Cd73 Is a Critical Regulator of Hepatocytes in Homeostasis and Disease
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CD73 IS A CRITICAL REGULATOR OF HEPATOCYTES IN HOMEOSTASIS AND DISEASE Karel Pastor Alcedo A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology and Physiology in the School of Medicine. Chapel Hill 2021 Approved by: Natasha T. Snider Patrick Brennwald Jay Brenman Hong Jin Kim Yuliya Pylayeva-Gupta © 2021 Karel Pastor Alcedo ALL RIGHTS RESERVED ii ABSTRACT Karel Pastor Alcedo: CD73 Is a Critical Regulator of Hepatocytes in Homeostasis and Disease (Under the direction of Natasha T. Snider) The enzymatic dephosphorylation of 5´-nucleotides like adenosine 5´-monophosphate (AMP) is a key step in purine salvage and purinergic signaling. This reaction occurs inside the cell or in the extracellular space. Ecto-5´-nucleotidase (CD73) is the major enzyme catalyzing the formation of extracellular adenosine from AMP. CD73-generated adenosine controls tissue homeostasis and responses related to inflammation, ischemia, fibrosis, and cancer. However, the specific functions of CD73 in liver homeostasis and diseases have not been fully elucidated. The objectives of the study were to 1) define the cell-specific role of CD73 in the liver, 2) determine how hepatocyte CD73 regulates alcohol-induced liver injury, and 3) examine CD73 regulation in human hepatocellular carcinoma. We generated and characterized a hepatocyte-specific CD73 knockout mouse (CD73- LKO) model using serological, biochemical, and histological assays. We found that CD73- LKO mice developed spontaneous liver injury in a sex- and age-dependent manner, which was associated with hypoactivation of AMPK, the key regulator of cellular energy metabolism. Furthermore, CD73-generated extracellular adenosine mediated AMPK activity upon cellular uptake through nucleoside transporters. Given that hepatocyte CD73 and adenosine exerts basal hepatoprotection, we then determined its role in alcohol-induced liver injury, a leading risk factor for chronic liver diseases. The loss of hepatocyte CD73 predisposed mice to more severe liver injury. In contrast, alcohol exposure in WT mice iii resulted in mild injury, concomitant with CD73 induction and enhanced enzymatic activity. In vitro assays revealed a non-enzymatic CD73 binding protein function for bioactive lipids, which are generated upon alcohol exposure. These data suggest CD73-mediated dual protection in alcoholic liver. In humans, chronic alcohol-induced liver injury leads to hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide. Because hepatocyte CD73 is critical in normal and injured liver, we examined its regulation in HCC. CD73 was expressed in tumor and non-tumor liver tissues. However, tumor-specific CD73 altered N- linked glycosylation led to its cytoplasmic redistribution and a reduction in the enzymatic activity, suggesting a potential impairment in CD73-mediated liver protection. In conclusion, these studies provide further understanding of CD73 biology in the liver and has implications for the potential therapeutic use of CD73 in chronic liver diseases. iv I dedicate this to Jerry and Nerie. Napuno nak ti pinag-agyaman ko para kinya yo nga dua – diyay kinaado ti pinagsakripisyo yo para kinyami nga agkakabsat, tapno laeng mumayat diyay biyag mi. Detoy ti maisublik kinya yo nga dua – pinagpayso ti pinagadal ko. v ACKNOWLEDGMENTS I want to acknowledge my mentor, Natasha Snider, for her exemplary commitment to my scientific and personal growth, for the opportunities and experiences that she has afforded me, for her guidance and encouragement in my endeavors beyond my scientific training, and for the support and endorsement in my career decisions. I attribute a huge part of my success to her while in graduate school. I also want to acknowledge all members of the Snider Lab for their guidance and assistance, most specially to Morgan Rouse, Gloria Jung, and Dr. Dong Fu. I want to thank the members of my thesis committee – Drs. Patrick Brennwald, Yuliya Pylayeva-Gupta, Hong Jin Kim, and Jay Brenman for their invaluable guidance and support throughout my time in the Cell Biology and Physiology program. Thank you to all of the collaborators who have significantly contributed to the completion of this project. I am also very thankful to the Cancer Cell Biology Training Program – to Drs. Adrienne Cox, Ben Major, William Kim, and to all the trainees – for providing a community that was vital in my development as a scientist for cancer research. I want to acknowledge my solid support group of family and friends for their endless love and encouragement, and their continued support as I navigated graduate school. Lastly, I am thankful to Kenneth Alarcon Negy for being my pillar. This dissertation was funded by pre-doctoral fellowships from the Cancer Cell Biology Training Program and UNC Graduate School. vi TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………...x LIST OF FIGURES…………………………………………………………………………xi LIST OF ABBREVIATIONS…………………………………………………….………..xiii CHAPTER 1: CHRONIC LIVER DISEASE………………………………………………..1 Overview……………………………………………………………………………..1 Liver as a Metabolic Hub…………………………………………………………….2 Pathophysiology and Treatment of Chronic Liver Diseases…………………………4 HCC Development and The Role of Alcohol………………………………………..6 Tight Regulation of Cellular Energy Metabolism Maintains Liver Homeostasis……7 5’NT and Extracellular Adenosine in Physiological Adaptation and Disease Development………………………………………………………………...8 5’NT/CD73 in Chronic Liver Disease……………………………………………….9 Rationale and Objectives……………………………………………………………10 REFERENCES……………………………………………………………………...11 CHAPTER 2: THE ELEGANT COMPLEXITY OF 5’-NUCLEOTIDASE (CD73)……...19 Overview……….…………………...……………………………………………....19 From 5’-NT to CD73: Changing Nomenclature and Shifting Research Directions………………………………………………………………...20 Genetic Deficiency of CD73 and Cross-Talk Between Purinergic Signaling and Gene Regulation…...………………………………………………...22 The Emerging Importance of Age and Sex in the Physiological Functions of CD73 and Adenosine…………………………………………………23 vii Cellular Uptake-Mediated versus Receptor-Mediated Effects of CD73-Derived Adenosine………………………………………………………..24 The Importance of Location: Zonal Distribution of CD73 in Digestive Epithelia………………………………………………………………….25 CD73 and Intestinal Epithelial Homeostasis………………………………………..26 CD73 and Liver Homeostasis…………………………………………………….....28 Complex Regulation of CD73 mRNA, Protein, and Enzymatic Activity…………..29 Development of CD73 Inhibitors and Other Tools to Support Further Research…..31 REFERENCES……………………………………………………………………...33 CHAPTER 3: CD73 MAINTAINS HEPATOCYTE METABOLIC INTEGRITY AND MOUSE LIVER HOMEOSTASIS IN A SEX-DEPENDENT MANNER………………...43 Introduction…………………………………………………………………………43 Materials and Methods……………………………………………………………...45 Results………………………………………………………………………………54 Discussion…………………………………………………………………………..66 REFERENCES……………………………………………………………………...73 CHAPTER 4: HEPATOCYTE CD73 IS A TARGET OF ETHANOL AND PROTECTS DURING ALCOHOL-INDUCED LIVER INJURY…………………………80 Introduction…………………………………………………………………………80 Materials and Methods……………………………………………………………...81 Results………………………………………………………………………………86 Discussion…………………………………………………………………………..94 REFERENCES……………………………………………………………………...99 CHAPTER 5: TUMOR-SELECTIVE ALTERED GLYCOSYLATION AND FUNCTIONAL ATTENUATION OF CD73 IN HUMAN HEPATOCELLULAR CARCINOMA……………………………………………………104 viii Introduction………………………………………………………………………..104 Materials and Methods…………………………………………………………….106 Results……………………………………………………………………………..110 Discussion……………….………………………………………………………...123 REFERENCES…………………………………………………………………….128 CHAPTER 6: CD73 AS A THERAPEUTIC TARGET OF HEPATIC AND EXTRAHEPATIC DISEASES……………………………………………………..133 Overview…………………………………………………………………………..133 CD73 as a Metabolic Gatekeeper in the Liver…………………………………….134 CD73 as a Novel Lipid Binding Protein…………………………………………..135 CD73 as a Marker of Hepatocyte Stress…………………………………………..135 Sex-Dependent Differences in HCC and a Potential Role of CD73………………136 Conclusions and Future Perspectives……………………………………………...137 REFERENCES…………………………………………………………………….141 ix LIST OF TABLES Table 3.1 – Primers for Genotyping and Quantitative Polymerase Chain Reaction…….….51 Table 6.1 – Outstanding Questions on CD73 Function and Expression……………….….140 x LIST OF FIGURES Figure 1.1 – Metabolic Zonation in the Liver………………………………………………..3 Figure 1.2 – Sequential Progression of Chronic Liver Disease……………………………...5 Figure 2.1 – CD73 is an Essential Component of Purinergic Signaling and a Novel Disease Target..……………………………………………………. 21 Figure 2.2 – Zonal Expression of CD73 Supports Tissue-Specific Homeostasis…………..27 Figure 2.3 – Molecular Regulation of CD73………………………………………………. 30 Figure 3.1 – CD73 is Expressed Primarily on Hepatocytes in Normal Mouse Liver…..…..55 Figure 3.2 – Generation of Liver-Specific CD73-LKO Mice………………………………56 Figure 3.3 – CD73-LKO Mice Develop Normally and Do Not Show Major Liver Abnormalities Up to 21 Weeks of Age……………………………………………….58 Figure 3.4 – CD73-LKO Mice Develop Spontaneous Liver Injury After 21 Weeks of Age in a Sex-Dependent Manner……………….………………………………...60 Figure 3.5 – Metabolic Imbalance and Increased Steatosis in Male CD73-LKO Mouse Hepatocytes and Livers………………………………………………...62 Figure 3.6 – Inhibition of CD73 Enzymatic Activity Promotes Lipid Accumulation in Hepatocytes…………………………………………………………….....64 Figure 3.7 – CD73-Generated Extracellular Adenosine Activates AMPK in