MOLECULAR AND PHYSIOLOGICAL RESPONSES TO HYPOXIA by HOI I (QUEENIE) CHEONG, B.S. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine CASE WESTERN RESERVE UNIVERSITY May, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/ dissertation of Hoi I (Queenie) Cheong Candidate for the degree of Ph.D. Committee Chair Kingman P. Strohl, M.D. Committee Members Mitchell Drumm, Ph.D. George R. Stark, Ph.D. Sathyamangla V. Naga Prasad, Ph.D. Serpil C. Erzurum, M.D. (Thesis Advisor) Date of Defense February 27th, 2017 * We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION To my parents, Lai Wa Lam and Kuok Kin Cheong. For they are the reason for whom I have become today. For their strength and love to support me to leave home since 2003 to broaden my vision and knowledge. To my mentor, Serpil C. Erzurum. For providing such wonderful learning opportunities and experience for science, medicine, collaborative spirits and leadership. To my husband, Emir Charles Roach. For his continuous love and kind support. For his intellectual curiosity to stir daily scientific discussions. For his excitements and encouragements. Table of Contents List of Tables iii List of Figures iv Acknowledgements vi List of Abbreviations viii Abstract ix Chapters 1. Introduction I. Hypoxia 1 II. HIF-1 in Hypoxia Sensing Ø History of discovery 1 Ø Function 2 Ø Regulation 3 III. Beta-Adrenergic Receptors and Hypoxia Responses Ø βAR subtypes, expression, structure and history 4 Ø Ligand binding 5 Ø β2AR signaling 5 Ø βAR function 6 Ø βAR under hypoxia 8 IV. Nitric Oxide: an Adaptive Response at High Altitude Ø Background 10 Ø Sources of nitric oxide 10 Ø Mechanism of Action 12 Ø Nitric Oxide at High Altitude 13 i 2. Hypoxia Sensing through Beta-Adrenergic Receptors Ø Abstract 15 Ø Introduction 16 Ø Results 19 Ø Figures 25 Ø Tables 34 Ø Supplemental Tables 35 Ø Discussion 38 3. Alternative Hematological and Vascular Adaptive Responses to High-Altitude Hypoxia in East African Highlanders Ø Abstract 55 Ø Introduction 56 Ø Materials and Methods 58 Ø Results 61 Ø Figures 65 Ø Discussion 70 4. Discussion and Future Directions Ø A novel role of βAR in hypoxia sensing 76 Ø Does hypoxia induce GRK activity? 76 Ø Is β-arrestin the molecular that mechanistically link GRK and HIF-1? 78 Ø Translational aspects of GRK in pulmonary arterial hypertension 78 Ø Nitric oxide-mediated flow based pathway as an alternative hypoxia response to erythropoiesis 80 Ø What is/ are the potential mechanism(s) of higher hemoglobin affinity with hypoxic adaptation? 81 Bibliography 83 ii List of Tables Table 1. Quantitative analyses of β2AR phosphorylation sites 34 Supplemental Table 1. Examples of hypoxia-inducible genes, whose expression levels are reversed by propranolol 35 Supplemental Table 2. KEGG pathway analysis of transcripts reversed by propranolol under hypoxia 36 Supplemental Table 3. Percent phosphorylation of β2AR sites at 21% or 2% oxygen, or with isoproterenol (ISO) 37 iii List of Figures Chapter 2 Figure 1. β-Blocker blunts HIF-mediated erythropoiesis under hypoxia in vivo. 25 Figure 2. β-Blocker attenuates hypoxia responses in vitro. 26 Figure 3. β-Agonist promotes HIF-1α accumulation under normoxia. 28 Figure 4. Hypoxia or β-agonist mediated HIF-1a accumulation depends on phosphorylation of beta-adrenergic receptor (βAR) by G protein-coupled receptor kinase (GRK). 29 Figure 5. Hypoxia induces a unique beta-adrenergic receptor (βAR) phosphorylation barcode in the absence of agonist binding. 31 Figure 6. Working Model. 33 Chapter 3 − Figure 1. Elevated urinary levels of nitrate (NO3 ) and cyclic guanosine monophosphate (cGMP) in high-altitude Amhara but not Oromo. 65 − Figure 2. Higher NO3 level is associated with higher cGMP in urine samples of Amhara and Oromo. 66 Figure 3. Diastolic blood pressures of Amhara were lower than Oromo at high altitudes. 67 iv Figure 4. Oxygen-saturated hemoglobin levels do not change with altitudes in Amhara and Oromo, whereas the increase in deoxyhemoglobin from low to high altitude is much greater in Oromo than Amhara. 68 Fig. 5. Increased affinity of hemoglobin for oxygen in high-altitude samples, relative to hypothetical subjects derived from the standard oxygen dissociation curve (ODC). 69 v Acknowledgements July 2011 marked the beginning of my Ph.D. training. The first year of coursework was a well-planned curriculum by the Molecular Medicine Ph.D. program, directed by Jonathan Smith, Ph.D. and administered by Marcia Jarrett, Ph.D. Robert Fairchild, Ph.D. has also been very supportive of students. I would like to thank them for nurturing students’ growth. I would also like to thank the faculty who has taught the classes, and the Howard Hughes Medical Institute who has conceptualized the Med-into-Grad Initiative and funded this program to make the training possible. In July 2012, I started my thesis with the mentorship of Serpil C. Erzurum, M.D., who has been instrumental in guiding my development, both in research and career. I sincerely thank Dr. Erzurum for providing many opportunities to grow inside and outside of lab, and for continuously encouraging me to explore and pursue my interests. I am very grateful to my thesis committee, composed of Kingman Strohl, M.D., Raed Dweik, M.D., Mitchell Drumm, Ph.D., George R. Stark, Ph.D. and Sathyamangla V. Naga Prasad, Ph.D. Throughout the years, they have provided valuable advice on research, writing and oral presentation skills, which serve as the foundation for my career. The inception of the project “hypoxia sensing through beta- adrenergic receptors” was based on the work of Weiling Xu, M.D. on vi pulmonary hypertension. I thank Weiling for orienting me in the lab in the very beginning and for training me different techniques, which enable me to take the project to the next level. Other lab members, including Adrianna Garchar, Olivia Stephens, Kewal Asosingh, Ph.D., Kimberly Queisser, Suzy Comhair, Ph.D. and Lori Mavrakis have been very supportive throughout my training. The high altitude adaptation project would not have been possible without the guidance of Cynthia Beall, Ph.D. and Allison Janocha. They were the people in the field in East Africa, and had shared valuable insights and experience with me. Lastly, there are sincere and true friendships cultivated throughout the years that made me strong and feel supported. Thank you very much, Marybeth Boyle, Francis Enane and Josephine Dermawan! vii List of Abbreviations HIF-1, Hypoxia Inducible Factor-1 βAR, beta-adrenergic receptors cGMP, cyclic guanosine monophosphate cAMP, cyclic adenosine monophosphate PKA, protein kinase A GRK, G protein-coupled receptor kinases - NO3 , nitrate NO, nitric oxide VHL, von Hippel-Lindau ODDD, oxygen-dependent degradation domain CAD, COOH-terminal transactivation domain RACK1, Receptors for activated C-kinase NOS, nitric oxide synthases 2,3-DPG, 2,3-diphosphoglyceric acid OSC, oxygen saturation curve viii Molecular and Physiological Responses to Hypoxia Abstract by HOI I (QUEENIE) CHEONG Life-sustaining responses to hypoxia rely on the transcription factor, Hypoxia Inducible Factor-1 (HIF-1). Under hypoxia, HIF-1 accumulates and regulates multifaceted cellular responses. However, many underlying mechanisms of HIF-1 regulation are incompletely understood. Previous studies suggest a link between HIF-1 and beta-adrenergic receptors (βAR). Here, we interrogated the role of βARs in hypoxia responses by β- blocker treatment of mice with hypoxia-inducible erythropoiesis. β-blocker suppressed renal accumulation of HIF-1α, erythropoietin production and the generation of erythroid progenitor cells. Likewise, β-blocker treatment of human endothelial cells attenuated HIF-1α accumulation and binding to target genes under hypoxia, and subsequent downstream gene expression. Consistently, β-agonist increased HIF-1α accumulation in a dose- and time-dependent manner, an effect that was blocked by both β1- and β2-blockers, indicating a general property of this receptor class. βAR signal transduction involves cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) and G protein-coupled receptor kinases (GRK). Direct activation of cAMP/ PKA pathways did not increase ix HIF-1α accumulation, and inhibition of PKA did not suppress HIF-1α by hypoxia. In contrast, pharmacological inhibition of GRK, or genetic mutation of βAR that impairs GRK phosphorylation, blocked hypoxia- mediated HIF-1α accumulation. Mass spectrometry analyses revealed a unique hypoxia βAR phosphorylation barcode different from the classical agonist. These findings identify an unknown role of βAR in hypoxia responses. Another determinant of HIF-1 regulation is nitric oxide, a potent vasodilator. A natural experiment of genetically similar Ethiopians at high altitude (>3000 m), the Amhara and Oromo, revealed a dampened hemoglobin response in Amhara compared to Oromo. We hypothesized that Amhara highlanders offset their dampened hemoglobin response with the vascular nitric oxide response. We identified high levels of urinary nitrate and its bioactive signal molecule cyclic guanosine monophosphate (cGMP) in high-altitude Amhara, but not Oromo. Consistently, high-altitude Amhara have lower diastolic blood pressure than Oromo, an indicator of vasomotor tone. Both Amhara and Oromo maintained the amount of oxyhemoglobin at high altitudes, but the high-altitude Oromo suffered a much higher deoxyhemoglobin level. In conclusion, high-altitude Amhara offset a dampened hemoglobin response with the vasodilatory nitric oxide, whereas the Oromo mount a bigger hemoglobin response at the cost of circulating deoxyhemoglobin. x Chapter 1 Introduction I. Hypoxia Hypoxia is low oxygen. There are two major ways of experiencing hypoxia, acute and chronic hypoxia exposure. An example of acute hypoxia is rapid ascent to high altitude. One can imagine mountaineers that climb the Mount Everest. An example of chronic hypoxia is lifelong residence at high altitude, such as the Tibetan highlanders.