif in the TM domain V of ZIP proteins. Another important similarity found between ZIP and LZT was a conserved HS motif in TM IV of the ZIP s and a HNF motif in TM IV of LZT proteins. The HS motif in the TM IV and the HXXXE motif in TM V of ZIP proteins a re thought to be part of the intramembrane metal binding site, which forms the pore region. Therefore, these sequence similarities suggested a zinc transport functio n for the LZT subfamily members, wi th one exception being Zip14 since Zip14 had a glutamic acid replacement of the first histidine residue of the conserved HEXPHE motif of LZT proteins. This replacement may allow for the transport of ions other than zinc ( 190 ). In fact, our lab has shown p reviously that Zip14 transports iron in Zip14 overexpressed HEK 293H cells ( 191 ). Cadmium and manganese transport function of Zip14 was also reported in transfected MFF cells ( 192 ). In this study transpo rt efficiency of two transcript variant s was tested. These transcript variants of Zip14 are the result of alternative splicing of either exon 4A or exon 4B. Mouse Zip14 exons 4A and 4B are both 170 bp long and share a 67% nucleotide identity. Transcripts of ZIP14A and ZIP14B encode two different proteins, both having 489 amino acids but dif ferent molecular masses. R esult s showed that Zip14A transfected cells had more efficient cadmium and manganese transport than Zip14B transfected cells. D iffering expression of exons 4A and 4B between normal and tumor samp les was also reported ( 193 ). Even
91 though there was no difference in total Zip14 expression, the 4A/4B ratio was significantly lower in adenomas and carcinomas when compared to normal tissues. The f irst attempt to show a zinc transport function of Zip14 in transfected CHO cells revealed no change in intracellular zinc, unlike LIV 1(Zip6) transfected CHO cells ( 185 ). However, in later studies from the same research group showed that intracellular zinc concentration was increased in the Zip14 transfected CHO cells when they provided extracellular zinc to the cells ( 1194 ). This finding complies with the plasma membrane localization of Zip14 ( 190 194 and 82 ). The z inc transport ability of the Zip14 transfected cells was temperature dependent, suggesting a car rier mediated transport process. In the same study (Taylor et al. 2005) Zip14 was shown to run as a trimer in an SDS PAGE when reducing conditions were not present. This ability to form complexes is consistent with the formation of ion channels ( 195 ). C oncurrently, the z inc transport function of the mouse counterpart of Zip14 was shown by our lab in the transfected HEK 293H cells in the presence of 10 M extracellular zinc ( 82 ) In the same study the plasma membrane localization of Zip14 was also confirm ed Besides revealing zinc transport function and cell ular localization of Zip14, this study showed for the first time that Zip14 is a cytokine regulated gene. The idea was driven from the fact that hypozincemia was one of the consequences of an acute inf lammatory response ( 196 ). The hypozincemia i s thought to be a part of host defense mechanism. The physiological role of the hypozincemia is not known. Two possibilities are deprivation in the availability of zinc for any pathogenic microorganisms ( 197 ) or the provision of more zinc for cellular needs. During acute inflammation, acute phase proteins are synthesized by the liver. Redistribution of hepatic zinc and increased
92 hepat ic MT were observed in cytokine injected rats ( 198 ) suggesting that serum zinc d epletion was caused by hepatic zinc uptake during inflammation. To investigate this hypothesis, mice were injected with LPS since it has long been know n that LPS lead s to production of acute phase response proteins in the liver ( 199 ). Hepatic MT levels wer e significantly higher in LPS injected mice in comparison to PBS injected control mice, suggesting hepatic zinc uptake was increased. To delineate which transporters were responsible for hepatic zinc uptake differential expression of ZIP and ZnT transport ers were measured in the liver. The most responsive zinc transporter was found to be Zip14, having a 3.1 fold increase in response to the LPS injection. Even though the LPS injection causes increased production of many acute ph ase response proteins IL6 i s viewed as the main proinflammatory cytokine responsible ( 200 ). Therefore, further experiments were done with IL 6 KO mice. Higher hepatic Zip14 and MT expressions and lower serum zinc were not detected in the IL6 KO mice in response to the LPS injection. These results suggested two important conclusions: Zip14 was an IL6 regulated gene, and Zip14 was the main transporter to facilitate hepatic zinc uptake during inflammation since MT up regulation and serum zinc down regulation were diminished in the IL6 K O mice. C ytokine regulation of Zip14 expression was supported by a recent study i n sheep pulmonary artery endothelial cells (SPAEC) ( 201 ). When SPAEC were treated with LPS a 5 fold increase in Zip14 expression was observed. The initial observations with the cytokine regulation of Zip14 and being the main transporter responsible for the hepatic zinc uptake during an inflammatory response ( 197 ) initiated the hypothesis that Zip14 may have a role in the priming step of the hepatocyte proliferation in LR s ince it is a cytokine dependent process. This idea was
93 supported by the results that were presented in chapter 3 on Zip14 up regulation in response to PHx. Therefore in this chapter a specific role for Zip14 in the LR process and the regulation of Zip14 e xpression were investigated. Results To investigate a specific role of Zip14 in hepatocyte proliferation, two approaches were taken: Zip14 overexpression and a ZIP14 KO mouse. Overexpressed Zip14 E nhances H epatocyte P roliferation For the overexpression s tudies, AML12 hepatocytes were transiently transfected with either pCMV Sport6 Zip14 or pCMV Sport6 empty vector for 96 hours. Overexpression of Zip14 wa s confirmed by qPCR (p<0.001) and western blot analyses (Figure 5 1B). To test the effect of Zip14 over expression on hepatocyte proliferation, the following treatments were applied during the 96 hours of trans fection: hepatocytes were serum starved for 20 hours starting at 24 hours of transfection, and then hepatocytes were incubated with HGF for 48 hours. At the end of 96 hours, hepatocytes were harvested and PCNA expression was measured. Higher PCNA expression was detected in the ZIP14 overexpressed cells when compare to the empty vector transfected control cells (Fig 5 1C). For the further investigatio n of hepatocyte proliferation, a BrDU incorporation assay was conducted. Results showed significantly higher (p<0.001) BrDU incorporation in ZIP14 overexpressed cells when compare to empty vector transfected control cells (Figure 5 1A). The se results sugge sted that Zip14 overexpression enhanced AML12 hepatocyte proliferation. The p hysiological consequence of Zip14 overexpression was an increased intracellular zinc concentration ( 82 ). With the similar approach that I took in chapter 3, I hypothesized that th e overexpressed Zip14 facilitated an increase in intracellular zinc
94 that may ha ve a role in PTP1B inhibition of HGF c Met pathway during hepatocyte proliferation. To test this hypothesis after 96 hours of Zip14 transfection hepatocyte s were harvested and membrane fractions were isolated. A phosphatase activity assay was conducted with these membrane fractions and a phosphopeptide substrate. The result showed a 40% decrease in PTP1B activity in the Zip14 overexpressed conditions (Figure 5 2A). With the supp ort of this result I further hypothesized that PTP1B inhibition may cause enhanced phosphorylation of tyrosine residues of the c Met kinase domain. To test this hypothesis, AML12 hepatocytes were incubated with HGF for 30 minutes after the 96 hours transf ection Thereafter, hepatocytes were harvested and proteins were analyzed by SDS PA GE. Enhanced phosphorylations of c Met kinase domain and of ERK1/2 were detected wh en Zip14 was overexpressed (Figure 5 2B). ZIP KO C aused D ecrease in H epatocyte P roliferati on A more concrete approach to test the role of the gene in a specific cellular event is to use a KO mouse model when available. Fortunately, a ZIP14 KO mouse became available during my dissertation research; consequently the contribution of Zip14 to the L R process was investigated directly Genotyping of the mice was conducted with two different sets of primers. Primers were specifically design to amplify either WT or mutant DNA (Figure 5 3). After genotyping, the ZIP14 KO was confirmed at both the protein and mRNA level. Intestine and liver tissues were chosen for the confirmation since these two tissues abundantly express Zip14 ( 194 82 ). Zip14 protein (Figure 5 3A) and mRNA (Figure 5 3B) showed a gradual decrease in the HET and KO mice when compare to W T mice. Next, PHx surgeries were conducted with ZIP14 KO and WT mice. In these sets of experiments, comparisons were made between prePHx and postPHx conditions
95 instead of between sham and postPHx, because of the limitation in the number of ZIP14 KO mice. For that same reason, female mice were included in this experimental setting as well. There were two female and two male mice in total. At 24 hours postPHx, ZIP14 was up regulated at the mRNA and protein level in WT mice (Figure 5 4A). However, Zip14 was not detectable in either prePHx or postPHx liver (Figure 5 4A). To test the hypothesis that Zip14 might be responsible for the hepatic zinc increase during liver regeneration, MT expression (Figure 5 4B) and the hepatic zinc concentration (Figure 5 4C) we re measured by qPCR and AAS, respectively. A significant increase in hepatic MT expression in response to PHx in the WT mice was observed and that increase was significantly diminished in ZIP14 KO mice. Similar results were obtained from the hepatic zinc m easurement. Total hepatic zinc was significantly (p<0.001) increased in the WT in response to PHx. However, that increase in hepatic zinc was not present in ZIP14 KO mice, strongly suggesting that ZIP14 was the main zinc transporter that facilitates zinc m obilization to the liver during LR. Therefore, I hypothesized that the enhancement of regeneration that was caused by increased hepatic zinc may diminish in ZIP14 KO mice as a result of impairment in hepatic zinc transport. To test this hypothesis, PCNA ex pression was measured in prePHx and at 24 hours postPHX liver tissue. Zip14 mRNA was significantly (p<0.001) increased in WT mice and that increase was significantly diminished (p<0.01) in ZIP14 KO mice (Figure 5 6A). Similar results were observed in the p rotein level of ZIP14. The f emale mice had a 30% and male mice had a 60% decreases in PCNA expression in comparison to the WT mice at 24 hours postPHx (Figure 5 6B). Relative densitometry
96 values of PCNA that were normalized to the tubulin controls from fou r mice were averaged and presented in Figure 5 6C. Transcriptional R egulation of Zip14 during liver regeneration The si gnificance of Zip14 in LR led to an investigation of the transcriptional regulation of Zip14 during LR. A DNA pull down assay was use d to identify possible transcription factors that regulate Zip14 transcription. In this assay 2 kb of the Zip14 purified afterwards (Figure 5 7A). Purified DNA was incubated with nuclear lysates from either partially hepatectomized or sham operated mice. DNA bound proteins were isolated by magnetic ally labeled streptavidin beads and run in SDS PAGE (Figure 5 7B). Even though there was a difference in the band pattern between hepatec tom ized mice and sh am control, these data are at the preliminary level and needed to be improved in terms of specificity. When conditions are optimized DNA bound proteins will be sent to the mass spectrometry facility for the determination of transcription factors that regu late Zip14 transcription during LR. Discussion The main findings of this chapter were (i) overexpressed Zip14 enhanced liver regeneration via inhibition of PTP1B phosphatase activity; (ii) c Met phosphorylation at the active site residues Tyr 1234 and Ty r 1235 were enhanced in Zip14 transfected hepatocytes; (iii) enhanced c Met activation caused increased phosphorylation in ERK1/2; (iv) ZIP14 KO mice had decreased hepatic zinc after PHx when compare d to WT mice ; (v) ZIP14 KO mice had decreased proliferati on in comparison to WT mice. LPS induction of Zip14 up regulation in SPA EC was reported ( 201 ). In that study they showed that LPS treatment caused the cells to become zinc deficient when
97 measured by the zinc s elective fluorescent probe, FluoZin 3 The res earchers claimed that in order to compensate for zinc deficiency, Zip14 was up regulated They further supported this claim by showing an up regulation of Zip14 in the TPEN treated zinc deficient SPAEC. Even though hypozinc emia is one of the hallmarks of L R after PHx, there have not been any reports (including this dissertation study) regarding liver tissue zinc deficiency to date. Therefore, I concluded in this dissertation that the Zip14 up regulation was to provide required zinc for proliferation of hepa tocytes rather than compensating for liver tissue zinc deficiency. This conclusion was supported with the data from the dietary zinc study (chapter 3). In this study no change in Zip14 expression was observed between ZnD and ZnA fed mice in either prePHx or postPHx samples (data not shown). However, there was a significant decrease in Zip14 expression in the ZnH fed mice when compared to ZnA fed mice after PHx (data not shown). One of the most important finding s of this chapter is the decreased prolifera tion in ZIP14 KO mice. This decrease possibly resulted from impaired hepatic zinc transport since, unlike WT mice, no increase was observed in hepatic zinc between pre and post hepatectomy samples from ZIP14 KO mice. Additionally, when hepatic zinc concent rations were compared between WT and ZIP14 KO mice before PHx, there was no significant change between the two groups, suggesting that the ZIP14 KO genotype did not cause liver tissue zinc deficiency. This result could be from compensation by other ZIP tra nsporters, since four other transporter s were significantly up regulated during LR (Zip1, Zip3, Zip7, and Zip10 ) besides Zip14.
98 Ev en though ZIP14 KO did not show zinc deficiency in liver tissue, proliferation was still imp aired, raising the idea that Zip 1 4 may have an additional role in the zinc transport function during LR. One of the possible roles for Zip14 could be related to adipogenesis during LR. Markedly increased hepat ocellular fat was detected at 12 to 24 hours post PHx mice. ( 202 ). To evaluate t he functional significance of hepatic adipogenic changes d uring the regenerative response, in that study the adipogenesis was suppressed either pharmacologically by leptin injection or by using hepatocyte specific glucocorticoid receptor ( GR ) KO mice. Both strategies led to a disruption of adipogenesis. L ess BrDU incorporation was detected after PHx, w hen adipogenesis was disrupted. Interestingly, Zip14 participation in the zi nc uptake during adipogenesis ha s been reported ( 203 ). Zip14 was up regulated in the early stages of adipocyte differentiation in 3T3 L1 cells. The time of Zip14 up regulation (10 hours post PHx) during LR, the time of the hepatocellular fat accumulation during (12 24 hours post PHx) LR and lastly Z ip14 participation in early stages of adipogenesis collectively suggest a role for Zip14 in early adipogenesis during LR. Another possible role for Zip14 in LR could be metallopro tease activity. Hepatic biomatrix degradation by metalloproteases is essential for the initiation of LR. Zip14 bel ongs to the LZT subfamily of the ZIP transporter family of proteins. The LZT subfamily proteins share the conserved HEXPHE motif. This motif is similar to consensus sequence for the catalytic zinc binding site of metalloprateases (HEXXH). In conventional m etalloproteases Zn 2+ co ordinates with two histidines, a water molecule f rom the first glutamic acid, and other residues downstream, depending on the subgroup ( 204 205 ). Even though in Zip14, the first histidine residue was replaced by glutamic
99 acid resid ue in the conserved motif, there is still possibility for Zip14 to have metalloprotease activity depending on the substrate. Lastly, contribution of Zip14 to the LR process could be related to G protein coupled receptors (GPCR). Concurrently with the prep aration of this dissertation, Zip14 essentiality to systemic growth via control of GPCR was reported ( 206 ). They generated ZIP14 KO by deletion of exons 5 8 while the ZIP14 KO mouse that was used in this project was generated by deletion of exons 3 5. E ven though deletions were made in different exons, in both ZIP14 KO mice growth retardation was common feature. Their result showed that Zip14 plays important roles in mammalian growth and energy metabolism by regulating GPCR cAMP CREB signaling, suggestin g another zinc/zinc transporter regulated signaling pathway to study in LR.
100 Figure 5 1. Effect of Zip14 overexpression on AML12 hepatocyte proliferation. AML12 hepatocyte s were transiently transfected with either an empty vector or the pCMV Sport Zip1 4 for 48 hours, then serum starved for 20 hours. Next, cells were incubated either with HGF and BrDU or HGF alone for 24 hours. (A) BrDU incorporation was measured at absorbance 450 nm. (B, C) Cells that were incubated with HGF alone were harvested, and RN A and cell lysates were prepared for measurement of Zip14 mRNA and PCNA expression, respectively. Values shown are means SD (n=3 5).
101 Figure 5 2. Effect of Zip14 overexpression on c Met signaling pathway. AML12 hepatocyte s were transiently transfect ed with either an empty vector or the pCMV Sport Zip14 for 96 hours. (A) Total cell membranes were isolated and used for the phosphatase assay. The phosphate release was measured at absorbance 650 nm. (B) Cells were serum starved in the last 20 hours of t ransfection, and then were incubated with HGF for 30 minutes. Total cell lysates were used for the western blot analysis. Values shown are means SD (n=3).
102 Figure 5 3. Genotyping of mouse tail samples. Genomic DNA was isolated from the mouse tail, an d 150 ng of the total DNA was used for the PCR reaction. The two sets of primers that were specifically design to amplify either WT or mutant DNA were used for the determination of mouse genotype.
103 Figure 5 4. Validation of Zip14 knock out Intestine and liver tissues were colle cted from WT, HET and KO mice. T issue lysates and total RNA were prepared for western blot (A), and qPCR (B), respectively.
104 Figure 5 5. Effect of Zip14 Knock out on hepatic zinc in response to partial hepatect omy PHx was performed on WT and Zip14 KO mice. Liver tissues were collected at the time of surgery from the resected part (PrePHx) and 24 hours after surgery (PostPHx). Total RNA and tissue lysates were isolated and used for Zip14 (A) and MT (B) mRNAdetec tion. (C) Tissues were digested by nitric acid and total zinc concentrations were measured by AAS. Values shown are means SD (n=4).
105 Figure 5 6. Effect of Zip14 knock out in liver regeneration PHx was performed on WT and Zip14 KO mice. Liver t issues were collected at the surgery time from the resected part (PrePHx) and 24 hours after surgery (PostPHx). Total RNA and tissue lysates were isolated and used for qPCR (A) western blot (B) analyses, respectively. (C) The average relative densitometry values were obtained from individual mice (n=4).
106 Figure 5 7. Transcription al regulation of Zip14 during liver regeneration (A) Zip14 Amplified DNA was purified and incubated with h epatic nu clear extracts that were isolated from either hepatectomized or sham operated mice. (B) DNA with bound proteins was isolated by magnetically labeled streptavidin beads and proteins were separated in SDS PAGE. Bound protei ns were visualized by silver stai ni ng.
107 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The general aim of this research was to gain a better understanding of the involvement of zinc and zinc transporters in the LR process. To accomplish this task three specific aim s were proposed: Determin ation of changes in zinc transporter expression and which of those are zinc respo nsive during liver regeneration; exploration of a role for zinc in the HGF pathway during hepatocyte proliferation ; and d elineation of the specific role and regulation of ZIP1 4 during hepatocyte proliferation. To investigate these specific aims 70% partial hepatectomy was chosen as an in vivo model In parallel, in vitro AML12 hepatocytes were used for some experiments. As summarized in the model (Figure 6 1), t he results that were obtained from both in vivo and in vitro experiments revealed that increased hepatic zinc during LR, enhanced hepatocyte proliferation This increase in hepatic zinc was mainly facilitated by the most up regulated zinc transporter, Zip14 as confirmed with a ZIP14 KO mouse The hepatic zinc increase in response to PHx was not present in the ZIP14 KO mice. Zip14 up regulation was mainly caus e d by IL6 however; presence of the other non parenchymal cells produced factors was required To investigate the specific role for zinc and Zip14 in LR, the HGF c Met pathway was chosen. The inhibitory effect of zinc was detected on PTP1B phosphatase activity. PTP1B is the enzyme responsible for dephosphorylation of the c Met kinase domain. Zinc inhibition of PTP1 B enhanced c Met ERK1/2 pho sphorylation resulting in enhanced hepatocyte proliferation. The importance of the results in my dissertation is their applicability for the treatment of liver diseases. T hree decades a go, whole liver transplantation was the only o ption for those who suffer ed from chronic liver diseases. However, the field of liver
108 transplantation is limited by the availability of donor organs. Therefore, new approaches to ta ke advantage of the regenerative capacity of the liver have been develop ed for the treatment of chronic liver diseases (208 ) These approaches include partial hepatectomy split liver tran splantation (SLT) and live donor liver transplantation (LDLT) The most important factor for survival after these surgeries is the regenerative capacity of the remnant liver ( 208 209 ) as it is reported in human (210, 211) and animal models (212 214 ). Enhanced liver regeneration is vital for the survival of the recipient a nd also for the donor in LDLT. Besides the success of these surgical appro aches, enhancement of LR is also required for the success of pharmacological approaches that aim to reverse fibrosis to treat liver diseases. Although PHx, S LT and LDLT offer the alternative curative option s many patients develop postoperative complicati ons because the remnant livers or grafts are too small or of poor quality to sustain sufficient organ function ( 208 ) This phenomenon is for In a partial liver graft mouse model, almost complete failure of hepatocyte proliferation was observed in the small graft (30%) receiving mice when compare d to 50% graft receiving mice (208 ) Similar observations were obtained from human cases ( 208 ) These o bservations led to the conclusion that defective liver regeneration is th e central mechanism of SFSS and focus should turn toward the relevant pathways of regeneration. Zinc may be a factor upon which to develop new therapies to enhance LR. Zinc supplementation was used in a few clinical trials and was shown to have a positiv e effect in liver function (108 113 ) All of the liver health assessments in those trials were done by the measurement of serum ALT levels. Thus, it is not clear if zinc has any
109 effect on proliferation of hepatocytes. Proliferation of hepatocytes is the ma in event in LR (10). Zinc is involved in cell proliferation both as a structural element ( 91,215 219 ) and as a regulatory element in many mitogenic s ignaling pathways (71, 72 ). The HGF c Met pathway is essential in LR. In this dissertation, specific role s for zinc an d Zip14 in HGF c Met pathway were shown. In this dissertatio n, zinc and Zip14 were proposed as potential therapeutic targets to treat liver diseases since enhancement of liver regeneration by zinc was shown in response to PHx. However, unlike m urine PHx model s in human disease settings, there are important parameters to consider including functional mass of remnant liver, the age of the patient, and the presence of pre existing liver diseases. Depending on the type of pre existing liver disease the effect of zinc may change For instance if a patient has chronic hepatitis caused by hepatitis C virus (HCV) infection, zinc may have an effect on both enhancement of regene ra tion and clearance of the HCV at the same time. Inhibition of genome length HCV RNA replication by zi nc supplementation was shown in the HCV inoculated HuH 7 cell line ( 220 ) These in vitro data were supporte d with a number of clinical data s howing improved hepatic outcomes of chronic hepatitis C and liver cirrhosis when patients were supplemented with zinc along with drug therapy ( 112 ) Significantly lower cumulative incidence of hepatocellular carcinoma was also reported in the zinc responder patients when compared to non responder patients. Another important parameter to consid er for the determination of the right strategy for the LDLT is patient age Impaired liver regeneration in old livers was shown in basic and clinical studies (221, 222 ) Interestingly, zinc deficiency in the elderly was reported (223 ) The results that wer e presented here indicated that zinc was a required
110 factor for regeneration. These observations may suggest that zinc supplementation for the older donors and recipients before liver surgeries may have a beneficial effect. To test this, zinc supplementatio n and PHx surgeries should be conducted with older mice in the future. In this dissertation project the main focus was zinc and Zip14. However there wer e five more ZIP transporter s that were significantly up regulated during LR. The specific role of these transporters in LR should be address ed in future studies. Additionally, in the ZIP14 KO m ouse model, expression of the rest of the ZIP transporters should be m easured to determine if there i s a compensatory mechanism for the maintenance of hepatic zinc lev el s d uring LR. In the ZIP14 KO model, alternative roles for Zip14 during LR (discussed in chapter 5) should be further investigated.
111 Figure 6 1. Shematic representation of inv olvement of zinc and Zip14 in liver regeneration Kupffer and innate immune s ystem cells produced IL6 upregulates Zip14 at the priming step of LR. Zip14 up regulation causes an increase in hepatic zinc. Increased hepatic zinc enhances hepatocyte proliferation by enhanced c Met ERK1/2 phos phorylation via inhibition of PT P1
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129 BIOGRAPHICAL SKETCH Tolunay Beker Aydemi r was born and raised in Ankara Turkey Tolunay attended the Ankara University and acquired her Bachelor of Science degree in b iol ogy in 1999. In 2000, Tolunay began her master studies at the Ankara University, College of Medicine. During her master studies she came to Emory University with the student exchange program and continued her research there for two years. In 2004, sh e defended her master research in Turkey and moved to Gainesville Florida. At the end of 2004, she started to work in Dr. Cousins Lab as a biological scientist. In 2006, she was accepted to the Interdisciplinary PhD program (IDP) in the College of Medi cine. After a one year rotation in the IDP program, she joined to Dr. Cousins Lab again to pursue her degree in biochemistry and molecular b iology an advance concentration in IDP.