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

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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

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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

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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

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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!

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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

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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.

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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. Both examples are, in common, hypobaric hypoxia ⎯ oxygen partial pressures drops with atmospheric barometric pressure at high altitude. This is in contrast with normobaric hypoxia, in which hypoxia is introduced by reducing oxygen concentration in air.

II. HIF-1 in Hypoxia Sensing

History of discovery

HIF-1 was first cloned in 1995 and identified to consist of heterodimers, alpha (HIF-1α) and beta (HIF-1β) subunits (Wang et al.).

The alpha subunit contains a basic-helix-loop-helix domain, two PAS domains and two transcriptional activation domains. The beta subunit is identical to the Aryl hydrocarbon receptor nuclear translocator. Two years after this discovery, HIF-1α was identified to be a member of the PAS superfamily 1, and its mRNA expression in human tissues was the highest

1

in the kidneys and heart (Hogenesch et al.). It is a highly conserved protein. The human and mouse protein sequences share 90% identity

(Wenger et al.).

Function

When oxygen is available, HIF-1 expression is low. However, when oxygen is limited (hypoxia), HIF-1 expression increases and regulates the transcription of genes to mediate adaptive responses such as erythropoiesis (Wang and Semenza; Maxwell, Pugh and Ratcliffe), cell survival (Bruick), angiogenesis (Forsythe et al.) and energy metabolism

(Firth, Ebert and Ratcliffe; Semenza "Hif-1 and Human Disease: One

Highly Involved Factor").

Mice with complete Hif1a genetic deficiency do not survive beyond midgestation. They exhibit cardiovascular deformities and myeloid cell loss

(Yu et al.). Mice heterozygous for Hif1a are viable. However, they show delayed onset of hemoglobin response, pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy compared with their wild-type counterparts when exposed to 10% oxygen for one to six weeks (Yu et al.). Interestingly, Hif1a heterozygotes maintain acute ventilatory responses to 3-day hypoxia via vagal afferents with loss of neural activity in carotid bodies, while the wild-type counterparts rely mainly on carotid bodies (Kline et al.).

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Regulation

The alpha subunit of the HIF-1 heterodimer, HIF-1α, is targeted for proteasomal degradation by direct interaction with the tumor suppressor gene product von Hippel-Lindau (VHL) in an oxygen-dependent manner

(Maxwell et al.). This interaction between VHL and HIF-1α is iron- dependent, and can be disrupted by iron chelation or cobaltous ions

(Maxwell et al.). Genetic ablation of VHL or the inhibition of the VHL-HIF-

1α interaction results in constitutive stabilization of HIF-1α and subsequent increased expression of angiogenic and glycolytic factors

(Maxwell et al.). The specific protein sequence of HIF-1α which binds VHL was identified to contain a hydroxylated proline residue, also known as the oxygen-dependent degradation domain (ODDD) (Ivan et al.; Jaakkola et al.). Consistently, the proline hydroxylation is both oxygen- and iron- dependent (Ivan et al.; Jaakkola et al.). In mammalian cells, isoforms of prolyl hydroxylase for HIF-1α were determined to be PHD1, PHD2 and

PHD3 (Epstein et al.). The abundance level of PHD1 and PHD3 is regulated by SIAH1 and SIAH2 (Nakayama et al.).

Another domain of HIF-1α, the COOH-terminal transactivation domain (CAD), could be induced under hypoxia by preventing hydroxylation of an asparagine within the domain (Lando et al.). Under normoxic conditions, asparagine hydroxylation prevents the binding of

HIF-1α, to its coactivator p300/ CBP (Lando et al.). Thus, HIF-1 is fully

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activated only when both proline and asparagine hydroxylation were abrogated within the ODDD and CAD domains under hypoxia (Lando et al.).

In addition to oxygen-dependent post-translational modification,

HIF-1α is also regulated in an oxygen-independent manner. Receptors for activated C-kinase (RACK1) competes with the heat shock protein HSP90 that stabilizes HIF-1α, and tags HIF-1α for ubiquitination and consequently proteasomal degradation (Y. V. Liu et al.).

III. β-Adrenergic Receptor (βAR) and Hypoxia Responses

βAR subtypes, expression, structure and history

βARs belong to the superfamily of G protein-coupled receptors that are characterized by seven hydrophobic transmembrane domains spanning the plasma membrane. Known βAR subtypes include β1AR,

β2AR and β3AR. β1AR is predominantly expressed in the heart, β2AR in the in the lung, vasculature, and kidneys, and β3AR in the adipose tissues

(Rockman, Koch and Lefkowitz). This dissertation will mainly focus on

β2AR due to its ubiquitous expression in the peripheral tissues.

Human β2AR was successfully cloned in 1987 and identified to be composed of 413 amino acid residues (Kobilka et al.). Human β2AR shared 87% protein sequence identify with the hamster β2AR. The regions that are most highly conserved are the hydrophobic transmembrane

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helical and the cytoplasmic domains. The ADRB2 gene that encodes for

β2AR is located on Chromosome 5 and contains no introns (Kobilka et al.;

Sheppard et al.). In the 5’ flanking region, the promoter region consists of mRNA cap site, TATA box, CAAT box, and GC-rich elements; in the 3’ flanking region, sequence homologous to the glucocorticoid-response elements was identified (Emorine et al.).

Ligand binding

β2AR bind the endogenous ligand epinephrine, which is both a hormone and a neurotransmitter. The site of β2AR accessible for ligand binding is located at the second extracellular loop (Cherezov et al.). The binding of ligand stabilizes the receptor in an active conformation

(Rosenbaum et al.), which alters the structure of the heterotrimic G protein

Gs (Chung et al.).

β2AR signaling

Agonist-activated β2AR are coupled to G proteins, which result in the dissociation of the alpha and beta-gamma subunits. The alpha (Gα) subunit mediates the release of cyclic adenosine monophosphate (cAMP) by adenylyl cylcase, which activates protein kinase A (PKA) (Daaka,

Luttrell and Lefkowitz). The beta-gamma (Gβγ) subunit recruits a protein complex that contains activated c-Src (a non-receptor tyrosine kinase

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often implicated in cancer), mediated via the binding of the adaptor protein beta-arrestin-1 (Luttrell et al.). The protein complex targets the receptor to clathrin-coated pits, resulting in the activation of the MAP kinases Erk1 and Erk2. One of the ultimate effector signals by β2AR is the class C L- type calcium channel CaV1.2, which regulates the release of hormone and neurotransmitter, muscle contraction and other cellular processes

(Amberg and Navedo). β2AR directly interacts with CaV1.2 in a signaling complex that comprises a G protein, adenylyl cyclase, PKA and the counterbalancing protein phosphatase 2A (PP2A) (Davare et al.).

Another consequence of β2AR stimulation with agonist is rapid ubiquitination of both the receptors and the regulatory protein beta-arrestin

2 (Shenoy, McDonald, et al.). Beta-arrestin ubiquitination leads to β2AR internalization and degradation, as demonstrated by genetic knockout of the ubiquitin ligase MDM2 (Shenoy, McDonald, et al.).

βAR function

Cardiovascular responses.

Adrb1 and Adrb2 genetically deficient mice are viable (Rohrer et al.). Analysis of Adrb1-/-, Adrb2-/- and Adrb1-/- Adrb2-/- mice demonstrated that cardiac chronotropy is mostly mediated by β1AR, while the vascular tone and metabolic rate are regulated by all βAR subtypes (Rohrer et al.).

The double knockout mice (Adrb1-/- Adrb2-/-) have blood pressure, cardiac

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function, and metabolic rate similar to wild-type controls at baseline.

However, stimulation with catecholamines or exercise revealed significant impaired chronotropy, oxygen consumption and carbon dioxide production in the double knockout mice (Adrb1-/- Adrb2-/-) (Rohrer et al.).

Bone remodeling.

The investigation of Adrb2 genetically deficient mice showed that

β2AR, as part of the sympathetic nervous system, promotes bone resorption, a process by which osteoclasts break down bone tissues and release the minerals to the blood (Elefteriou et al.). This process is mediated by increasing the expression of the osteoclast differentiation factor Rankl in osteoblast progenitor cells, and requires PKA phosphorylation of the osteoblast differentiation transcription factor ATF4.

Lipolysis.

With the use of subcutaneous infusion of βAR subtype-specific and non-specific agonists and the measurement of glycerol outflow, Barbe et al. showed that β1AR, β2AR and β3AR (to a lesser extent) participate in the regulation of lipid mobilization in human subcutaneous abdominal adipose tissues (Barbe et al.). In a clinical research study, the β2AR common polymorphism gln27 to glu is significantly associated with obesity in women (Large et al.). In an experimental model of high-fat diet, mice

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lacking Adrb1, Adrb2 and Adrb3 gained remarkable body weight and developed obesity compared to wild-type mice, despite similar food intake

(Bachman et al.).

βAR under hypoxia

In an experimental rat model of chronic hypoxia, five-week exposure of hypobaric pressure chamber at 450 Torr (comparable to 4250 m) led to the hypertrophy of the right ventricles and an increase in hematocrit (Voelkel et al.). βAR density, assessed by the binding of [125I] iodohydroxybenzylpindolol, markedly decreased in right ventricles, which could be blocked by daily injection of propranolol (Voelkel et al.). In a rodent experiment of acute hypoxia exposure of similar hypobaric pressure, tissue norepinephrine decreased after 7 days of hypoxia, and adenylate cyclase activity stimulated by isoproterenol in right ventricles decreased after 21 days (Kacimi et al.). Another study showed that chronic hypoxia diminished heart rate response to βAR stimulation by agonist (Maher et al.). These findings are in variance of a recent in vitro study, which showed that hypoxia limits the ubiquitination of βAR by prolyl hydroxylase, thus stabilizing βAR and increasing receptor density (Ward et al.).

A canine study of acute hypoxia in which dogs breathed in 7% oxygen for 7 minutes demonstrated that hypoxia activated βAR, which led

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to increased heart rate and cardiac output (Kontos and Lower). Such responses are independent of elevated circulating level of catecholamines, as evidenced by similar hypoxia responses after bilateral adrenalectomy

(Kontos and Lower).

βAR activation mediates vasodilation of the pulmonary and systemic vasculature (Burnett et al.). In an experimental rat model, seven days of hypobaric hypoxia exposure resulted in decreased βAR density in both pulmonary and systemic arteries (Bredt et al.). Adenylyl cyclase activity was significantly higher in systemic, but not pulmonary, arteries

(Bredt et al.). In another experiment, pre-treatment of rabbits with the β- blocker propranolol prior to eighteen hours of hypoxia exposure marked decreased erythropoietic responses (Thomas et al.). However, how and why βAR blockade suppressed erythropoietic responses were not explored. In Chapter 2, we describe experiments that tested the idea that

βAR plays an important role in hypoxia sensing, addressing the knowledge gap in the field.

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IV. Nitric Oxide, an Adaptive Response at High Altitude

Background

Nitric oxide was discovered by Furchgott, Zawadzki, Rapoport and

Murad (Geller et al.; Janssens et al.). Nitric oxide is a colorless gaseous molecule important for cellular signaling produced in many organisms, including plants, fungi, prokaryotes and eukaryotes (Lowenstein et al.).

Nitric oxide, also known as the endothelium-derived relaxing factor, is a potent vasodilator with a half-life of 3-5 seconds (Charles et al.). It increases the accumulation of cyclic guanosine monophosphate (cGMP).

It is inhibited by superoxide anion and oxyhemoglobin, and stabilized by superoxide dismutase (Charles et al.). Nitric oxide also reacts with hemoglobin to form nitrosylhemoglobin (Charles et al.).

Sources of nitric oxide

Endogenous production.

Nitric oxide is endogenously produced by nitric oxide synthases

(NOS), namely endothelial (NOS3), inducible (NOS2) and neuronal

(NOS1) nitric oxide synthases. They synthesize nitric oxide and L-

Citrulline (a co-product) from L-Arginine in the presence of oxygen and

NADPH (Marsden et al.). All NOS share similar requirements for NADPH,

FMN, and tetrahydrobiopterin. The endothelial and neuronal forms of NOS are constitutively active and dependent on calcium and calmodulin.

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However, NOS2 activity is induced and not dependent on calcium or calmodulin (Lowenstein et al.).

The neuronal form of nitric oxide synthase, NOS1 or nNOS, shares similar sequence identity to cytochrome P450 reductase (Bredt et al.). Its expression is high in mammalian skeletal muscles. It increases blood flow by inhibiting sympathetic vasoconstriction for muscle contraction (Thomas et al.). NOS1 also has a physiological role in regulating erectile function

(Burnett et al.). Under hypoxia, NOS1 protein expression increases in rat aorta, mesenteric arterioles, pulmonary arteries, brain and diaphragm, and in human aortic smooth muscle cells. It antagonizes vasoconstriction induced by hypoxia (Ward et al.).

NOS2, also known as iNOS, was originally cloned from macrophage in 1992 (Lowenstein et al.), and later in chrondrocytes

(Charles et al.) and hepatocytes (Geller et al.). The mRNA expression is markedly induced two to six hours after endotoxin exposure, but otherwise quiescent in macrophages or spleen (Lowenstein et al.). Likewise, NOS2 mRNA expression is induced twenty-fours hours after Interleukin-1 beta exposure in chrondrocytes (Charles et al.).

The endothelial form of nitric oxide synthase, NOS3, was cloned in the same year as NOS2 (Janssens et al.; Marsden et al.). NOS3, or eNOS, is directly phosphorylated and activated by the protein kinase AKT, resulting in the production of nitric oxide (Fulton et al.). In the vasculature,

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the local increases in blood flow creates a shear stress sensed by the endothelium that activates the Phosphoinositide 3-kinases and AKT, which in turn stimulates the production of nitric oxide (Dimmeler et al.).

The nitrate-nitrite-nitric oxide pathway.

This pathway elevates nitric oxide independent of NOS. Dietary

- nitrate (NO3 ) derived from plant-based food could increase nitric oxide level in the body. This is accomplished by sequential reduction of dietary

- - NO3 to NO2 by commensal bacteria in the oral cavity and gastrointestinal

- tract. NO2 can be further reduced for nitric oxide production in blood and tissues in the presence of acidity, ascorbate and polyphenols (Lundberg,

Weitzberg and Gladwin). Under hypoxia, this process is potentiated by xanthine oxidoreductase, deoxygenated myoglobin, enzymes of the mitochondrial chain and protons (Lundberg, Weitzberg and Gladwin). The

- cardiovascular protective property of dietary NO3 from beetroot juice was

- evidenced by elevated plasma level of NO2 and substantial decrease in blood pressure (Webb et al.).

Mechanism of Action

A well-established mechanism of action by nitric oxide is the activation of the soluble guanylate cyclase, which leads to cGMP production (Archer et al.). cGMP in turn activates protein kinase G, which

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increases calcium uptake and open calcium-activated potassium channel.

The lower calcium concentration impairs the phosphorylation of myosin by the myosin light-chain kinase, resulting in vasodilation (Archer et al.).

Nitric oxide also mediates post-translational modification of proteins, namely S-nitrosylation of thiols, and nitrosylation of transition metal ion, which alters protein functions (Hess et al.). A prominent example is hemoglobin. Oxygen induces the transfer of NO group that is bound to haem to thiol, which occurs in parallel when hemoglobin switches from the T state (deoxyhemoglobin) to the R state (oxyhemoglobin) (Gow and Stamler). When the R state hemoglobin from the lung reaches the systemic capillary bed, the allosteric transition to the T state triggers the release of oxygen and (S)NO release (Gow and Stamler). The transfer of

NO to glutathione and smooth muscles promotes vasodilation and oxygen delivery in local tissues. Hemoglobin S-nitrosylation has recently been shown to confer protection against myocardial infarction and heart failure

(Zhang et al.).

Nitric Oxide at High Altitude

Acute exposure to high-altitude could lead to the development of cerebral and pulmonary syndromes six to ten hours after rapid ascent to high altitude (> 2500 m) (Hackett and Roach). Other symptoms that are frequently observed include loss of appetite, insomnia, dizziness and

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fatigue (Hackett and Roach). Individuals susceptible to high-altitude pulmonary edema have been shown to have lower pulmonary nitric oxide production (Busch et al.). Inhalation of nitric oxide significantly relieves pulmonary vasoconstriction and improves arterial oxygenation (Scherrer et al.; Anand et al.)

Chronic exposure to high altitude, in some cases, allows long-term opportunities for adaptation. This is evidenced by the study of mountain dwellers Tibetans at 4200 m and Bolivian Aymara at 3900 m, whose exhaled nitric oxide is much higher than low-altitude individuals and is directly correlated with higher pulmonary blood flow (Beall, Laskowski, et al.; Hoit, Dalton, Erzurum, et al.).

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Chapter 2 Hypoxia Sensing through β-Adrenergic Receptors

Hoi I Cheong, Kewal Asosingh, Olivia R. Stephens, Kimberly A. Queisser,

Weiling Xu, Belinda Willard, Bo Hu, Josephine Kam Tai Dermawan,

George R. Stark, Sathyamangla V. Naga Prasad, Serpil C. Erzurum.

Published January 4, 2017

Citation Information: JCI Insight. 2017;1(21):e90240. doi:10.1172/jci.insight.90240.

Abstract

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by hypoxia-inducible factors (HIF), but the underlying mechanisms by which cells sense hypoxia are not completely understood.

Based on prior studies suggesting a link between the beta-adrenergic receptor (βAR) and hypoxia responses, we hypothesized that the βAR mediates hypoxia sensing and is necessary for HIF-1α accumulation. β- blocker treatment of mice suppressed hypoxia-induction of renal HIF-

1α accumulation, erythropoietin production and erythropoiesis in vivo.

Likewise, β-blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes, and the downstream hypoxia-inducible gene expression. In mechanistic studies, cyclic adenosine monophosphate (cAMP)-activated

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protein kinase A (PKA) and/or G protein-coupled receptor kinases (GRK), which both participate in βAR signal transduction, were investigated.

Direct activation of cAMP/ PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a

GRK phosphorylation-deficient βAR mutant in cells, blocked hypoxia- mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a unique hypoxia-mediated βAR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings identify that the βAR is fundamental to the molecular and physiological responses to hypoxia.

Introduction

Oxygen delivery to tissues is essential for life. Under hypoxia, adaptive responses take place over the course of hours and days, to generate erythrocytes and create new blood vessels. These responses have been identified as the downstream effects of Hypoxia-inducible factors (HIFs) (Y. Liu et al.; Semenza and Wang; Kapitsinou et al.; Beall,

Cavalleri, et al.). HIF-1 is a master transcription factor for oxygen sensing.

It is composed of an oxygen-regulated alpha (HIF-1α) subunit and a constitutively expressed beta subunit (HIF-1β) (Huang et al.; Jaakkola et al.; Wallace). Under hypoxia, HIF-1a is stabilized, dimerizes with HIF-1β

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and translocates to the nucleus to promote transcription of genes such as erythropoietin EPO and vascular endothelial growth factors VEGF, to orchestrate greater capacity of oxygen delivery to tissues (Y. Liu et al.).

However, the determinants of HIF-1α regulation are incompletely understood in the complex physiologic environment of cells (Semenza

"Hypoxia-Inducible Factors in Physiology and Medicine"; Cash, Pan and

Simon; Schumacker).

More than forty years ago, long before the discovery of HIF and erythropoietin, beta-adrenergic receptor (βAR) blockade was shown to diminish the erythropoietic response to hypoxia in animal models, but the mechanisms of effects have been unexplored (Thomas et al.; Zivny et al.).

Since that time, there has been a wealth of studies that have detailed the downstream signal transduction events from activation of the βARs. βARs belong to a large family of G protein-coupled receptors (GPCRs). βARs are ubiquitously expressed throughout the body, with β1AR as the major subtype expressed in the heart, and β2AR in the lung, vascular endothelium, and kidneys (Rockman, Koch and Lefkowitz; Hedberg,

Minneman and Molinoff; Krief et al.). When an agonist, such as the endogenous ligands noradrenaline and adrenaline, binds to βAR, the receptor is stabilized in an active conformation, and couples to the heterotrimeric G proteins, resulting in dissociation of Gα and Gβγ subunits.

These molecular events rapidly propagate complex intracellular signals.

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For instance, the dissociation of Gα subunit initiates the release of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA)

(Daaka, Luttrell and Lefkowitz), while Gβγ subunits recruit GPCR kinases

(GRKs). Although both PKA and GRK participate in βAR phosphorylation, the level of phosphorylation at distinct PKA and GRK sites depends on the type and concentrations of agonists (Hausdorff et al.).

Phosphorylation ultimately diminishes βAR responsiveness to agonist stimulation, also known as receptor desensitization, and modulates receptor trafficking and turnover. The diminished responsiveness to an overload of noradrenaline is the cardinal feature of congestive heart failure (Lefkowitz, Rockman and Koch; Thomas and

Marks; Bristow, Ginsburg, Minobe, et al.). Effective therapies with β- blockers, such as carvedilol, have resulted in this class of drug being widely prescribed for left and right heart failure (Bristow, Gilbert, et al.;

Quaife et al.). Elevation of HIF-1α levels is also implicated in the pathophysiology of the failing heart (Moslehi et al.). We hypothesized that the βAR is part of the pathway for hypoxia sensing and necessary for HIF-

1α accumulation. We tested the hypothesis using in vivo models, genomic, pharmacologic and molecular approaches, and detailed barcoding of the phosphorylation status of the βAR.

18

Results

Decreased hypoxia responses in mice with β-blocker treatment. To determine whether βAR blockade prevents hypoxia-mediated erythropoietic responses via HIF-1α in vivo, mice were orally administered vehicle or the β-blocker carvedilol, followed by exposure to hypoxia (10% oxygen) (Figure 1A). Under hypoxia, HIF-1α accumulated in kidneys, and was significantly decreased by carvedilol (Figure 1B). In addition, HIF-1α- specific binding to the Epo gene increased, and was blocked by carvedilol

(Figure 1C). Moreover, hypoxia increased erythropoietin mRNA and protein (Cahan et al.), both of which were suppressed by βAR blockade

(Figure 1, D and E). Since erythropoietin is known to induce the proliferation of erythroid progenitor cells (Malik et al.), we quantified the percentages of proerythroblasts, basophilic and polychromatic erythroblasts in the bone marrow with flow cytometry (J. Liu et al.). Under hypoxia, the erythroid progenitor cell population increased by 35%, and the effect was blunted by carvedilol (Figure 1F). These findings indicate that βAR blockade by carvedilol suppresses the classical hypoxia erythropoietic responses through HIF-1α.

Decreased HIF responses in hypoxic cells with β-blocker treatment.

Next, we tested the in vitro effects of βAR blockade on HIF-1α accumulation in hypoxic human endothelial cells isolated from umbilical cord veins (HUVEC). HUVEC were exposed to escalating doses of

19

propranolol, and then exposed to hypoxia (2% oxygen). Western blot analyses of nuclear proteins revealed that hypoxia increased HIF-1a levels when compared to normoxia, and propranolol dose-dependently decreased HIF-1α (Figure 2A). To confirm βAR blockade inhibitory effects on HIF-1α, chromatin immunoprecipitation (ChIP) studies were performed to test whether βAR blockade decreases the binding of HIF-1α to the promoters of its classical target genes, like chemokine stromal cell– derived factor-1 (SDF-1 or CXCL12), hexokinase 2 (HK2) and vascular endothelial growth factor A (VEGFA). Quantitative polymerase chain reaction revealed that hypoxia significantly increased HIF-1α-specific binding to promoter sequences, an effect that was blocked by propranolol

(Figure 2B).

To evaluate the entire spectrum of signaling pathways impacted by

β-blockers under hypoxia, we performed transcriptome-profiling studies that tested 47,000 probes representing well-characterized genes, gene candidates, and splice variants. HUVEC were exposed to diluent or propranolol alone, and then exposed to 24-hour hypoxia (accessible at

GEO repository GSE86793). Hypoxia induced changes in RNA expression levels relative to normoxia, corresponding to 1247 probes, 434 of which were reversed by propranolol (at a false discovery rate of 0.05) (Figure

2C; Supplemental Table 1). Pathway analyses and functional annotation clustering of the identified transcripts revealed the biological significance

20

of βAR in regulating hypoxia-inducible phenotypes including focal adhesion, glycolysis, p53 signaling pathways, angiogenesis, and cell migration (Figure 2D; Supplemental Table 2) (Huang da, Sherman and

Lempicki "Systematic and Integrative Analysis of Large Gene Lists Using

David Bioinformatics Resources"; Huang da, Sherman and Lempicki

"Bioinformatics Enrichment Tools: Paths toward the Comprehensive

Functional Analysis of Large Gene Lists").

β-Agonist increases HIF-1α accumulation under normoxia. To investigate if βAR activation can increase HIF-1α levels, dose-response and time-course studies with the β-agonist isoproterenol were performed in HUVEC under normoxia. Isoproterenol increased nuclear HIF-1α in a dose- and time-dependent manner, with a maximum response at 2-hours

(Figure 3, A and B). To test the possibility that stabilization of HIF-1α is mediated via a specific βAR subtype 1 or 2 (β1AR or β2AR), HUVEC were exposed to CGP-20712A (a β1-blocker) or ICI-118551 (a β2-blocker) followed by isoproterenol stimulation. Isoproterenol stabilization of HIF-1α was dose-dependently decreased by both β1- and β2- blockers (Figure

3C). This suggests that isoproterenol stabilization of HIF-1α can be mediated by β1AR or β2AR, and excludes off-target effects.

HIF-1α accumulation under hypoxia relies on GRK phosphorylation of βAR. To establish the mechanistic link between βAR and HIF-1α, we investigated classical downstream signal pathways of βARs. Agonist

21

binding to βAR stabilizes the receptor in an active conformation, and leads to coupling to the heterotrimeric G proteins. This results in production of cyclic adenosine monophosphate (cAMP) by adenylate cyclase that activates protein kinase A (PKA) which, in turn, phosphorylates the βAR

(Rockman, Koch and Lefkowitz). To test if hypoxia activates the βAR canonical downstream signal transduction, we measured intracellular cAMP level in HUVEC exposed to hypoxia (2% oxygen) for 10-15 minutes.

Similar to isoproterenol, forskolin (which activates adenylate cyclase) and dibutyryl (a cAMP analog), hypoxia increased cAMP levels (Figure 4A).

However, forskolin and dibutyryl did not lead to HIF-1α accumulation

(Figure 4B). Further, PKA inhibition H89 did not affect HIF-1α accumulation by isoproterenol (Figure 4B). Thus, hypoxia can activate

βAR signaling, but activation of the PKA/ cAMP does not lead to HIF-1α accumulation.

G-protein-coupled receptor kinases (GRKs) are the other classical pathway known to phosphorylate βARs following receptor activation

(Rockman, Koch and Lefkowitz; Luttrell et al.; Rapacciuolo et al.;

Belmonte and Blaxall). Evaluation of GRK phosphorylation pathway using pharmacological inhibition of GRK2 in HUVECs revealed dose-dependent decrease in β-agonist mediated HIF-1α accumulation (Figure 4C).

Likewise, GRK inhibition, but not PKA, significantly attenuated hypoxia- induced HIF-1α levels (Figure 4D). The results suggest that hypoxia-

22

mediated HIF-1α accumulation is dependent on GRK phosphorylation of

βAR. To test this idea, we evaluated the hypoxia response of human embryonic kidney cells (HEK293), which overexpress wild-type β1AR

(WTβ1AR) or mutant β1ARs lacking either the phosphorylation target sites

− − of PKA (PKA β1AR) or GRK (GRK β1AR) (Figure 4E) (Rapacciuolo et al.).

− Hypoxia significantly induced HIF-1α levels in WTβ1AR and PKA β1AR

− cells but not in GRK β1AR cells (Figure 4F). These results indicate that

HIF-1α accumulation occurs in response to hypoxia in a manner that requires βAR-GRK signal transduction.

Hypoxia-specific βAR phosphorylation barcode. Because agonist binding to bAR results in unique receptor phosphorylation patterns that correlate with distinct downstream signals (Nobles et al.), we investigated whether hypoxia imparts a specific receptor phosphorylation signature, i.e. a unique barcode. HEK293 overexpressing β2AR, were exposed to the b- agonist isoproterenol (10 µM) or hypoxia (2% oxygen). β2ARs from the plasma membranes were enriched via binding to alprenolol and subjected to mass spectrometry analyses, which successfully identified nine key phosphorylation sites of the receptor (Table 1; Supplemental Table 3).

Similar to canonical phosphorylation seen with isoproterenol, hypoxia

246 decreased β2AR phosphorylation at Ser by 2-fold and increased phosphorylation at Ser396 by 6.7-fold (Table 1; Figure 5, A and B). Ser246 on β2AR is known to be phosphorylated by ataxia telangiectasia mutated

23

protein kinase (ATM) and Ser396 by GRK2. In contrast to isoproterenol,

261 262 hypoxia did not alter β2AR phosphorylation at Ser and Ser , which are known PKA sites (Figure 5, C and D). Although isoproterenol and hypoxia increased Ser355 and Ser356 phosphorylation, the level of increase was substantially different (hypoxia 1.6-fold versus isoproterenol 153-fold)

(Figure 5, E and F). These findings are consistent with western blot analysis of protein extracts from hypoxic HUVEC. Hypoxia increased

Ser355 and Ser356 phosphorylation, an effect that was blunted by GRK inhibition (Figure 5, G and H). Altogether, these findings identify a distinct hypoxia-specific β2AR phosphorylation signature, which provides a definitive biochemical link between hypoxia sensing and activation of the

βAR (Figure 5I).

24

Figures

A B 21% O2 10% O2 C 21% O2 Kidneys 10% O β-blocker 2 HIF-1 α 3 * Oral gavage vehicle/ β-blocker Lamin B * t ) r 0 1 2 3 4 5 6 7 n e e

Kidneys c m

n 2 h 10 a c i Time (Day) 21% or 10% O * h r

2 n n e e B o d n l p i 1 o

2 hour or 24 hour: E m ( F • HIF-1 in kidneys a α L • Erythropoietin in kidneys 5 /

and serum α 0 1 • Erythroid progenitor cells - β-blocker F I

in bone marrow H IgG HIF-1α Ab 0 β-blocker

D Kidneys E Serum F Bone marrow * 8 * * 30 ** 1000 s

TER 119+ TER 119+ l

A * ** l ) ** e N L III II c R E m I C m I P I /

* T - - I g I 9 n F p i 4 - 20 1 t ( e 1 c I 4 g 4 A O 500 a D t R / P o S C E E p T E % m u

r CD44-FITC FSC-A

0 e 10 β-blocker S β-blocker 0 β-blocker

Figure 1. β-Blocker blunts HIF-mediated erythropoiesis under hypoxia in vivo. (A) Mice orally administered vehicle or carvedilol (β- blocker), followed by exposure to 21% or 10% oxygen. Kidneys and serum were harvested after 2 hours, and bone marrow was collected at 24 hour

(n = 2-5/ group). (B) Expression of HIF-1α and Lamin B in nuclear extracts of kidneys, detected by Western blot. (C) HIF-1α occupancy at the erythropoietin gene Epo, detected by chromatin immunoprecipitation with control IgG or HIF-1α Ab and quantitative PCR. Fold enrichment was

Figure 1 percent input (treatment) divided by average percent input (normoxia). (D)

Expression of Epo and Actin messenger RNA in kidneys, detected by reverse transcription- quantitative PCR. (E) Serum erythropoietin by

25

enzyme-linked immunosorbent assay. (F) Flow cytometry for erythroid progenitor populations. Proerythroblasts (I) defined by CD44hiTER119lo and basophilic (II) and polychromatic erythroblasts (III) defined by

TER119hi as well as size and CD44 expression. Data are mean ± SD.

***P<0.0005; **P<0.005; *P<0.05. Student’s t test.

IgG 21% O A B 2 100 2% O 2 β-blocker 2% O + -blocker (100 M) t 2 β µ n 0 ( M) r 0 µ e 0 0 e

0 0 3 1 3 1 t m o h m c 50 HIF-1 i

α o r r n p e n

Lamin B d l o o F 21% O 2% O 0 2 2 CXCL12 HK2 VEGFA 120 * HIF-1α Ab B g

n 100 i n * i n m i t a a n L 60 r / e m e t e α m o 1 R h - m c 50 F i I o r % r n H p e

0 n d l o

0 3 10 30 100 o F β-blocker (µM) 0 CXCL12 HK2 VEGFA

C 2% O2 D β-blocker (100 µM) Focal adhesion ECM-receptor interaction Small cell lung cancer Glycolysis/ gluconeogenesis NOD-like receptor signaling p53 signaling pathway Dilated cardiomyopathy

Glutathione metabolism Log [fold change 2 Arginine and proline 21% O ] relative to 2 metabolism -1 0 1 2 0 1 2 3 4 Enrichment score

Figure 2. β-Blocker attenuates hypoxia responses in vitro. (A)

Expression of HIF-1α and Lamin B in HUVEC, treated with escalating doses of propranolol, followed by 5-hour hypoxia (2% oxygen) (n = 2-7/ condition). Data are mean ± SEM. *P<0.05. Student’s t test. (B) HIF-1α occupancy at target genes in HUVEC treated with diluent or propranolol,

26 Figure 2

followed by 12-hour hypoxia. Chromatin immunoprecipitation with control

IgG or HIF-1α Ab and quantification of promoter regions of CXCL12, HK2 and VEGFA by quantitative PCR. Fold enrichment was percent input

(hypoxia with or without β-blocker) divided by average percent input

(normoxia) (n = 3). *P<0.05. ANOVA. (C) Heat map showing the levels of differentially expressed transcripts of HUVEC treated with diluent (-) or propranolol (+), followed by 24-hour hypoxia. The levels of expression relative to normoxia are represented on a continuous scale from blue

(lowest) to pink (highest) (n = 5 biological replicates), FDR=0.05. (D) Kyoto

Encyclopedia of Genes and Genomes (KEGG) pathway analysis of transcripts reversed by propranolol under hypoxia in HUVEC. Enrichment score is –log [P-value].

27

A B β-Agonist .5 4 4 0 0 2 4 1 2 (h) Duration 0 0 0 3 0 0 0 (µM) 0 3 1 3 9 HIF-1α HIF-1α Lamin B Lamin B 10 3 *** * B n B i n 2 m i a m L / a 5 L α / 1 1 - α F 1 I - H F I

H 0 0 -1 0 .5 2 4 4 4 0 1 2 0 3 0 0 0 β-Agonist 3 0 0 0 Duration of β-agonist 1 3 9 (µM) treatment (h)

C β1-blocker β2-blocker 150 ** -blocker 0 0 β1 0 0 0 0 0 0 0 0 0 0 (nM) 0 0 1 1 1 1 1 1 1 1 B β2-blocker g n i n 100 i n HIF-1 m α i a a L / m e Lamin B α 1 R - 50 F I β-Agonist % H

0 0 1 10 100 1000 Concentration (nM)

Figure 3. β-Agonist promotes HIF-1α accumulation under normoxia.

(A) Expression of HIF-1α and Lamin B in HUVEC treated with diluent or isoproterenol (33-900 µM) for 2 hours. (n = 3-6/ condition). Data are mean

± SEM. (B) Expression of HIF-1α and Lamin B in HUVEC, treated with

300 µM isoproterenol from 0.5 to 24 hours (n = 3-4/ condition). Data are mean ± SD. (C) Expression of HIF-1α and Lamin B in HUVEC, treated with β1-blocker CGP-20712A or β2-blocker ICI-118551 (1-1000 nM) for 45 minutes followed by 2-hour isoproterenol (n = 3). Data are mean ± SD. FIGURE 1 ***P<0.0005; **P<0.005; *P<0.05; ANOVA.

28

GRK inhibitor A B HIF-1α C

* 5 Lamin B 5 2 ( M) 0 0 0 1 5 2 1 µ 2.5 * * 21% O2 30 *** HIF-1α 2.0 2% O ** ) * 2 Lamin B

P ** B M n 1.5 i 20 A β-Agonist c m ( a g

L 120 o 1.0 / * L α 1 - 10 ) F B 0.5 I g n H i n

i 90 n m i a 0.0 a L / 0 m e forskolin α 1 β-agonist R - 60 F

dbcAMP I PKA inhibitor % H β-agonist forskolin ( dbcAMP 30 CoCl2 0 1 5 25 125 GRK inhibitor (µM)

R R D 21% O 2% O E F R - A - A 2 2 A β β 1 β AR Tβ 1 A 1 K 1 W PK GR GRK inhibitor PKA inhibitor 21 2 21 2 21 2 (% O2) HIF-1α HIF-1α

Lamin B PKA Lamin B 2 S S 4 S S/A 21% O S * * 2 T S B B 2% O 21% O

n * n 2 T * 2 i i ** S m m T 2% O a a T GRK 2 L L 1 T 2 T / / α

α S/A 1 1 S - -

T/A F I F

I S H

H S S S S 0 0 R R R GRK inhibitor A - A - A Tβ 1 β 1 β 1 PKA inhibitor W A K PK GR

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). (A) Intracellular cAMP in

HUVEC 10-15 minutes after stimulation with factors shown (n = 3-6). (B)

Expression of HIF-1α and Lamin B in HUVEC treated with 300 µM β- agonist isoproterenol with or without 10 µM PKA inhibitor H89, or forskolin, dibutyrylFigure 4 cAMP (dbcAMP) for 2 hours. Cobalt Chloride (CoCl2) shown as positive control (n = 3). (C) Expression of HIF-1α and Lamin B in HUVEC exposed to GRK inhibitor (1-125 µM) for 45 minutes followed by 2-hour isoproterenol (n = 2). (D) Expression of HIF-1α and Lamin B in HUVEC

29

exposed to a GRK (125 µM) or protein kinase A (PKA) inhibitor H89 (10

µM), followed by 5-hour hypoxia (2% oxygen) (n=3). (E) Map of β1AR

Serine and Threonine phosphorylation sites by PKA and GRK, mutated to

Alanine. (F) Expression of HIF-1α and Lamin B in human embryonic kidney cells (HEK293), overexpressing β1AR (WTβ1AR) or mutants lacking

− − PKA (PKA β1AR) or GRK (GRK β1AR) sites as shown in (E), exposed to normoxia or 5-hour hypoxia (n = 3). Data are mean ± SD; Student’s t test.

***P<0.0005; **P<0.005; *P<0.05.

30

A B S246 100 12 21% O +2 2 [M+2H] - H PO 3 4 pS QVEQDGR 2% O FHVQNL n 2 o i

80 t a e l c y r n o a h

d 60 6 p n s u o b h a

40 P e v i % t a l

e 20

R 0 β-agonist 0 200 600 1000 1400 m/ z C D S261, S262 2.5 100 +2 [M+2H] - H PO 3 4 n o 2.0 i +3 t

80 [M+3H] - 2H PO a l e 3 4 R pS pS KFCLK y c r

n 1.5 o a h d

60 p n s u

o 1.0 b h a

40 P e v % i 0.5 t a l 20 e

R 0 0 β-agonist 300 500 700 900 1100 m/ z E F S355, S356 100 16 +3 [M+3H] - H PO 3 4

AYGNGY pS pS NGNTGEQSGYHVEQEK n o

80 i e t c a l n y a r d 60 o n +3 h 0.4 u [M+3H] - 2H PO p b

3 4 s a 40 o h e P v i t a % l 20 e R 0 0 400 800 1200 1600 2000 β-agonist m/ z

0.4 Hypoxia-specific β AR barcode G H I 2

21% O2 2% O2 R A

GRK inhibitor 2 β / 0.2 pβ AR R 2 A 2 β

p S355 β AR S246 2 S356

0.0 S396 GRK inhibitor

Figure 5. Hypoxia induces a unique beta-adrenergic receptor (βAR) phosphorylation barcode in the absence of agonist binding. Human Figure 5 embryonic kidney cells (HEK293), overexpressing β2AR were exposed to

β-agonist isoproterenol or 5-hour hypoxia. β2AR were enriched by alprenolol for quantitative mass spectrometry analysis of phosphorylated

31

peptides. 21% and 2% O2 (n = 3); β-agonist (n = 2). (A) Spectrum for the pS246-containing peptide. The mass difference between the y7 and y8 ions is consistent with phosphorylation at S246. (C) Spectrum for the pS261 and pS262-containing peptide. The masses of the y5, y6, and y7 ions are consistent with phosphorylation at S261 and S262. (E) Spectrum for the pS355 and pS356-containing peptide. The masses of the y16, y17, and y18 ions are consistent with phosphorylation at S355 and S356. (B, D, F) Dot plots showing abundance of each phosphorylated peptide at 21% or 2% oxygen, or with β-agonist. (G, H) Expression of phosphorylated β2AR at

S355/ S356 and total β2AR in HUVEC with 21% or 2% oxygen (vehicle or

GRK inhibitor at 125 µM) (n=2). (I) Hypoxia-specific βAR phosphorylation barcode with increased (pink) and decreased (blue) phosphorylation at unique sites.

32

Hypoxia

βAR

GRK

Nuclear translocation Gene transcription

Nucleus HIF-1

Figure 6. Working Model. Hypoxia induces a unique beta-adrenergic receptor (βAR) phosphorylation barcode that is G protein-coupled receptor kinase (GRK)-dependent, which drives hypoxia-inducible factor-1 alpha

(HIF-1α) accumulation.

33

Tables

Table 1. Quantitative analyses of β2AR phosphorylation sites

Site Enzyme Peptide Fold changeΨ

2% O2 ISO

S246 ATM/R FHVQNLpSQVEQDGR ↓ 2.0 ↓ 1.7

S262 PKA RSpSKFCLK NC NC

S261+S262 PKA RpSpSKFCLK NC ↑ 4.9

S346 PKA RRSpSLKAYGNGYSSNGNTGEQSGYHVE NC NC

QEK S355,S356 GRK6 AYGNGYpSpSNGNTGEQSGYHVEQEK ↑ 1.6 ↑153

S355,S356,T36 GRK2/6 AYGNGYpSpSNGNpTGEQSGYHVEQEK NI ISO only

S355,S356,S360 GRK2/6 AYGNGYpSpSNGNTGEQpSGYHVEQEK NI ISO only

S3964 GRK2 LLCEDLPGTEDFVGHQGTVPpSDNIDSQG ↑ 6.7 ↑ 6.7

R¶

Ψ Fold change was normalized to untreated 21% O2 control. β2AR enriched from HEK293 β2AR with alprenolol were digested with protease, followed by liquid chromatography- tandem mass spectrometry analysis. 21% and

2% O2, n=3; ISO, n=2.

ISO, 10 µM isoproterenol; NC, no change; NI, not identified

¶ Identified in two of three independent experiments.

Hypoxia-related phosphorylation sites in bold

34

Supplemental Tables

Supplemental Table 1. Examples of hypoxia-inducible genes, whose expression levels are reversed by propranolol

Illumina ID Gene Gene Name

Symbol

ILMN_2135798 NR2C2AP nuclear receptor 2C2-associated protein

ILMN_1733863 Fam100a family with sequence similarity 100, member A

ILMN_2325168 ARRB1 arrestin, beta 1

ILMN_1705753 C3orf26 chromosome 3 open reading frame 26

ILMN_1704571 FAM53B family with sequence similarity 53, member B

ILMN_1686664 MT2A metallothionein 2A

ILMN_1776147 C21orf59 chromosome 21 open reading frame 59

ILMN_1660554 VWA1 von Willebrand factor A domain containing 1

ILMN_2409642 Tro trophinin

ILMN_1775823 POFUT2 protein O-fucosyltransferase 2

ILMN_1766264 PI16 peptidase inhibitor 16

ILMN_2205350 LOC100131 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,

149 assembly factor 4; similar to HSPC125

ILMN_2205350 Ndufaf4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,

assembly factor 4; similar to HSPC125

ILMN_1698020 DLC1 deleted in liver cancer 1

ILMN_1803772 POLD4 polymerase (DNA-directed), delta 4

ILMN_1702363 Sulf1 sulfatase 1

ILMN_2391150 FILIP1L filamin A interacting protein 1-like

ILMN_2126832 Sec24a SEC24 family, member A (S. cerevisiae)

ILMN_1767542 THAP10 THAP domain containing 10

ILMN_2133799 Acat2 acetyl-Coenzyme A acetyltransferase 2

ILMN_1682015 GAL galanin prepropeptide

35

ILMN_1776157 SEPT4 septin 4

ILMN_1705814 KRT80 keratin 80

ILMN_1743402 Six4 SIX homeobox 4

ILMN_1844593 MST131 MSTP131

ILMN_2104356 COL1A2 collagen, type I, alpha 2

ILMN_1675612 BLCAP bladder cancer associated protein

ILMN_1779257 CD40 CD40 molecule, TNF receptor superfamily member 5

ILMN_1755711 C17orf68 chromosome 17 open reading frame 68

Supplemental Table 2. KEGG pathway analysis of transcripts reversed by propranolol under hypoxia

KEGG Pathway Count -log(P- P-Value Genes

Term Value)

Focal adhesion 16 3.79 1.62E-04 ACTB, COL4A2, COL3A1, MYLK2,

CAPN2, BIRC2, MYL9, ACTG1,

CCND1, LAMA3, ITGA6, CCND2,

ITGAV, COL1A2, PDGFC, THBS1

ECM-receptor 8 2.27 0.005363926 COL4A2, LAMA3, ITGA6, ITGAV,

interaction COL3A1, COL1A2, THBS1, SDC2

Small cell lung cancer 8 2.27 0.005363926 CCND1, COL4A2, LAMA3, ITGA6,

ITGAV, RARB, BIRC2, MYC

Glycolysis / 6 1.75 0.017959703 TPI1, LDHA, LOC100133042,

Gluconeogenesis PFKP, BPGM, GAPDH, GAPDHL6,

ALDH3A2, TPI1P1

NOD-like receptor 6 1.69 0.020440137 IL8, MAPK14, CXCL2, RIPK2,

signaling pathway TNFAIP3, BIRC2

p53 signaling pathway 6 1.53 0.029192106 CCND1, CCND2, GADD45G,

GADD45B, THBS1, IGFBP3

36

Dilated 7 1.53 0.029586555 ACTG1, ACTB, ADCY4, ITGA6,

cardiomyopathy ITGAV, TPM1, TGFB2

Glutathione 5 1.42 0.037989004 ODC1, G6PD, GGCT, IDH1,

metabolism MGST1

Arginine and proline 5 1.34 0.045586458 ODC1, ASS1, NAGS, AMD1,

metabolism ALDH3A2

Hypertrophic 6 1.18 0.065483522 ACTG1, ACTB, ITGA6, ITGAV,

cardiomyopathy TPM1, TGFB2

(HCM)

Vascular smooth 7 1.18 0.066068909 KCNMA1, ADCY4, CALD1, MYLK2,

muscle contraction PLCB1, ITPR3, MYL9

hsa05219:Bladder 4 1.04 0.091190186 CCND1, IL8, THBS1, MYC

cancer

Regulation of actin 10 1.01 0.097991534 ACTG1, ACTB, ITGA6, ITGAV,

cytoskeleton SSH2, MYLK2, RRAS, PDGFC,

MYH9, MYL9

Cell cycle 7 1.00 0.099667653 CCND1, CCND2, GADD45G,

PCNA, GADD45B, MYC, TGFB2

Supplemental Table 3. Percent phosphorylation of β2AR sites at 21% or

2% oxygen, or with isoproterenol (ISO)

Site Peptide % Phosphorylated Fold change

21% O2 2% O2 ISO 2% ISO

O S246 FHVQNLpSQVEQDGR 8±2.3 4.0±1.6 4.6±1.7 ↓ 2.02 ↓ 1.7

S262 RSpSKFCLK 99±0 98±0.6 98±1.4 NC NC

S261+S262 RpSpSKFCLK 0.45±0.2 0.53±0.19 1.6±0.42 NC á 4.9

9 S346 RRSpSLKAYGNGYSSNGNTGE 1.02±0.7 1.53±1.72 0.65±0.9 NC NC

QSGYHVEQEK 8 2

37

S355,S356 AYGNGYpSpSNGNTGEQSGYH 0.098±0. 0.16±0.04 15±0.6 á 1.6 á153

VEQEK 003

S355,S356, AYGNGYpSpSNGNpTGEQSGY 0 0 0.39±0.2 NI ISO

T360 HVEQEK 5 only

S355,S356, AYGNGYpSpSNGNTGEQpSGY 0 0 0.079±0. NI ISO

S364 HVEQEK 006 only

S396 LLCEDLPGTEDFVGHQGTVPpS 0.045±0. 0.3±0.2 0.3 á6.7 á6.7

DNIDSQGR¶ 064

NC, no change; NI, not identified

¶ Identified in two of three independent experiments.

Data are mean±S.D.

Discussion

Physiologic responses are rapidly initiated within minutes of hypoxia. Catecholamines activate the β1AR in the cardiac muscles resulting in increased cardiac output (Adachi et al.; Calbet; Hansen and

Sander), while the peripheral vasculature dilates to increase the perfusion of blood to oxygen-limited tissues (Michiels). Secondary to these immediate events, adaptive responses take place over the course of hours and days, to generate erythrocytes and create new blood vessels in order to provide greater oxygen delivery to tissues. These late responses have been identified as the downstream effects of hypoxia inducible factors (Y.

Liu et al.; Semenza and Wang). HIF-1 is the primary intracellular mediator of the myriad physiological processes for adaptation. This study provides a biochemical connection between the catecholamine pathway and

38

hypoxia responses. Here, we identify that hypoxia, like catecholamine agonists, can activate the βAR and lead to HIF accumulation and adaptive responses. Prior work has suggested a close relationship between HIF and βAR, i.e. both are hydroxylated by prolyl hydroxylase 3 under hypoxia

(Ward et al.). However, to our knowledge, this is the first connection of hypoxia to activation of βAR.

In early studies of β-blockers in the 1970’s, intraperitoneal injection of rabbits with the subtype-nonspecific β-blocker propranolol prior to eighteen-hour exposure to hypobaric hypoxia, resulted in decreased erythropoietic responses. In another study, rats pre-injected with metipranolol (another subtype-nonspecific β-blocker) had less erythropoiesis (Zivny et al.). There were some discrepant findings based on type of β-blocker used, but the studies suggested the possibility that

βAR may regulate hypoxia-inducible erythropoiesis. Many years after these studies, Semenza et al. identified HIF-1 in the molecular mechanisms that lead to production of erythropoietin, and the subsequent erythropoiesis associated with hypoxia (Semenza and Wang). However, the effect of βAR on HIF-1 accumulation under hypoxia has been untested until now.

In this study, mice were orally administered the readily absorbed b- blocker carvedilol, which has a longer half-life (six to ten hours) as compared to propranolol (three to six hours) (De et al.). The mice given

39

carvedilol had lower levels of renal HIF-1α and consequently less binding of the transcription factor to Epo in vivo. Messenger RNA of erythropoietin in the kidneys, the principal site of production, and serum erythropoietin levels were significantly lower with carvedilol treatment. Consistent with these results, mice with carvedilol had less numbers of erythroid progenitor cells in the bone marrow. Importantly, β-blocker decreased HIF-

1α levels and its binding to target genes, and reversed ~30% (equivalent to 434 identified transcripts) of the hypoxia-inducible changes in expression. These transcripts mapped to known signal transduction pathways that contribute to cancer biology and dilated cardiomyopathy. In this context, HIF-1a levels are increased in heart tissues obtained from patients with ischemic cardiomyopathy (Moslehi et al.). It is interesting to speculate that benefits of β-blockers in heart failure may be related to suppression of HIF-1α.

To define the molecular mechanisms that link βAR and HIF-1α, the cAMP-PKA pathway was evaluated using pharmacological approaches and genetic mutants. Under normoxia, simply increasing the intracellular cAMP levels in cells could not cause HIF-1α accumulation. These findings are slightly in variance from a recent study, which suggests that isoproterenol can increase HIF-1α protein levels (Bullen et al.). In that study, treatment of human cardiomyocytes with the beta-agonist isoproterenol, or cervical cancer cells with forskolin, increased HIF-1α

40

protein abundance, but forskolin did not increase HIF-1α transcriptional activity (Bullen et al.). In this study, pharmacological inhibition of PKA in primary human endothelial cells, or the expression of a PKA phosphorylation-deficient βAR mutant in human kidney embryonic cells, does not alter hypoxia-mediated HIF-1α accumulation. This is consistent with the prior study where a stable knockdown of the catalytic subunit of

PKA did not decrease hypoxic accumulation of HIF-1α (Bullen et al.).

Unlike PKA, pharmacological inhibition of GRK, or genetic mutation of the

βAR that impairs GRK phosphorylation, profoundly decreased hypoxia- mediated HIF-1α accumulation in this study. These results support the idea that hypoxia-mediated HIF-1α accumulation is dependent on GRK phosphorylation of βAR. In a study by Baloğlu, et al., pre-exposure to 24 hours of hypoxia decreased β-agonist induction of cAMP in alveolar epithelial cells (Baloglu et al.). Here, 10-minute hypoxia exposure of human endothelial cells, rapidly increased cAMP. The findings suggest that the βAR under hypoxia produces higher basal levels of cAMP, but is overall less responsive to agonist stimulation. Another possibility for the different findings in this study is that cAMP responses to hypoxia are dependent on specific cell types and/ or times of exposure.

Carvedilol and propranolol were both employed in experiments performed in this study, and distinctions should be noted in these β- blockers. Carvedilol, but not propranolol, induces phosphorylation at

41

GRK6 sites within β2AR and initiates beta-arrestin-mediated signaling

(Nobles et al.; Wisler et al.). Thus, carvedilol might exert some of its effects independent of receptor antagonism. Shenoy et al. previously found that beta-arrestin 1 interacts with HIF-1α, which could be one mechanistic link between the hypoxia barcode and HIF-1α stabilization

(Shenoy, Han, et al.). Studies are needed to test the possible roles of beta-arrestins, receptor internalization, and other mechanisms through which GRK phosphorylation may induce HIF.

Human β1AR and β2AR protein sequences share 54% sequence identity, contain phosphorylation sites that are targeted by PKA and GRK, but have different expression patterns and activate different signaling pathways (Lefkowitz, Rockman and Koch; Rapacciuolo et al.). One limitation of the current study is that β1AR mutants were used in evaluation of hypoxia signal transduction, but β2AR was used to investigate effects of hypoxia on the phosphorylation barcode. Effects of

β2AR PKA and GRK mutants were not studied. Likewise, phosphorylation barcode of β1AR or its GRK/ PKA mutant forms were not studied. In addition, expression levels of β1AR or β2AR were not quantitated in vitro or in vivo. Studies delineating differences and similarities between β1AR and

β2AR under hypoxia will be important to understand mechanisms and cell- specific effects. Abundance and membrane localization of the β1AR and

42

β2AR under hypoxia will also be important to understand regulatory mechanisms of the receptors under hypoxia.

Quantitative mass spectrometry analyses of β2AR peptides, enriched from alprenolol binding, indicate highly differential percent phosphorylation of known serine and threonine amino acid residues, ranging from 0.045% at Ser396 to 99% at Ser262 (Supplemental Table 3).

Our method identified nine β2AR phosphorylated peptides with isoproterenol treatment, compared to thirteen, that was previously reported using the stable isotope labeling with amino acids in cell culture with phosphopeptide enrichment (Nobles et al.). Despite differences in methodologies, we identified overall similar level of phosphorylation change from isoproterenol stimulation at Ser246 (1.7-fold decrease compared to previously reported 2-fold), at Ser396 (6.7-fold increase compared to previously reported 3.5-fold), and at Ser261 and Ser262 (4.9- fold compared to 15.5-fold). Strikingly, hypoxia alone induces decreased phosphorylation at Ser246 and increased phosphorylation at Ser396 at a comparable level to isoproterenol. No change in the phosphorylation level at PKA sites is observed with hypoxia. These findings point to a unique hypoxia-specific β2AR phosphorylation signature, perhaps mediating a different pattern of signal transduction for HIF-1α regulation.

Our results collectively support a model in which hypoxia activates a unique βAR phosphorylation barcode that allows for distinct downstream

43

signals, which are essential for HIF-1α accumulation (Figure 6). The agonist-independent hypoxia-driven βAR activation is mediated by GRK phosphorylation, which results in a unique phosphorylation signature that mediates HIF-1α stabilization.

Although βARs were first identified for their roles in the fight-or-flight response as part of the sympathetic nervous system, this study identifies a previously unknown and unsuspected essential role of βAR in hypoxia sensing in renal hypoxia-induced accumulation of HIF in vivo and in endothelial and epithelial cells in vitro. However, β-blockers are used principally in the context of cardiovascular disease, such as heart failure.

Further work is needed to define whether or not βAR-dependent hypoxic signaling is operative in the heart and, in a clinically relevant model such as ischemic heart failure.

Methods

Animal Model

8-week old female balb/c mice from the Jackson Laboratory (Bar

Harbor, Maine) were orally administered with vehicle or carvedilol (C3993,

Sigma-Aldrich, St. Louis, MO), dissolved in 20% DMSO/ water at 15 mg/kg/day for 7 days, followed by exposure to hypoxia (10% O2). Heart rates were monitored consciously with the MouseOx pulse oximeter.

44

Flow Cytometry

Bone marrow was isolated from the femur, tibia, and vertebrae.

Non-specific binding sites were blocked by incubation of cells with 10% normal goat serum in PBS for 15 minutes at room temperature. Cells were washed with 1% BSA in PBS and incubated with APC conjugated anti-

CD45 (eBioscience) at 1:100 (2 µg/mL), PE conjugated TER119

(eBioscience) at 1:50 (4 µg/mL), and FITC conjugated anti-CD44

(eBioscience) at 1:100 (5 µg/mL) for 30 minutes on ice. All antibodies were diluted in 1% BSA in PBS. Cells were washed twice with 1% BSA in

PBS and resuspended in FACS Flow containing the dead cell marker 7-

AAD (BD Biosciences) at 1:200. Samples were run on a LSRII flow cytometry (Becton Dickenson) and data were analyzed using Flowjo vX.0.7 (Tree Star Inc.). Aggregates, cell debris and dead cells were gated out. Leukocytes were excluded using CD45. Three populations were defined based on size and CD44 and TER119 levels. Proerythroblasts were defined as CD44hiTER119lo, and basophilic and polychromatic erythroblasts were defined as TER119hi and selected based on decreasing size and CD44 expression. Proerythroblasts, basophilic and polychromatic erythroblasts were defined such that the percentage of cells in each population fit the expected ratio of 1:2:4 (J. Liu et al.).

45

Enzyme-linked immunosorbent assay

Blood obtained from cardiac puncture was clotted at 37°C for at least 30 minutes. Serum was isolated after centrifugation at 10000g for 10 minutes. Mouse Erythropoietin Quantikine ELISA Kit (MEP00B, R&D

Systems, Minneapolis, MN) was used to quantify serum erythropoietin concentration.

Cell culture

Human umbilical vein endothelial cells (HUVEC) were cultured in

Endothelial Basal Medium-2 (EBM-2) basal medium, supplemented with

Endothelial Cell Grow Medium-2 (EGM-2) bulletkit (Lonza, Walkersville,

MD). They were cultured up to passage 6 for experiments. All kidney embryonic cells (HEK293) were cultured in minimum essential media with

10% heat-inactivated fetal bovine serum and 1% penicillin/ streptomycin.

Cells were serum-starved 4 hours or overnight prior to any experiment.

For hypoxia studies, cells were incubated in a sealed chamber at 37oC with 2% O2, 5% CO2, balanced with 93% N2. For treatment of cells, propranolol (P0884, Sigma-Aldrich, St. Louis, MO), βARK1/ GRK2 inhibitor

(125 µM) (182200, EMD Millipore, Billerica, MA) and PKA inhibitor H89

(10 µM) (B1427, Sigma-Aldrich, St. Louis, MO) was added to cells for 45 minutes prior to normoxia or hypoxia. HUVEC were generous gifts from

Dr. Terenzi and Dr. Fox at Cleveland Clinic, Cleveland, Ohio; HEK293

− overexpressing β1AR (WTβ1AR) or mutants lacking PKA (PKA β1AR) or

46

− GRK (GRK β1AR) sites from Dr. Howard Rockman at Duke University in

Durham, North Carolina; and HEK293 overexpressing β2AR were from Dr.

Robert Lefkowitz at Duke University in Durham, North Carolina.

Western blot

For HIF-1α protein quantification, nuclear protein extracts were prepared according to the manufacturer’s protocol of the nuclear extraction kit (Affymetrix, Santa Clara, CA). Proteins were subjected to

SDS-PAGE in 4-15% or 18% Tris-HCl gel (Bio-rad, Helcules, CA) and transferred to polyvinylidene difluoride membranes. Antibodies for human

HIF-1α (610959, BD Biosciences, San Jose, CA), mouse HIF-1α (sc-

10790), phosphorylated β2AR (sc-22191-R), β2AR (sc-569) and Lamin B

(sc6216) from Santa Cruz Biotechnology Inc., Dallas, TX were used for detection.

Isolation of RNA

Cells were lysed using QIAzol buffer. RNA was isolated based on the manufacturing protocol of the miRNeasy Mini Kit (Qiagen, Valencia,

CA). The quality of RNA was assessed with the 2100 BioAnalyzer (Agilent

Technologies, Santa Clara, CA). Samples with RNA Integrity number higher than 7 were used for genome-wide expression assay.

Genome-wide expression assay

The HumanHT-12 v4 Expression BeadChip (BD-103-0204,

Illumina, San Diego, CA) was used. The experiment was performed on

47

five biological replicates. Data have been deposited in GEO repository with accession number GSE86793.

Chromatin Immunoprecipitation- quantitative polymerase chain reaction

HUVEC were treated with propranolol, isoproterenol or hypoxia for

12 hours. Chromatins were prepared according to the manufacturing protocol of the Chromatin Immunoprecipitation Assay Kit (17295, EMD

Millipore, Billerica, MA). Rabbit anti- HIF-1α antibody (sc10790, Santa

Cruz Biotechnology Inc., Dallas, TX) was used to pull down chromatin bound by HIF-1α; Rabbit IgG (sc2027, Santa Cruz Biotechnology Inc.,

Dallas, TX) was used as control. DNA primers used for qPCR to quantify

HIF-1α-bound promoters are as follows. SDF-1 forward: 5’-

TCTAACGGCCAAAGTGGTTT-3’; SDF-1 reverse: 5’-

GCCACCTCTCTGTGTCCTTC-3’. HK2 forward: 5’-

CACATTGTTGCATGAAACTCC-3’; HK2 reverse: 5’-

GACCTCTCCGATTCACAGG-3’. VEGFA forward: 5’-

TCTTCGAGAGTGAGGACGTGTGT-3’; VEGFA reverse: 5’-

AAGGCGGAGAGCCGGAC-3’ (Kim et al.). Epo forward: 5’-

TCTCTTCATGACTGTACACACC-3’; Epo reverse: 5’-

TGACAGCCACATAGAATAAAAG-3’ (Yeo et al.). Quantitative polymerase chain reaction was performed using iQ™ SYBR® Green Supermix (170-

8880, Bio-rad, Hecules, CA).

48

Measurements of cyclic adenosine monophosphate

Cyclic adenosine monophosphate (cAMP) was measured according to the manufacturing protocol of the CatchPoint cAMP Fluorescent Assay

Kit (R8089, Molecular Devices, Sunnyvale, CA).

Reverse transcription- quantitative polymerase chain reaction

Messenger RNA was prepared according to manufacturing protocol of miRNeasy kit (217004, Qiagen, Venlo, Limburg). Messenger RNA was reverse-transcribed using SuperScriptTM First-Strand Synthesis System

(11907, Invitrogen, Carlsbad, CA). DNA primers used for qPCR to quantify

EPO and ACTIN cDNA were as follows. EPO forward: 5’-

GAGGCAGAAAATGTCACGATG-3’; EPO reverse: 5’-

CTTCCACCTCCATTCTTTTCC-3’. ACTIN forward: 5’-

CTAGGCACCAAGGTGTGAT-3’; ACTIN reverse: 5’-

CAAACATGATCTGGGTCATC-3’.

Pathway analysis and annotation clustering

DAVID v6.7 (Database for Annotation, Visualization and Integrated

Discovery) was used for Kyoto Encyclopedia of Genes and Genomes

(KEGG) pathways analysis and annotation clustering of genes that show significant differential expression in cells treated with hypoxia, isoproterenol, and hypoxia with propranolol pretreatment.

49

β2AR alprenolol beads purification

Kidney embryonic kidney cells which overexpress β2AR

(HEK293 β2AR) were serum starved for at least 4 hours prior to exposure to isoproterenol (10 µM) for 5 minutes or hypoxia (2% oxygen) for 5 hours.

After cell lysis with hypotonic solution, plasma membranes were solubilized with 1% n-Dodecyl β-D-maltoside, supplemented with Halt

Protease and Phosphatase Inhibitor Cocktail (78440, Thermo Fisher

Scientific, Waltham, MA). Alprenolol beads (CM29101, CellMosaic, Inc.,

Woburn, MA)(Shorr, Lefkowitz and Caron) was incubated with plasma membranes at 4°C overnight. Receptors were eluted with loading buffer for gel electrophoresis.

Mass Spectrometry

The protein samples were subjected to in-gel digestion in which the bands were cut from the gels and washed in 50% ethanol, 5% acetic acid.

The gel pieces were then dehydrated in acetonitrile, dried in a Speed-vac, and digested by adding 5 µL 10 ng/µL of trypsin or chymotrypsin, in 50 mM ammonium bicarbonate, followed by incubation overnight. The peptides were extracted into two portions of 30 µL each 50% acetonitrile,

5% formic acid. The combined extracts were evaporated to <10 µL in a

Speed-vac and then re-suspended in 1% acetic acid to make up a final volume of ~30 µL for liquid chromatography–mass spectrometry (LC-MS) analysis. The LC-MS system was a Finnigan LTQ-Obitrap Elite hybrid

50

mass spectrometer system. The high performance liquid chromatography

(HPLC) column was a Dionex 15 cm x 75 µm id Acclaim Pepmap C18,

2µm, 100 Å reversed-phase capillary chromatography column. Five µL volumes of the extract were injected and the peptides, eluted from the column in an acetonitrile, 0.1% formic acid gradient at a flow rate of 0.25

µL/min, were introduced into the source of the mass spectrometer on-line.

The digest was analyzed in both a survey manner and a targeted manner. The survey experiments were performed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. The LC-

MS/MS data was searched with the programs Mascot and Sequest against both the full human reference sequence database and specifically against the sequence of β2AR. The parameters used in this search include a peptide mass accuracy of 10 ppm, fragment ion mass accuracy of 0.6 Da, carbamidomethylated cysteines as a constant modification, and oxidized methionine and phosphorylation at S, T, and Y as a dynamic modification. The results were filtered based on Mascot ion scores and

Sequest XCorr scores. All positively identified phosphopeptides were manually validated. The targeted experiments involve the analysis of specific β2AR peptides including the phosphorylated and unmodified forms of several tryptic and chymotryptic peptides. The chromatograms for

51

these peptides were plotted based on known fragmentation patterns and the peak areas of these chromatograms were used to determine the extent of phosphorylation.

Statistics Analysis

JMP PRO 10 was used for analysis. Analysis of Variance or

Student’s t test was used to test the statistical significance for the observed differences as described in figure legends. The p-value threshold chosen for statistical significance was 0.05.

For mouse kidney ChIP, data outside 95% confidence interval were excluded from analysis.

For genome-wide expression array, the raw expression data were log-2 transformed with force positive background correction and were then quantile normalized. Differential expression analysis was carried out between controls and each group of hypoxia, propranolol, and hypoxia with propranolol pre-treatment. For each two-group comparison, unexpressed and unannotated probes were first excluded from differential analysis. Probes with differential expression levels were identified using the “limma” package in R. False discovery rate was used to account for multiple comparisons. Significant probes were identified as those with

FDR<0.05. All analyses were conducted using R-3.1.3 (cran.r- project.org).

52

Study Approval

All animal experiments were approved by the Cleveland Clinic

Institutional Animal Care and Use Committee at the Lerner Research

Institute in Cleveland, Ohio.

Author contributions

H.C. performed research, collected, analyzed and interpreted data, and wrote the manuscript. K.A., O.R.S., and K.A.Q. collected data and analyzed data for the animal model. B.W. collected and analyzed data for mass spectrometry. B.H. contributed to statistical analysis. W.X. and

J.K.T.D. contributed to data collection and analysis. G.S. and S.V.N.P. provided research design and directions. S.C.E. designed and performed research, analyzed and interpreted data, and wrote the manuscript.

Acknowledgments

This work is supported by National Institutes of Health grants

(HL115008, HL103453, HL081064 and HL60917). H.C. is in the Molecular

Medicine Ph.D. Program of the Cleveland Clinic and Case Western

Reserve University, funded in part, by the Med into Grad initiative of the

Howard Hughes Medical Institute. S.C.E. is supported in part by the Alfred

Lerner Memorial Chair in Innovative Biomedical Research. The Perinatal

Clinical Research Center at the Cleveland Metrohealth Hospital

53

(UL1TR000439) supported this work through the harvest of HUVEC. The

Orbitrap Elite instrument was purchased via an NIH shared instrument grant, 1S10RR031537-01. We thank Dr. Rockman for the generous gift of

HEK293 b1AR wild type and mutant cells, Dr. Lefkowitz for HEK293 b2AR cells, Dr. Aldred for the detailed methods of RNA isolation, Dr. Beall, Dr.

Strohl and Dr. K Gupta for their scientific input, Dr. Terenzi, Dr. Comhair and D. Mavrakis for providing HUVEC, Dr. Semenza for the detailed methods for nuclear extract preparation and David Schumick for the graphical illustration of the working model.

54

Chapter 3 Alternative hematological and vascular adaptive responses to high-altitude hypoxia in East African Highlanders

Hoi I Cheong, Allison J. Janocha, Lawrence T. Monocello, Adrianna C.

Garchar, Amha Gebremedhin, Serpil C. Erzurum1, Cynthia M. Beall

Published December 15, 2016

Citation Information: American Journal of Physiology-Lung Cellular and

Molecular Physiology. https://doi.org/10.1152/ajplung.00451.2016

Abstract

Elevation of hemoglobin concentration, a common adaptive response to high-altitude hypoxia, occurs among Oromo, but is dampened among Amhara highlanders of East Africa. We hypothesized that Amhara highlanders offset their smaller hemoglobin response with a vascular response. We tested this by comparing Amhara and Oromo highlanders at

3700 m and 4000 m to their lowland counterparts at 1200 m and 1700 m.

To evaluate vascular responses, we assessed urinary levels of nitrate

− (NO3 ) as a readout of production of the vasodilator nitric oxide and its downstream signal transducer cyclic guanosine monophosphate (cGMP), along with diastolic blood pressure as an indicator of vasomotor tone. To evaluate hematological responses, we measured hemoglobin and percent

55

oxygen saturation of hemoglobin. Amhara highlanders, but not Oromo,

− − had higher NO3 and cGMP compared to their lowland counterparts. NO3 directly correlated with cGMP (Amhara R2=0.25, P<0.0001; Oromo

2 − R =0.30, P<0.0001). Consistent with higher levels of NO3 and cGMP, diastolic blood pressure was lower in Amhara highlanders. Both highland samples had apparent left shift in oxyhemoglobin saturation characteristics, and maintained total oxyhemoglobin content similar to their lowland counterparts. However, deoxyhemoglobin levels were significantly higher, much more so among Oromo than Amhara. In conclusion, the

Amhara balance minimally elevated hemoglobin with vasodilatory response to environmental hypoxia, whereas Oromo rely mainly on elevated hemoglobin response. These results point to different combinations of adaptive responses in genetically similar East African highlanders.

Introduction

The amount of oxygen delivered to tissues is related to the product of percent oxygen saturation, total hemoglobin and blood flow. A universal response to acute high-altitude hypoxia and lowered percent of oxygen saturation is elevated hemoglobin. Elevated hemoglobin facilitates higher arterial oxygen content and delivery to tissues. This response occurs to different degrees in genetically closely related Ethiopian highlanders of

56

Amhara and Oromo ethnicity, who have resided above 2500 m for over

5000 years, and for about 500 years, respectively (Lundgrin et al.; Alkorta-

Aranburu et al.; Scacchi et al.; Tartaglia, Scano and DeStefano). Percent oxygen saturation is much lower in Oromo compared to Amhara highland natives (Lundgrin et al.). To compensate for this drop, Oromo highland natives have more hemoglobin than Amhara to maintain oxygen delivery.

Population differences in the magnitude of the hemoglobin response suggest the existence of alternative routes of adaptation.

Vasodilation is a potential adaptive mechanism enabling increased oxygen delivery. For example, Tibetan highlanders have a dampened hemoglobin response with high blood flow and nitric oxide levels (Beall,

Brittenham, et al.; Erzurum et al.; Hoit, Dalton, Erzurum, et al.; West). To determine whether Amhara, like the Tibetans, offset a dampened hemoglobin response with the synthesis of vasodilatory molecules such as nitric oxide (Lundgrin et al.), we quantified the end product of nitric oxide in

− the body, nitrate (NO3 ) along with the downstream signaling molecule cyclic guanosine monophosphate (cGMP) both in urine, (Arnold et al.;

Archer et al.), and evaluated the vasomotor tone by measuring the diastolic blood pressures of Amhara and Oromo lowland and highland natives.

To compare hematological responses, we quantified the amount of oxygen-saturated and –unsaturated hemoglobin, i.e. oxy- and deoxy-

57

hemoglobin levels. In this study, we tested the hypothesis that the Amhara adapt to high-altitude hypoxia through the vascular nitric oxide - cGMP pathway to offset a modest hematological response while the Oromo mount a robust hemoglobin response.

Materials and Methods

Study participants

Study participants and sample collection have previously been described (Alkorta-Aranburu et al.; Hoit, Dalton, Gebremedhin, et al.;

Lundgrin et al.). All individuals were agropastoralists born and raised at the altitude of residence. High-altitude participants had not traveled to altitudes below 2500 m in the previous six months while low-altitude participants had not traveled to altitudes above 2500 m in the same timeframe. They had normal body iron levels and no infection or inflammation, as determined by human malarial plasmodium DNA and C- reactive protein levels (McNamara et al.). At the time of study, environmental conditions were recorded as follows: study group, altitude, average barometric pressure in early morning, temperature and relative humidity. High-altitude Oromo: 4000 m, 469mm Hg, 3°C, 40.5%; low- altitude Oromo: 1700 m, 635 mm Hg, 15°C, 32%; high-altitude Amhara:

3700 m, 498 mm Hg, 6°C, 36%; low-altitude Amhara: 1200 m, 659 mm

Hg, 22°C, 36%.

58

Study approval

Institutional Review Boards at Case Western Reserve University and Cleveland Clinic, Addis Ababa University Faculty of Medicine, and the

Ethiopian Science and Technology Commission Ethics Committee approved the study protocols. All participants provided informed consent.

Sample collection

Venous blood was collected into heparinized tubes with peripheral venipuncture. Hemoglobin level was measured in duplicate with the cyanmethemoglobin method (HemoCue®, Ängelholm, Sweden). Spot urine samples were collected, shipped frozen in liquid nitrogen to

Cleveland and stored at -80°C. Blood pressure was the mean of three measurements taken while participants rested in a seated position.

Percent oxygen saturation was measured with pulse oximetry (Criticare

Model 503 and SpO2; Criticare Systems, Waukesha, WI), and the reported value was the mean of six readings taken ten seconds apart. C- reactive protein levels were quantified by ELISA (R&D Systems,

Minneapolis, MN).

Equations

���ℎ��������� ! = ������� ������ ���������� ∗ ℎ��������� ! / !" !"

100

�����ℎ��������� ! = !"

! (100 − ������� ������ ����������) ∗ ℎ��������� / 100 !"

59

�!�! ���� = 0.21 × ���������� �������� − 47 ���� − 40 ����/

0.8

Measurements of urinary nitrate and cGMP

All measurements of urine samples were normalized by urine creatinine concentrations to account for differences in hydration and urine volume. Creatinine concentrations were measured with a Jaffe reaction- based colorimetric assay (Cayman Chemical, Ann Arbor, MI). All samples

− tested negative for nitrite using the Griess reaction. Nitrate (NO3 ) was measured by chemiluminescence as previously described (Hoit, Dalton,

Gebremedhin, et al.). cGMP was quantified by diluting urine samples by

10-fold according to the manufacturer’s protocol of the cGMP parameter assay (KGE003, R&D Systems, Minneapolis, MN). cGMP/ creatinine levels in this study were within the previously reported reference range

(0.002 to 1.28 mmol/g) (Jakob et al.).

Statistics

Data points more than three standard deviations from the mean were considered outliers and excluded from statistical analysis. JMP PRO

10 was used for analysis. Student’s t-tests were used to test the statistical significance for the observed differences between groups. P-value less than 0.01 was considered statistically significant to account for multiple testing.

60

Results

Study participants

113 men and 44 women of Amhara, and 100 men and 37 women of

Oromo participated in the study (Table 1). The study samples were healthy by self-report and medical examination, non-anemic, non-smoking, normotensive and not pregnant. Amhara and Oromo highlanders had similar ages, but Oromo lowlanders were overall younger than the other

− groups. Study outcomes including NO3 , cGMP, systolic blood pressure, diastolic blood pressure, oxyhemoglobin and deoxyhemoglobin did not correlate with age.

− Elevated NO3 and cGMP levels in Amhara highlanders

− Amhara highland natives had 34% higher in urinary NO3

(P=0.0015) and 43% higher in cGMP (P<0.0001) compared to their lowland counterparts; in contrast, Oromo samples did not differ (Fig. 1, A

− and B). At high altitudes, NO3 and cGMP levels were 86% and 46% higher in Amhara than Oromo (P<0.0001). These data demonstrate that the nitric oxide/ cGMP pathway was activated in Amhara, but not Oromo highlanders.

− Higher urinary NO3 levels associated directly with higher cGMP (Amhara:

R2=0.25, P<0.0001; Oromo: R2=0.30, P<0.0001) (Fig. 2, A and B), consistent with the known NO – cGMP signal transduction pathway

(Arnold et al.).

61

− Dietary NO3 from liquid samples

To assess possible dietary contribution to urinary nitrate, we measured the nitrate level of beverage samples collected from the households of study participants, including water, beer, tea and coffee.

The average nitrate levels of Amhara highlanders’ beverages were higher than lowlanders’ beverages (highland Amhara, μM: 464.7 ± 78.4 (n=9); lowland Amhara, μM: 102.4 ± 40.6 (n=10); highland Oromo, μM: 86.8 ±

23.0 (n=3); lowland Oromo, μM: 259 (n=1)).

Lower diastolic blood pressure in Amhara highlanders

Systolic and diastolic blood pressures are indicators of sympathetic activity and the peripheral vasomotor tone respectively. Systolic blood pressure was similar across the study groups (Fig. 3A), but at high altitudes, diastolic blood pressure was, on average, 3.7 mmHg lower among Amhara than Oromo (P<0.0001) (Fig. 3B).

Oxyhemoglobin concentration was similar at low and high altitudes, while deoxyhemoglobin was higher.

As previously reported (Lundgrin et al.), percent oxygen saturation of hemoglobin was lower at high altitude in both Amhara and Oromo, but the altitude difference among Oromo was 1.8-fold larger than Amhara

(P<0.0001) (Fig. 4A). Similarly, the hemoglobin level was higher at high altitude among both ethnicities, but the Oromo hemoglobin response was

1.9-fold greater (P<0.0001) (Fig. 4B).

62

Neither Amhara nor Oromo showed altitude differences in oxyhemoglobin levels, although Oromo had about one g/dL higher oxyhemoglobin levels than Amhara at both altitudes (Amhara-Oromo mean difference at low-altitude: P=0.0004; at high-altitude: P<0.0001)

(Fig. 4C). These findings indicate that both high-altitude Amhara and

Oromo both successfully maintained levels of oxygen-saturated hemoglobin, despite severe hypoxia at high altitudes.

In contrast to oxyhemoglobin, both Amhara and Oromo had higher deoxyhemoglobin with high altitude, but high-altitude Amhara deoxyhemoglobin concentration was triple that of low-altitude Amhara, while Oromo deoxyhemoglobin was quadruple (P<0.0001). Oromo highlanders had 1.7-fold higher deoxyhemoglobin levels than Amhara highlanders (P<0.0001) (Fig. 4D).

To consider the potential role of oxygen affinity of hemoglobin in these East African highlanders, we plotted the measured percent oxygen saturation of hemoglobin and estimated oxygen partial pressures of low- and high-altitude samples, alongside the standard oxygen dissociation curve of hypothetical subjects derived from the Hill’s equation (Fig. 5)

(Dash and Bassingthwaighte). The average measured oxygen saturations of high-altitude Amhara and Oromo lie on the left side of the standard oxygen dissociation curve. The results suggest a greater affinity of hemoglobin for oxygen in highland natives than predicted from the

63

standard curve at low oxygen partial pressures. Interpolations based on

Oromo low- and high-altitude oxygen saturations estimate lower than actual values for the Amhara highlanders at comparable oxygen partial pressures. This suggests a greater affinity for oxygen among high-altitude

Amhara than Oromo. Oromo show marked increase in hemoglobin and modest increase in oxygen affinity; Amhara show increased vasodilation and marked increase in oxygen affinity.

64

Figures

A P<.0001 B P<.0001

) .0015 6 P= ) 3

g P<.0001 g / l / l o o m m µ m ( ( 4 2 e e n n i i n n i i t t a a e e r r 2 1 C C / / e P t a M r t G i c N 0 0 1200 3700 1700 4000 Altitude (m) 1200 3700 1700 4000 Altitude (m) Amhara Oromo Ethnicity Amhara Oromo Ethnicity

− Fig. 1. Elevated urinary levels of nitrate (NO3 ) and cyclic guanosine monophosphate (cGMP) in high-altitude Amhara but not Oromo. Low- altitude (open circles) and high-altitude (close circles) Amhara and Oromo healthy volunteers (men in blue and women in pink) were recruited.

− Amhara have significantly higher NO3 (A) and cGMP (B) levels at high vs. low altitudes, whereas Oromo do not. Black line indicates sample mean, while grey line represents grand mean of all study groups.

Figure 1 65

A B 3 Amhara 3 Oromo P<.0001 P<.0001 R2=0.25 R2=0.30 ) ) M

M 2 2 µ µ ( ( P P M M G G

1 c 1 c

0 0

0 1.5 3.0 4.5 0 1.5 3.0 4.5 Nitrate (mM) Nitrate (mM)

− Fig. 2. Higher NO3 level is associated with higher cGMP in urine samples of Amhara and Oromo. Least square regression analysis shows positive

− correlation between the vasodilator NO3 and the secondary messenger

− cGMP. (A) 25% variation in cGMP is explained by NO3 in Amhara. (B)

− 30% variation in cGMP is explained by NO3 in Oromo.

Figure 2

66

A B ) )

g 160 100 P=.34 g P<.0001 H H m m m

m 90 ( 140 ( e e r r u u 80 s s s s

e 120 e r r p p 70 d d o o o 100 o l l

b 60 b c c i i l l o o t 80 t 50 s s

y Altitude (m) Altitude (m)

1200 3700 1700 4000 a 1200 3700 1700 4000 i S Amhara Oromo Ethnicity D Amhara Oromo Ethnicity

Fig. 3. Diastolic blood pressures of Amhara were lower than Oromo at high altitudes. (A) Systolic blood pressures are similar among all groups.

(B) At high altitudes, Amhara have lower diastolic blood pressures than

Oromo. Group mean shown in black line, and grand mean of all study groups in grey.

Figure 3

67

A P<.0001 B P<.0001 P<.0001 P<.0001 100 24 P<.0001

) P=.0002 L ) d % / (

g P=.0003 ( n o n i i

t 90 20 b a r o l u g t o a s m n 80 e 16 e H g y x O 70 12 1200 3700 1700 4000 Altitude (m) 1200 3700 1700 4000 Altitude (m) Amhara Oromo Ethnicity Amhara Oromo Ethnicity

C D P<.0001 P<.0001 22 5 P<.0001 ) ) L L d

P=.0004 d / / 4 g g ( ( P<.0001 n n i i b b 3 o o l l g

16 g o o

m 2 m e e h h y y x x 1 o O e 10 D 0 1200 3700 1700 4000 Altitude (m) 1200 3700 1700 4000 Altitude (m) Amhara Oromo Ethnicity Amhara Oromo Ethnicity

Fig. 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. (A) Percent oxygen saturation is lower at high altitude in both Amhara and Oromo, but the drop is much greater in Oromo than Amhara. (B) Hemoglobin levels increase with altitudes in both Amhara and Oromo, but the increase in hemoglobin from low to high altitude is much greater in Oromo than Figure 4 Amhara. (C) Oxyhemoglobin levels do not change with altitudes, but

Oromo have higher oxyhemoglobin levels than Amhara at low and high

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altitudes. (D) Deoxyhemoglobin levels increase with altitudes in Amhara and Oromo, but the increase is much greater in Oromo than Amhara.

100

80

60 Amhara 1200 m Amhara 3700 m 40 Oromo 1700 m Oromo 4000 m 20

0 20 40 60 80 100 Oxygen partial pressure (mmHg)

Fig. 5. Increased affinity of hemoglobin for oxygen in high-altitude samples, relative to hypothetical subjects derived from the standard oxygen dissociation curve (ODC). The ODC is mathematically derived from the Hill’s equation, with a P50 of 26.8 mmHg and the Hill coefficient of

1.3922*10-4 mmHg-2.7 (Dash and Bassingthwaighte). The depicted values of alveolar oxygen partial pressure in Amhara and Oromo are estimated based on the assumption of 47 mmHg water vapor pressure, 40 mmHg arterial partial pressure of carbon dioxide, and 0.8 respiratory exchange ratio (Sánchez-Godoy).

69 Figure 5

Discussion

Amhara and Oromo at 3700 m and 4000 m have adapted to survive lifelong hypoxic stress over generations, but the underlying mechanisms remain unclear (Alkorta-Aranburu et al.; Hoit, Dalton, Gebremedhin, et al.;

Lundgrin et al.). The major finding of this study is that indigenous Amhara highlanders produce more nitric oxide and its bioactive signal molecule

− cGMP. The positive correlation between NO3 and cGMP is consistent with previous studies, which showed that nitric oxide increases the accumulation of cGMP (Arnold et al.; Archer et al.). In Amhara, elevated nitric oxide-cGMP enable vasodilation, lower diastolic blood pressure and, by extension, increase blood flow, which offset their relatively lower hemoglobin response. Unlike Amhara, Oromo highland natives produce a much greater hemoglobin response to maintain sufficient oxygen-carrying capacity at the cost of lower body iron stores (Lundgrin et al.) and higher level of circulating deoxyhemoglobin. This evidence suggests different balances of hemoglobin (Oromo) and vascular (Amhara) adaptive responses to hypoxia among East African high-altitude natives.

Plotting percent oxygen saturation and estimated partial pressure of oxygen for study participants, along with the standard oxygen dissociation curve, illustrates that East African high-altitude natives have a higher than predicted affinity of hemoglobin for oxygen. This is consistent with the

70

general pattern of high-altitude native vertebrates but opposite to acutely exposed people (Storz; Aste-Salazar and Hurtado). Lower body temperature or carbon dioxide levels, increased pH and decreased 2,3- bisphosphoglycerate are factors that could have caused the leftward shift of the oxygen dissociation curve (Jacquez). The oxygen saturation value of high-altitude Amhara is higher than estimated from interpolations based on Oromo low- and high-altitude samples, perhaps related to nitric oxide production (Hsia; Jia et al.; Stamler et al.). High-altitude Amhara rely on nitric oxide-based vasodilation to offset their dampened hemoglobin response, and their hemoglobin pick up oxygen more efficiently in the lung. In contrast, high-altitude Oromo mainly rely on hemoglobin response of much greater magnitude, and their hemoglobin give up oxygen more readily.

Vasodilation presents an alternative approach to high-altitude adaptation, as evidenced in Tibetans (Erzurum et al.; Hoit, Dalton,

Erzurum, et al.), and now among Amhara on a different continent. Urine

− sampling of NO3 and cGMP allows non-invasive global measurements of these biological molecules produced in the systemic vasculature. Urinary nitrate levels reflect the net result of dietary intake, total body biosynthesis and metabolic loss of nitric oxide (Green et al.). To evaluate whether the

~4.5 fold higher nitrate level in the beverages of the highland Amhara contributed significantly to their relatively high urinary nitrate, we estimated

71

the daily liquid consumption that might affect total body nitrate.

Tannenbaum, et al. showed that approximately 60% of ingested nitrate is recovered as urinary nitrate. Ingestion of 3500 µmol nitrate in the

Tannenbaum paper led to, on average, 1865 µmol of urinary nitrate

(Green et al.). Working from these data, high-altitude Amhara would need to ingest 2673 μmol nitrate, corresponding to a daily 6L of fluid ingestion.

Observations in the field do not support excessive fluid intake. Average fluid intake in U.S. is approximately 2L (Popkin, D'Anci and Rosenberg).

Furthermore, recorded dietary recalls for the day of the study show that leafy greens (a nitrate rich diet) were rarely consumed in study groups.

Thus, diet is unlikely to be the major contributor to the urinary nitrate level.

− A limitation of the current study is that NO3 , cGMP and blood pressures were measurements at one time point. Blood flow was not directly measured. However, many studies show that blood flow increases with increasing nitric oxide (Dejam et al.; Ferguson et al.; Petersson et al.).

− Thus, we infer that blood flow is higher due to the higher NO3 and lower vasomotor tone of high-altitude Amhara.

Another potential limitation is that our lowlander samples were collected at 1200 m and 1700 m. These sites provided a substantial contrast in partial pressures of oxygen and are similar to previous studies using Denver Colorado at 1600 m as a low-altitude reference group

(Palmer et al.; Moore et al.). An advantage of these lowland groups is the

72

absence of the endemic malaria found at lower altitudes in East Africa, which would impact hemoglobin. Our previous study found no erythropoietin or hepcidin response in the lowlander groups (Lundgrin et al.), further supporting their use as low-altitude comparison groups.

Further, nitrate levels accounted for only about 30% variation in cGMP in this study. These results suggest other possible upstream signal pathways, other than nitric oxide, that could lead to cGMP production. For example, atrial natriuretic peptides and brain natriuretic peptides, which are released in response to atrial stretch, bind to plasma-membrane guanylate cyclase receptors and in turn generate cGMP (Kuhn). Further investigation in the role of these pathways in high-altitude adaptation is warranted.

In conclusion, the discovery of elevated cGMP associated with

− NO3 among Amhara but not Oromo provides new evidence that chronically exposed populations may adapt differently to the same stress of high-altitude hypoxia. The two closely related ethnic groups have in common elevated oxygen affinity and maintenance of oxyhemoglobin levels. Oromo show substantially higher hematological responses.

Amhara activate the NO-cGMP vasodilation pathway. The balance between hematological and vascular adaptation may be essential for human adaptation and survival in extreme hypoxic conditions.

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Acknowledgments

We are grateful for the permission to conduct the research by the

Ethiopian Science and Technology Commission, the Amhara and the

Oromia Regional Governments and the Semien Mountains and Bale

Mountains National Parks. We thank the communities for their hospitality and participation in the study. Gezahegn Fentahun, M.D. and the late

Daniel Tessema of Addis Ababa University worked in both Ethiopian field sites. Lawrence T. Monocello is now a graduate student in the Department of Anthropology in University of Alabama.

Grants

This work was supported by the National Institutes of Health (HL60917)

(S.C.E.) and the National Science Foundation (BCS-0452326) (C.M.B.).

H.C. is a predoctoral student in the Molecular Medicine Ph.D. Program of the Cleveland Clinic and Case Western Reserve University, funded in part, by the Med into Grad initiative of the Howard Hughes Medical Institute.

S.C.E. is supported in part by the Alfred Lerner Memorial Chair in

Innovative Biomedical Research.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the author(s).

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Author Contributions

H.C. performed research, analyzed and interpreted data, and wrote the manuscript. A.J.J., L.T.M. and A.C.G. performed research and collected data. S.C.E. designed research and interpreted data, and wrote the manuscript. A.G. designed the research. C.M.B. designed and performed the research, collected data, analyzed and interpreted data, and wrote the manuscript.

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Chapter 4 Discussion and Future Directions

A novel role of βAR in hypoxia sensing

βARs were originally discovered for their role in the fight-or-flight response, and HIF-1 for its gene regulatory role in hypoxia sensing. Both the βAR and HIF-1 signal transduction pathways are fundamental to life and stress responses. They have been extensively studied for the past few decades. We now propose that these signaling pathways are interlinked under hypoxic stress.

Hypoxia, in the absence of the ligands catecholamines, puts a tag on the receptor, i.e. a hypoxia-specific phosphorylation barcode, which is distinct from classical catecholamine binding to the receptor. Moreover, when these tagging sites phosphorylated by GRK are mutated to impair phosphorylation, hypoxia-driven HIF-1 responses are blocked. Our findings provide insight into HIF-1 regulation under hypoxia, broaden the scope of βAR signaling, and expand our understanding of fundamental physiological responses to hypoxia.

Does hypoxia induce GRK activity?

How is GRK activated, resulting in βAR phosphorylation under hypoxia? This is an intriguing question. GRK is classically activated upon ligand binding to GPCR, but our data suggests that GRK is activated in the

76

absence of ligand binding. It will be important to first validate that GRK interact with βAR for phosphorylation under hypoxia in immunoprecipitation studies. Future studies can be performed to test if

GRK function or protein (or both) is increased under hypoxia in vitro and in cell cultures. These studies could address (1) if hypoxia alone could induce GRK activity, and (2) if intact cells are required to activate GRK under hypoxia.

If it is validated that hypoxia induces GRK activity, one can further investigate the mechanisms by which GRK activity is regulated under hypoxia. There are three classical ways of GRK activation, namely subcellular localization, intrinsic kinase activity and kinase expression level (Penn, Pronin and Benovic). Phospholipids can bind GRK, which promotes plasma membrane localization and increases the catalytic activity of GRK (Carman et al.; Onorato et al.; Pitcher et al.; Pronin,

Carman and Benovic). Interestingly, lipid rafts, which are plasma membrane microdomains consisting of lipids and membrane proteins, have been shown to increase under hypoxia (Botto et al.). These studies raise the possibility that hypoxia induces changes in the plasma membrane composition, which increases the chance of GRK phosphorylating βAR by altering regulatory proximity. An alternative hypothesis is that protein kinase C (Chuang, LeVine and De Blasi; Pronin and Benovic; Winstel et al.) or the tyrosine kinase Src phosphorylates

77

GRK (Sarnago, Elorza and Mayor), thereby increasing its intrinsic kinase activity. Lastly, it is possible that GRK has increased activity simply because of elevated expression, which could be directly tested by protein quantification in plasma membranes and cytosol compartments.

Is β-arrestin the molecule that mechanistically link GRK and HIF-1?

βAR activated by ligand binding is coupled to Gα and Gβγ subunits; the coupling leads to subunit dissociation. The Gβγ subunits bind and recruit GRK2/3 from the cytosol to the plasma membrane, which mediates phosphorylation of βAR. Mutation of the Gβγ-binding domain in GRK2 impairs the phosphorylation of βAR. Phosphorylated βAR recruits β- arrestin, which uncouples the receptor from G-proteins, resulting in receptor internalization. Since β-arrestin is central to βAR signal transduction and has been shown to interact directly with HIF-1 (Shenoy,

Han, et al.), one can test the hypothesis that GRK regulates HIF-1 level and activity via β-arrestin under hypoxia. One can test this, as a first step, by knocking down β-arrestin in hypoxia cells and assess HIF-1 level and activity.

Translational aspects of GRK in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease, characterized by persistent elevated

78

pulmonary arterial pressure, leading to right ventricular failure (Simonneau et al.). Pulmonary arterial endothelial cells (PAEC) isolated from the lungs of PAH patients express high level of hypoxia inducible factor-1 (HIF-1) and are hyperproliferative (Fijalkowska et al.; Masri et al.). In a chronic hypoxia rodent model, heterozygous deficiency of HIF-1α delays PAH development (Yu et al.). We recently found a link between beta-adrenergic receptors (βAR) and HIF-1 (Cheong et al.). In the failing right ventricles

(RV), cell surface βAR is low (Bristow, Ginsburg, Umans, et al.).

Phosphorylation results in βAR internalization in the endosomes (von

Zastrow and Kobilka). Preliminary data shows that PAH PAEC have more phosphorylated βAR and decreased cyclic adenosine monophosphate

(cAMP) level, suggesting endothelial abnormalities in βAR. Our work uncovered the finding that G-protein coupled receptor kinase (GRK) phosphorylation of βAR is required for HIF-1 expression under hypoxia

(Cheong et al.). In PAH rodent models, pharmacological blockade of the interaction between the G-protein subunits Gβγ and GRK2 improves cardiac functions (Piao et al.). In preliminary studies, we show that GRK2 expression is similar in control and PAH PAEC, but pharmacological inhibition of GRK2 suppresses HIF-1 in PAH. Thus, we hypothesize that

PAH endothelium has higher GRK2 activity, which phosphorylates βAR and increases HIF-1, which leads to the vascular pathobiology of PAH.

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Future studies will define the molecular and functional differences in subcellular localization of β2AR and GRK2 in control and PAH PAEC. We will investigate β2AR expression and function, and GRK2 activity and associated phosphorylated β2AR in subcellular compartments of control and PAH PAEC, to test the hypothesis that PAH has increased GRK2 activity, leading to phosphorylation of β2AR and internalization into endosomes.

It will also be important to identify if GRK2 regulates β2AR function and HIF-1, which alters the rate of cell proliferation in vitro, and cardiac and pulmonary vascular remodeling in vivo. Using in vitro models of human PAEC with overexpression or knockdown of GRK2, and in vivo study of Grk2 globally deficient mice (Grk2+/-) and endothelial lineage- deficient mice (Tie2-CRE Grk2flox/flox), we can test the sub-hypotheses that

GRK2 regulates (1) β2AR function, HIF-1 and its downstream gene products) and cell proliferation rate, and (2) right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodeling to determine physiological relevance in vivo.

Nitric oxide-mediated flow based pathway as an alternative hypoxia response to erythropoiesis

Elevated hemoglobin is a common response to high-altitude hypoxia. This response is dampened in Amhara, but not in genetically

80

closely related Oromo high-altitude natives. We discovered that Amhara high-altitude natives have higher systemic level of nitric oxide-cGMP, which is known to mediate vasodilation and blood flow. This phenomenon of the balanced response between increased hemoglobin and vasodilation in genetically similar highlanders suggests adaptive responses to lifelong hypoxia can be achieved in different ways.

Plotting oxygen saturation levels of participants and the estimated alveolar oxygen partial pressure from barometric pressures in the fields shows an apparent left shift in the oxygen saturation curve (OSC). The findings suggest a greater affinity of hemoglobin for oxygen in high-altitude natives compared to normal hemoglobin. Future studies of these high- altitude natives with arterial blood gas measurement will be valuable in understanding the operating factors that increase hemoglobin affinity.

What is/ are the potential mechanism(s) of higher hemoglobin affinity with hypoxic adaptation?

It is well appreciated that hemoglobin affinity depends on the following non-exhaustive list of factors, namely body temperature, 2,3- diphosphoglyceric acid (2,3-DPG), fetal hemoglobin, pH, arterial carbon dioxide partial pressure and methemoglobin.

• Lower body temperature increases the binding strength of hemoglobin

for oxygen (Barcroft and King).

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• 2,3-DPG is a glycolytic intermediate that binds stronger to

deoxyhemoglobin than oxyhemoglobin. The interaction of 2,3-DPG to

deoxyhemoglobin mediates oxygen release. Thus, lower 2,3-DPG

could cause a left shift in OSC (Dash and Bassingthwaighte).

• Fetal hemoglobin has higher affinity for oxygen than adult hemoglobin.

Therefore, higher circulating fetal hemoglobin may explain a left shift in

OSC (Winslow).

• The Bohr effect states the inverse relationship between the binding

affinity of hemoglobin for oxygen and acidity and arterial concentration

of carbon dioxide. Decreased acidity, or lower pH, and decreased

carbon dioxide concentration can cause a left shift in OSC.

• Increased methemoglobin causes a left shift in the OSC since it does

not unload oxygen, unless given enough time (Wright, Lewander and

Woolf).

Investigation of these operating factors for their contribution to increased oxygen binding affinity of hemoglobin in high-altitude natives will shed light on hypoxia adaptive responses.

82

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