Extracellular Regulation of Nitric Oxide Signaling Via Soluble Guanylate Cyclase

Extracellular Regulation of Nitric Oxide Signaling via Soluble Guanylate Cyclase

Item Type text; Electronic Dissertation

Authors Ramanathan, Saumya

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/223312 EXTRACELLULAR REGULATION OF NITRIC OXIDE SIGNALING VIA SOLUBLE GUANYLATE CYCLASE

Saumya Ramanathan

______

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2012 2

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Saumya Ramanathan entitled EXTRACELLULAR REGULATION OF NITRIC OXIDE SIGNALING VIA SOLUBLE GUANYLATE CYCLASE and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of PHILOSOPHY

______Date: April 11, 2012 Dr. William R. Montfort

______Date: April 11, 2012 Dr. Todd Camenisch

______Date: April 11, 2012 Dr. Roger Miesfeld

______Date: April 11, 2012 Dr. Tsu-Shuen Tsao

______Date: April 11, 2012 Dr. Ted Weinert Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: April 11, 2012 Dissertation Director: Dr. William R. Montfort

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or part maybe granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: SAUMYA RAMANATHAN

ACKNOWLEDGEMENTS Dr. Bill Montfort- without him I’d have no career. His enthusiasm for science inspires me. He welcomes people of different cultures and backgrounds into his lab, making it a truly enriching experience to work for him. For him, there is no good data or bad data, there is just interesting science. I loved working for you Bill!! J Committee members-Dr. Miesfeld, Dr. Weinert, Dr. Camenisch and Dr. Tsao- You all have been a part of my graduate career and I always felt that I could approach any of you without hesitation. Thank you for that and for timely advice on experiments, exams and life!! Dr. Scott Boitano-Thank you for all your help with the calcium experiments. I couldn’t have done any of this without your help. Montfort Lab-WOW!! You guys are the best. Jacquie-you keep the lab running like a machine, but more importantly, you care! In these five years, you have transitioned into someone so integral to my life that I do not know what I will do when I do not have your shoulder to lean on? Dr. Weichsel-Your wit and humor makes us all pale in comparison. Breezy Zegeer- for being the epitome of a powerful woman. Eman-you are the nicest addition to our lab. Thank you for being there for me during difficult times. The gang- Stacy, Jules, Bradley and Johnny- You are the best friends a girl can ask for. Good days, bad days, awful days and wonderful days-we’ve been through it all. I love you guys! MHW-Thank you!! You are a true cheerleader! Rahul- For all the apnapan in lab- Thank you! Aaron-You are one of the nicest kids around, please stay the same always (except stop making fun of me J). Marilyn Kramer-I wouldn’t be here in Tucson without your help. You helped me realize that I had options. Family-My parents-Amma and Appa-Thank you for making me the person I am, thank you for all the opportunities you have given me. Thank you for being the best parents in the world. My dear Annipanni-my blood brother-from that day in the hospital to now, its been 30 years and I can say for sure that you have never left my side and have been a part of every big decision I have made, good or bad. I wish all little girls had such elder brothers. My darling husband-Hemant-I love you. I am here today because of you. You have seen me at my best and my worst. You make me feel safe and inspire me to be a better human being-Thank you! My In-law’s-Amma and Appa Badgandi-Thank you for all your support in these last crucial months and for welcoming me into a your wonderful family!. Paro-my best friend, Thank you for welcoming me to Tucson with so much enthusiasm that I could not help but fall in love and thank you for being you.

DEDICATION

To Amma-for teaching me that unconditional love exists

To Appa-for teaching me the meaning of will-power by epitomizing it

To Annipanni-for never allowing me to feel like I am alone

To Hemant-Destiny brought me to you, love will keep me with you.

“The greatest glory in living lies not in never falling, but in rising every

time we fall.”

― Nelson Mandela

TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………….11 LIST OF FIGURES……………………………………………………………………...12 ABSTRACT……………………………………………………………………………...15 CHAPTER 1: INTRODUCTION………………………………………………………..16 1.1. Nitric oxide biology and synthesis……………………………………………….16 1.2. Soluble guanylate cyclase: The nitric oxide receptor protein…………………….17 1.2.1. Regulation of soluble guanylate cyclase……………………………………..20 1.2.1.1. Isoforms and splice variants…………………………………………….20 1.2.1.2. Transcriptional and post-transcriptional regulation……………………..20 1.2.1.3. Subcellular localization………………………………………………….21 1.2.1.4. Hetero-dimerization vs. homo-dimerization…………………………….22 1.2.1.5. Protein-protein interaction………………………………………………22 1.2.1.6. Phosphorylation…………………………………………………………23 1.2.1.7. Calcium………………………………………………………………….23 1.2.1.8. Allosteric regulators of sGC…………………………………………….24 1.3. Thrombospondin-1: Regulator of nitric oxide signaling…………………………26 1.3.1. Discovery of thrombospondin-1 as a modulator of NO signaling…………..26 1.3.2. Thrombospondins-A family of extracellular matrix proteins……………….27 1.3.2.1. Domain organization of TSP-1………………………………………….27 1.3.3. TSP-1 inhibits NO signaling via CD47……………………………………...28 1.4. CD47 (Integrin Associated Protein)……………………………………………...30 1.4.1. Ligands of CD47…………………………………………………………….30

1.4.1.1. Signal inhibitory receptor protein α (SIRPα)…………………………..30 1.4.1.2. Thrombospondin-1……………………………………………………...32

TABLE OF CONTENTS-continued 1.4.2. CD47: a non-canonical GPCR………………………………………………32 1.5. Research topic and dissertation outline…………………………………………..33 CHAPTER 2: DEVELOPMENT OF A LIVE CELL SYSTEM FOR STUDYING TSP-1 SIGNALING…………………………………………………….36 2.1. Defining the model cell line……………………………………………………..36 2.2. Materials and Methods…………………………………………………………..37 2.2.1. Materials…………………………………………………………………….37 2.2.2. Cell culture………………………………………………………………….37 2.2.3. Expression and purification of E3CaG1…………………………………….38 2.2.4. Cloning and transient transfection of human sGC…………………………..39 2.2.5. sGC activity and cGMP accumulation in intact cells……………………….40 2.2.6. Flow cytometry………………………………………………………………41 2.2.7. Expression and purification of the G1 domain of human TSP-1……………42 2.3. Results……………………………………………………………………………42 2.3.1. Expression of sGC and measurement of NO-inducible sGC activity……….42 2.3.2. CD47 expression on cell surface……………………………………………43 2.3.4. E3CaG1……………………………………………………………………..43 2.3.5. CD47 is necessary but insufficient for E3CaG1 binding to Jurkat T cells and inhibition of sGC……………………………………………………….49

2.3.6. Role of integrin αv in E3CaG1 signaling via CD47………………………..50 2.3.7. G1 domain of TSP-1………………………………………………………..56 2.4. Conclusions and Discussion……………………………………………………..56 CHAPTER 3: E3CaG1 AND ANGIOTENSIN II INHIBIT sGC VIA INCREASE IN INTRACELLULAR CALCIUM……………………………………59 3.1. Hypothesis………………………………………………………………………59

TABLE OF CONTENTS-continued 3.2. Materials and methods………………………………………………………….60 3.2.1. Materials……………………………………………………………………60 3.2.2. Cell culture………………………………………………………………….60 3.2.3. Calcium imaging……………………………………………………………61 3.2.4. Flow cytometry……………………………………………………………..61 3.2.5. sGC activity and cGMP accumulation in intact cells………………………62 3.3. Results…………………………………………………………………………..64

2+ 3.3.1. E3CaG1 induces an increase in [Ca ]i…………………………………….64

2+ 3.3.2. E3CaG1-dependent increases in [Ca ]i requires CD47……………………64

2+ 3.3.3. E3CaG1 mediates increase in [Ca ]i and inhibits sGC in human microvascular endothelial cells…………………………………………….65 3.3.4. Calcium inhibits NO-inducible sGC activity in Jurkat T cells……………..73 3.3.5. Angiotensin II and Phytohemoagglutinin inhibit NO-driven cGMP accumulation………………………………………………………………..73

2+ 3.3.6. E3CaG1 does not increase [Ca ]i via an IP3-dependent mechanism………76 3.4. Conclusions and Discussion…………………………………………………….81 CHAPTER 4: MECHANISM OF CALCIUM REGULATION OF sGC……………...83 4.1. Calcium regulation of sGC………………………………………………………83 4.2. Materials and Methods…………………………………………………………..84 4.2.1. Materials…………………………………………………………………….84 4.2.2. Cell culture………………………………………………………………….84 4.2.3. Phosphodiesterase activity………………………………………………….84 4.2.3.1. Jurkat T cells……………………………………………………………84 4.2.3.2. MCF-7 cells…………………………………………………………….85 4.2.4. Effect of compounds YC-1 and BAY 41-2272……………………………..85

TABLE OF CONTENTS-continued 4.2.4.1. Jurkat T cells…………………………………………………………..85 4.2.4.2. Immuno-precipitated sGC……………………………………………..86 4.2.5. Kinetics……………………………………………………………………..86 4.2.6. Cell-free inhibition of sGC…………………………………………………87 4.3. Results…………………………………………………………………………..87 4.3.1. Phosphodiesterases have minimal effect…………………………………..87 4.3.2. YC-1 and BAY 41-2272 overcome Ca2+-inhibition of sGC in live cells….88

4.3.3. Inhibited sGC exhibits an increase in Km………………………………….96 4.3.4. Inhibition of sGC requires a calcium-dependent tyrosine kinase…………96 4.4. Conclusions and Discussion……………………………………………………99 CHAPTER 5: IDENTIFICATION OF KINASE(S) REQUIRED FOR CALCIUM INHIBITION OF sGC………………………………………………...105 5.1. Human kinome………………………………………………………………..105 5.2. Materials and Methods………………………………………………………..106 5.2.1. Materials………………………………………………………………….106 5.2.2. Methods…………………………………………………………………..107 5.2.2.1. Cloning of human sGC alpha and beta subunit into lentiviral vector..107 5.2.2.2. Production of lentivirus………………………………………………107 5.2.2.3. Transduction of target cells…………………………………………..108 5.2.2.4. Inhibitor screening……………………………………………………109 5.2.2.5. Jurkat T cell lysate fractionation……………………………………..109 5.2.2.6. Depletion of candidate kinases……………………………………….110 5.2.2.7. Transient transfection of rat sGC……………………………………..110 5.2.2.8. Cell-free assay with rat sGC………………………………………….110 5.2.2.9. Bioinformatics………………………………………………………...111

TABLE OF CONTENTS-continued 5.3. Results………………………………………………………………………….111 5.3.1. Lentivirus production, titer determination and production of stable cell line HEK 293T-hsGC………………………………………………………………..111 5.3.2. Jurkat T cell lysate fractionation…………………………………………..116 5.3.3. Inhibitor screening…………………………………………………………116 5.3.4. Cell free assay with rat sGC……………………………………………….122 5.3.5. Bioinformatics……………………………………………………………..122 5.4. Conclusions and Discussion……………………………………………………122 CHAPTER 6: SUMMARY AND FUTURE DIRECTIONS………………………….126 6.1 Summary………………………………………………………………………..126 6.2 Future directions………………………………………………………………..128 6.2.1. Identification of kinase(s) regulating sGC………………………………..128 6.2.2. Identifying the site of sGC modification………………………………….129 6.2.3. Other pieces of the puzzle…………………………………………………129 REFERENCES………………………………………………………………………...132

LIST OF TABLES

TABLE 2.1. Relative cell surface expression of CD47………………………………..46 TABLE 4.1. Cellular calcium-induced changes in kinetic parameters of sGC………..98 TABLE 5.1. Summary of lentiviral expression studies………………………………115 TABLE 5.2. Summary of inhibitor screen……………………………………………119 TABLE 5.3. List of tyrosine kinases expressed in HEK 293T, Jurkat T and MCF-7 cells………………………………………………………...124

LIST OF FIGURES

FIGURE 1.1. Schematic overview of NO-mediated signaling……………………….18 FIGURE 1.2. Schematic representation of the domain organization of sGC………...19 FIGURE 1.3. Chemical structure of allosteric activators of sGC: YC-1 and BAY 41-2272…………………………………………………………25 FIGURE 1.4. Schematic diagram of full length TSP-1………………………………29 FIGURE 1.5. Predicted domain organization of CD47………………………………31 FIGURE 1.6. Schematic of the overall goal of the project…………………………..35

FIGURE 2.1. Western blot showing transient transfection of MCF-7 cells…………44 FIGURE 2.2. NO-induced sGC activity in different cell types………………………45 FIGURE 2.3. Schematic diagram of full length TSP-1 and the recombinant C-terminal fragment E3CaG1……………………………………...... 47 FIGURE 2.4. Gel showing purification of E3CaG1………………………………….48 FIGURE 2.5. Inhibition of sGC activity in Jurkat T cells as a function of E3CaG1 concentration...... 51 FIGURE 2.6. CD47 expression on young and old Jurkat T cells………..…………...52 FIGURE 2.7. E3CaG1 binding as measured by flow cytometry……………………..53 FIGURE 2.8. E3CaG1 inhibits sGC in young Jurkat cells but not in older cells…….54

FIGURE 2.9. Role of αv integrin in E3CaG1 signaling via CD47…………………..55 FIGURE 2.10. GST-G1 activity assay…………………………………………………57

FIGURE 3.1. E3CaG1 induces as increase in intracellular calcium………………….66 FIGURE 3.2. Calcium fluctuations with time…………………………………………67

LIST OF FIGURES-continued

2+ FIGURE 3.3. [Ca ]i over time of a typical Jurkat T cell following addition of E3CaG1 after preincubation with BAPTA-AM……………………….68

2+ FIGURE 3.4. [Ca ]i over time of a typical Jurkat T cell following addition of

Ionomycin in the presence of 2 mM extracellular CaCl2………………69

2+ FIGURE 3.5. E3CaG1-induced [Ca ]i is cell adhesion independent but requires CD47………………………………………………………………...... 70

2+ FIGURE 3.6. CD47 is necessary for E3CaG1-dependent increase in [Ca ]i…………71

2+ FIGURE 3.7. E3CaG1 induces increase in [Ca ]i in human microvascular endothelial cells and inhibits sGC………………………………………72

2+ FIGURE 3.8. Increased [Ca ]i leads to sGC inhibition in Jurkat T cells……………..74 FIGURE 3.9. Chelating cytosolic Ca2+ with cell permeable chelator BAPTA-AM reverses sGC inhibition by E3CaG1...... 75 FIGURE 3.10. Stimulation of calcium release by PHA results in sGC inhibition……..77

2+ FIGURE 3.11. Ang II increases [Ca ]i to the same levels as does E3CaG1…………..78 FIGURE 3.12. Stimulation of calcium release by Ang II results in sGC inhibition……79

2+ FIGURE 3.13. E3CaG1 does not mediate changes in [Ca ]i in an IP3-dependent mechanism………………………………………………………………80

FIGURE 4.1. Phosphodiesterases are minimally involved in the Ca2+-dependent lowering of cGMP in Jurkat T cells ……………………………………90 FIGURE 4.2. Phosphodiesterases are minimally involved in the Ca2+-dependent lowering of cGMP in MCF-7 cells……………………………………..91 FIGURE 4.3. YC-1 overcomes E3CaG1-dependent inhibition of sGC in Jurkat cells..92 FIGURE 4.4. BAY 41-2272 overcomes E3CaG1-dependent inhibition of sGC……...93

LIST OF FIGURES-continued FIGURE 4.5. Immuno-precipitated sGC remains inhibited but YC-1 and BAY 41-2272 overcome inhibition……………………………………94 FIGURE 4.6. YC-1 and BAY 41-2272 do not overcome direct Ca2+ inhibition of sGC…..………………………………………………………………95 FIGURE 4.7. Representative kinetic plots for immune-precipitated sGC from MCF-7 cells……………………………………………………………………..97 FIGURE 4.8. Cell-free assay…………………………………………………………100 FIGURE 4.9. Calcium/calmodulin-dependent kinases are not involved in Ca2+-dependent inhibition of sGC……………………………………..101 FIGURE 4.10. Genistein and alkaline phosphatase reverse inhibition of sGC………..102 FIGURE 4.11. Alkaline phosphatase reverses phosphorylation of sGC………………103

FIGURE 5.1. Illustration of the Tet-Off lentiviral expression system……………….113 FIGURE 5.2. Flowchart illustrating bioinformatics analyses………………………..114 FIGURE 5.3. Cell-free inhibition by calcium using fractionated Jurkat lysate………117 FIGURE 5.4. Depletion of pyk2 from MCF-7 lysate does not reverse calcium-mediated inhibition of sGC…………………………………..120 FIGURE 5.5. Depletion of c-src kinase from Jurkat lysate does not reverse calcium-mediated inhibition of sGC…………………………………..121 FIGURE 5.6. Rat sGC is inhibited by calcium……………………………………….123

FIGURE 6.1. Research summary……………………………………………………..131

ABSTRACT

Nitric Oxide (NO) regulates cardiovascular homeostasis by binding to soluble guanylate cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration

2+ ([Ca ]i) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, I show that a TSP-1 C- terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition

2+ requires an increase in [Ca ]i.. Using digital imaging microscopy on live cells, I further

2+ show that E3CaG1 binding results in a substantial increase in [Ca ]i, up to 300 nM.

2+ Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca ]i, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca2+ remains inhibited, while addition of kinase inhibitors staurosporine, genistein, PP1 or PP2 reverse inhibition, indicating inhibition likely involves a tyrosine kinase, more specifically, a src family kinase. Rat sGC is also inhibited by lysates supplemented with Ca2+, suggesting that the site of modification is at an evolutionarily conserved residue. Inhibition is through an increase in Km for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of

2+ NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca ]i in

2+ response to NO, inducing vasodilation, but is also inhibited by high [Ca ]i, providing a fine balance between signals for vasodilation and vasoconstriction.

CHAPTER 1

INTRODUCTION

1.1 Nitric oxide biology and synthesis

Nitric oxide (NO) regulates numerous vital functions in animal biology.

Endothelial cells of blood vessels produce NO to signal to the underlying smooth muscle cells to relax, resulting in vasodilation and increased blood flow. Macrophages generate nitric oxide as part of the immune response against invading bacteria and other pathogens. NO also functions as a neurotransmitter used by neurons to relay, amplify and modulate neuronal signals (2). Dysregulation of NO signaling contributes to cardiovascular disease, difficulties in wound healing, diabetes, asthma and aging. The localized concentration of NO determines the biological outcome of its signaling. In general low concentrations promote cell survival and proliferation whereas higher concentrations favor apoptosis, cell cycle arrest and senescence (3, 4).

NO is produced through a conversion of L-arginine to L-citrulline by nitric oxide synthase (5-7). Three isoforms of NOS are found in mammals, neuronal NOS (nNOS or

NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3). nNOS and eNOS are constitutively expressed and synthesize NO in response to higher Ca2+ levels or to calcium-independent stimuli such as shear stress. iNOS on the other hand is synthesized in response to proinflammatory signals. Each NOS polypeptide has an N- terminal oxygenase domain that binds heme, tetrahydrobiopterin (H4B) and L-arginine, a central calcium/calmodulin binding domain and a C-terminal reductase domain that binds

17 to cofactors FMN, FAD and NADPH. All three isoforms are homodimers in their active state (8).

The most well characterized effector of nitric oxide is its intracellular receptor protein, soluble guanylate cyclase (sGC). sGC catalyzes the conversion of GTP to cGMP and binding of NO results in 100-200 fold stimulation in its activity. NO production also leads to S-nitrosation of key proteins and a wide array of physiologically important responses, including changes in gene regulation, apoptosis, and angiogenesis. The factors governing these responses are less well characterized. Figure 1.1 outlines the outcome of NO signaling pathway.

1.2 Soluble guanylate cyclase: The nitric oxide receptor protein

Soluble guanylate cyclase is an obligate heterodimeric protein of ~150 kDa consisting of one α subunit and one heme-containing β subunit. The protein is subdivided into three functional domains based on sequence homology to adenylate cyclase and particulate guanylate cyclase (9, 10), as shown in Figure 1.2. The N-terminal region (heme NO oxygen binding (HNOX) domain) contains the heme moiety and binds to NO. The central portion of the protein consists of a Per-ARNT-Sim (PAS) domain and a coiled-coiled region that function as a dimerization domain. The C-terminus is the catalytic domain, a region that is highly conserved across the cyclase family (11). The catalytic domain possesses the binding site for substrate GTP and Mg2+ ion cofactor required for cGMP formation.

Figure 1.1. Schematic overview of NO-mediated signaling. Nitric oxide is synthesized by nitric oxide synthase and, being lipophilic, can cross cell membranes.

It binds to and activates its receptor protein soluble guanylate cyclase (sGC) in the same cell or in a target cell. This leads to increased production of cGMP, which acts as a second messenger and activates either protein kinase G (PKG) or cyclic nucleotide gated channels (CNGC) to bring about the various downstream physiological effects of NO signaling which include memory formation, smooth muscle cell relaxation and angiogenesis. The cGMP signal is degraded by phosphodiesterase (PDE) cleavage, yielding GMP. Some of the NO produced in the

- - - cell is oxidized to nitrite (NO2 ), nitrate (NO3 ) or peroxynitrite ions (ONOO ), which may further lead to S-nitrosothiol, nitrotyrosine and other protein or lipid modifications.

Figure 1.2. Schematic representation of the domain organization of soluble guanylate cyclase. sGC is a heterodimeric enzyme consisting of one α subunit and one heme- containing β unit. The protein is divided into an N terminal HNOX domain and central PAS and coiled-coiled region and a C terminal catalytic domain. NO binding to the HNOX domain leads to a conformational change in the protein, resulting in a

100-200-fold activation and increased synthesis of cGMP from GTP.

1.2.1. Regulation of soluble guanylate cyclase

1.2.1.1. Isoforms and splice variants

sGC is composed of one alpha subunit and one beta subunit. Two isoforms of each subunit are known: α1 and α2, and β1 and β2 (10). The most abundant and ubiquitous heterodimer in mammals is α1/β1, although α2/β1 is preferentially expressed in the brain and placenta. The pharmacological and kinetic properties of α1/β1 and α2/β1 are similar (12, 13).

Two shorter splice variants of the α1 subunit exist. These mRNA lack the predicted translation initiation sites and their expression correlates with lack of sGC activity (14). A catalytically inactive variant of α2 that has 31 extra amino acids at the C- terminus, α2i, is expressed selectively in certain human tissues (15). Similarly, splice variant of the β1 subunit with a deletion in the coding sequence, and a variant of the β2 subunit, β2b, with a deletion of 46 nucleotides at the C-terminus, have been detected.

The significance of the splice variants and the different isoforms is not fully understood, but could provide the cellular machinery with an additional level of regulation.

1.2.1.2. Transcriptional and post-transcriptional regulation

sGC subunit gene expression can be modulated by cytokines, second messengers and disease states (11). For example, steady state mRNA levels of α1 and β1 are reduced upon exposure to NO donors, estrogen, LPS and interleukin-1β (10). In addition, the mRNA levels of the α1 subunit correlate with mRNA levels of the β1 subunit. The principal transcription factors regulating sGC expression have been elucidated and

21 include CCAAT-binding factors, NF�B (p50) and NFAT (nuclear factor of activated T cells) (16).

The 3’ UTR region of both α1 and β1 mRNAs have AU rich regions which target the mRNA for rapid degradation by endonucleases. Under basal conditions, HuR

(Human antigen R or embryonic lethal abnormal visual [ELAV]-like RNA binding protein) binds to the 3’ UTR of both α1 and β1 mRNA stabilizing the message. Increase in cyclic nucleotides either by NO stimulation of sGC or via activation of adenylate cyclase decreases HuR expression and results in rapid degradation of sGC mRNA (17,

18).

1.2.1.3. Subcellular localization

sGC was initially thought to be an entirely cytosolic enzyme. However recent evidence indicates that in rat heart, the α1/β1 heterodimer is associated with the membrane fraction. The membrane association is labile and thought to be dependent on another membrane associating protein (19). The α2 subunit of the α2/β1 heterodimer is also associated with the membrane in neuronal cells. This association requires the PDZ domain containing post-synaptic density protein (PSD)-95 (13). In addition, sGC in endothelial cells is membrane associated whereas in vascular smooth muscle cells it is mostly cytosolic (20) and the translocation to the membrane requires association with heat shock protein 90 (hsp90). This leads to an attractive hypothesis that sGC forms a protein complex with eNOS in endothelial cells and movement to the membrane makes it more NO sensitive. The β2 subunit contains at its C-terminal domain an

22 isoprenylation/carboxymethylation consensus sequence (–CVVL–) that enables other proteins, such as ras, to be tethered to the membrane (21). However, there is no evidence that the β2 subunit is subject to this modification.

1.2.1.4. Hetero-dimerization vs. homo-dimerization

The obligate heterodimeric nature of sGC is evidenced by the observation that the expression of the α1, β1 or α2 alone does not lead to the formation of an active enzyme.

In addition, reduction in expression of either subunit in cells that express α1/β1 leads to decrease in basal and NO-stimulated activity (22, 23). In spite of this, α1/α1 and β1/ β1 homodimers do exist (24). It is not clearly understood if this is another form of regulation of the enzyme.

1.2.1.5. Protein-protein interaction

In rat brain homogenates the α2 subunit of sGC binds to PSD-95 via its C- terminus. PSD-95 interacts with sGC via one of its PDZ domains and via its other two

PDZ domains it interacts with neuronal NOS and the NMDA receptor (13). PSD-95 therefore acts as a molecular scaffold to bring together the receptor and its signaling effectors. Whether or not PSD-95 interaction with sGC is essential for NO signaling at the synapse is not yet understood.

sGC co-immunoprecipitates with heat shock protein 90 (hsp90). In endothelial cells there is evidence for the existence of an eNOS/sGC/hsp90-signaling complex (13).

This could allow for more efficient signaling by facilitating the transfer of NO from

23 eNOS to sGC before NO is scavenged in the cell. Yeast two hybrid screens have also revealed two other binding partners for sGC, CCTη and AGAP-1 (25, 26). Interaction with CCTη decreases NO-stimulated sGC activity. Again, the importance of these protein-protein interactions involving sGC have not been studied in much detail and whether or not they are employed in specific signaling pathways is not known.

1.2.1.6. Phosphorylation

Both the α and the β subunits of sGC possess multiple consensus phosphorylation sites for PKA, PKC, PKG and casein kinase II. In vitro experiments show that sGC is a

PKA substrate (27) and that this phosphorylation increases the activity of sGC. PKC phosphorylates sGC in PC12 cells leading to an increase in activity but the site and stoichiometry of this phosphorylation event is not known (28). PKG also phosphorylates sGC and decreases activity, in what is thought to be a classic biochemical feedback mechanism (29-31). sGC phosphorylation by c-src kinases leads to inhibition of activity

(32). Some of these studies were done in vitro and therefore not reflective of signaling pathways in the cell. In addition, the structure of sGC has not been solved, so predictions of phosphorylation sites and kinase binding motifs cannot be corroborated.

1.2.1.7. Calcium

Calcium, nitric oxide, and cGMP are intimately associated in controlling

2+ numerous cellular functions, especially vascular tone. High [Ca ]i levels lead to attenuation in NO-induced cGMP accumulation in transformed HEK 293 cells (33), in primary astrocytes (34) and in primary pituitary gland cells (35). In addition, micromolar

24 calcium concentrations can directly inhibit isolated sGC, in what is thought to be a competitive mechanism with Mg2+ that is required for catalysis (33, 36, 37).

1.2.1.8. Allosteric regulators of sGC

Nucleotides ATP, GTP and cGMP all alter sGC activity. ATP inhibits sGC by binding to an allosteric site (38, 39). In adenylate cyclase, there is only one active site at the interface between the two subunits. Forskolin an activator of the enzyme binds to the pseudo-symmetric site and activates it. Based on sequence homology, it can be hypothesized that nucleotides regulate sGC activity by binding to the pseudo-symmetric site in the cyclase domain.

NO-independent sGC stimulation was first demonstrated in 1994, when the synthetic benzylindazole compound YC-1 was shown to increase cGMP levels in platelets and inhibit their aggregation (40). This property of YC-1 is attributed to direct allosteric sGC stimulation (41). YC-1 causes a 10-20-fold increase in sGC activity by increasing the catalytic rate and by decreasing the Km for substrate GTP. In the presence of NO, the efficacy of YC-1 increases by 1-2 orders of magnitude (42). The molecular mechanism and site of YC-1 binding to sGC are still unknown. YC-1 was the starting point for designing chemical activators of sGC (Figure 1.3). It has led to the discovery of other chemical modifiers of sGC, such as BAY 41-2272. The mode of action of BAY

41-2272 is analogous to that of YC-1, but it demonstrates higher potency and specificity than its predecessor (43).

Figure 1.3. Chemical structure of allosteric activators of sGC: YC-1 and BAY 41-

2272

1.3. Thrombospondin-1: Regulator of nitric oxide signaling

1.3.1. Discovery of thrombospondin-1 as a modulator of NO signaling

Thrombospondin-1 (TSP-1), an extracellular matrix protein of ～450 kDa, is an inhibitor of NO signaling. Muscle tissue explants from TSP-1 null mice show enhanced vascular outgrowth in collagen matrix relative to wild type mice (44). This differential response is increased in the presence of L-arginine or a slow releasing NO donor, such as

DETA/NO. Alternatively, the vascular outgrowth is inhibited in the presence of L-

NAME an inhibitor of nitric oxide synthase (45). The NO-stimulated increase in endothelial cell proliferation, migration, and adhesion, which are of importance for angiogenesis, wound healing, and tumor progression, are potently blocked by picomolar concentrations of TSP-1. Additionally, endothelial cells from TSP-1 null mice display increased proliferation and adhesion when compared to cells from wild type mice. NO- stimulated cGMP accumulation in endothelial cells is TSP-1-sensitive, where picomolar concentrations block NO stimulation of sGC. Therefore, in endothelial cells, TSP-1 is an endogenous antagonist to NO signaling. TSP-1 also inhibits NO signaling in vascular smooth muscle cells (46). In these vascular smooth muscle cells, TSP-1 inhibits both short-term effects of NO such as vasorelaxation, and long-term effects such as proliferation, migration and adhesion (46, 47). The mechanisms behind TSP-1 attenuation of NO signaling are poorly understood but involve inhibition at multiple steps, including those involving vascular endothelial growth factor receptor-2 (VEGFR2), eNOS, sGC and protein kinase G (PKG) (48-50). Among these, inhibition of sGC is

27 particularly prominent and the focus of my research. TSP-1 is involved in a multitude of physiological processes signaling events by interacting with a diverse array of cell surface receptors via its many domains. The next section describes the domain organization of TSP-1 and the proteins with which they interact.

1.3.2. Thrombospondins-A family of extracellular matrix proteins

Thrombospondins are multi-domain, calcium-binding glycoproteins found in animals. TSP-1 is a prototypic member of this family and has multiple roles in platelet aggregation, cell adhesion, wound healing, tumor growth and inflammation. It first came to light as the first endogenous anti-angiogenic protein to be discovered (51). TSP-2 has similar functions and, like TSP-1, is a homotrimeric protein. The other TSPs, TSP-3, 4 and 5 are homopentamers. Gene knockout studies have revealed distinct and non- redundant roles for each of the thrombospondins. This is attributed to the different cell surface receptors that they each interact with and to differential spatio-temporal expression (52, 53).

1.3.2.1. Domain organization of TSP-1

A schematic diagram of the domain organization of TSP-1 is shown in Figure 1.4.

TSP-1 contains a globular N-terminal domain. The known receptors for this domain are syndecan, heparan sulphate proteoglycans (HSPG) and α3β1 integrin. This domain is involved in cell migration, spreading and attachment, endocytosis of TSP-1, platelet aggregation, disruption of focal contacts of cells and cell proliferation. An oligomerization domain follows the N-terminal domain. The procollagen homology

28 domain is involved in subunit assembly and inhibition of angiogenesis. The type-1 repeats inhibit angiogenesis, induce apoptosis and activate TGFβ. The known receptors for this domain are heparin, CD36 and β1 intergrin. The type 2 repeats bind to other soluble matrix proteins. The type 3 repeats are rich in aspartic acid residues and constitute the calcium-binding domain. Binding studies show that on an average one molecule of TSP-1 binds to 35 calcium ions. The type 3 repeats also possess RGD sequences that might facilitate binding of TSP-1 to various cell surface integrins (52, 53).

The globular C-terminal domain of TSP-1 binds to CD47, also known as integrin associated protein (54). This domain is mainly involved in cell binding, migration and attachment.

1.3.3. TSP-1 inhibits NO signaling via CD47

TSP-1 binds to both CD47 and CD36, two abundant transmembrane proteins, and binding to each inhibits NO signaling (55). Recombinant N-terminal fragments of TSP-1 did not block NO signaling in vascular cells (45). The exclusive role of CD36 was ruled out because muscle explants from CD36 null mice show similar inhibition in NO- stimulated growth by TSP-1. In contrast, NO stimulated the growth of muscle explants from CD47 null mice, but TSP-1 was unable to inhibit this growth, showing that while ligation of CD36 or CD47 by TSP-1 might inhibit NO signaling in vascular cells, CD47 was necessary. In addition, peptides derived from the CD47-binding region of TSP-1 and recombinant C-terminal fragments of TSP-1 were able to inhibit NO stimulation of sGC

Figure 1.4. Schematic diagram of full length TSP-1. An oligomerization domain (two bars indicate cysteines involved in disulphide linkage) and a procollagen homology domain follow the N-terminal domain. This is followed by three type 1 repeats and three tandem EGF-like type 2 repeats. There are seven calcium binding type 3 repeats. The globular carboxy-terminal domain binds to cell surface receptor CD47.

30 activity to the same extent as full length TSP-1 in both vascular smooth muscle cells and endothelial cells (56). The mechanism behind this inhibition was unknown prior to my studies and is the primary focus of this thesis.

1.4. CD47 (Integrin Associated Protein)

CD47 or integrin associated protein (54) was discovered originally as a membrane protein that co-purified with αvβ3 integrin from placenta and platelets. Monoclonal antibodies against CD47 prevent αvβ3 mediated cellular functions in polymorphonuclear leucocytes (PMN) and similarly, CD47 activating antibodies induce signaling identical to that resulting from ligation of αvβ3 integrin (57). CD47 forms stable interactions with integrin αIIbβ3 on platelets and with α2β1 on smooth muscle cells (58, 59).

CD47 is a member of the immunoglobulin (Ig) superfamily of membrane proteins. The N-terminal IgV-like domain of CD47 has been crystalized (60). A hydrophobic stretch with five membrane-spanning segments and an alternatively spliced intracellular region ranging in length from 3-36 amino acids are predicted to follow the

N-terminus (61).

1.4.1. Ligands of CD47

1.4.1.1. Signal inhibitory receptor protein α (SIRPα)

SIRPα is a single pass transmembrane protein that has three Ig-like domains at the extracellular region and that binds to tyrosine phosphatases SHP-1 and SHP-2 through its

Figure 1.5. Predicted domain organization of CD47. CD47 has an IgV like N- terminus. A hydrophobic segment that spans the membrane five times is predicted to follow. The C terminal region of the protein is alternatively spliced. Two known disulphide bonds in the protein are indicated by –S-S –.

32 cytoplasmic region. SIRPα is predominantly expressed in neurons, dendritic cells and macrophages. CD47/SIRPα interaction is implicated in regulating both immune homeostasis and neuronal network formation. The most well characterized function of the CD47/SIRPα complex is in phagocytosis of red blood cells (RBC’s) by macrophages.

Macrophages strive to maintain tissue integrity and get rid of old and apoptotic cells by phagocytosis. CD47 (on RBC’s) binds to SIRPα (on macrophages) and this results in inhibition of phagocytosis thereby determining the number and quality of the circulating

RBC’s (62). Extracellular domains of SIRPα and CD47 were crystallized and the structure of the complex reveals atomic details of the specificity of CD47/SIRPα interaction (60).

1.4.1.2. Thrombospondin-1

The many cell surface receptors of TSP-1 were elucidated by proteolytic cleavage of the protein or by the generation of recombinant fragments of the protein. The C- terminus of TSP-1 binds to cell membranes and generation of synthetic peptides (4N1-

RFYVVMWK) from this region showed that peptide sequence VVM is necessary for this activity. Affinity labeling of the peptide revealed that it bound to a 50 kDa membrane glycoprotein that was identified as CD47 (63-65).

1.4.2. CD47: A non-canonical GPCR

CD47 and its integrin binding-partners have been suggested to form non- canonical G protein coupled receptors (GPCRs) by mimicking the seven transmembrane receptor topology. CD47 co-immunoprecipitates with Giα and some of the biological

33 functions of CD47 can be blocked by treatment with pertussis toxin. Removal of cholesterol from the stable CD47/integrin and G-protein complex disrupts signaling.

This is an interesting observation because it is known that some G proteins are found in cholesterol-rich microdomains within the membrane (66-68). Moreover, integrins and G proteins were found to colocalize in three different cell types CD47 (61). Nonetheless, the mechanism(s) by which CD47 signals remains unknown and the role for G-proteins in this signaling unclear. For example, it is not known if CD47 can signal by itself or if it must be a part of a larger complex for signaling to occur. CD47 is also likely to form multiple complexes with different signaling outcomes. For example, CD47 associates with vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells but ligation of CD47 by TSP-1 or TSP-1 derived peptide is sufficient to prevent this association (48).

1.5. Research topic and dissertation outline

The research topic addressed in this thesis is the mechanism by which TSP-1 binding to CD47 leads to an inhibition in NO signaling via soluble guanylate cyclase.

Figure 1.6 illustrates the overall goal of the project. In chapter 2, I elaborate on how we chose our model system, the Jurkat T cells. I also describe our discovery that CD47 is necessary but not sufficient for TSP-1 signaling, requiring either a binding partner or specific modification for activity. Our central hypothesis was TSP-1 mediates inhibition of sGC via an increase in cytosolic calcium concentration. In Chapter 3, I describe experiments that tested our central hypothesis. I also discuss the discovery of calcium as

34 a modulator of sGC signaling in two pathways (TSP-1 and Angiotensin II). In Chapters 4 and 5, I describe the post-translational mechanism that regulates sGC function and finally in Chapter 6, I discuss my findings and describe experiments that are still needed to elucidate the signaling mechanism.

Figure 1.6. Schematic of the overall goal of the project. NO binds to and activated sGC leading to increased production of cGMP. TSP-1 inhibits NO signaling via sGC by binding to CD47. I set out to elucidate the mechanism of inhibition of sGC by

TSP-1. My central hypothesis was that TSP-1 mediates inhibition of nitric oxide signaling via sGC by increasing cytosolic calcium concentration.

CHAPTER 2

DEVELOPMENT OF A LIVE CELL SYSTEM FOR STUDYING TSP-1 SIGNALING

2.1. Defining the model cell line

In order to discover the molecular mechanism behind TSP-1 inhibition of sGC, I needed an experimental cell line that would have all the components of the signaling pathway we had chosen to study. This includes, NO inducible sGC, CD47, and the ability to respond to TSP-1. Since CD47 is ubiquitously expressed in most cell types

(69), I first measured sGC expression, more specifically NO-inducible sGC activity. To look for the ability of TSP-1 to inhibit sGC, we used full length TSP-1 purified from platelets and provided to us by our collaborator at the NIH, Dr. David D Roberts. TSP-1 is difficult to work with, leading to inconsistent results. So, we decided to use the more robust C-terminal fragment of TSP-1, E3CaG1 that retains the CD47 binding domain.

After surveying many different cell types, I chose Jurkat T cells, which are immortalized T lymphocytes. They have robust NO-inducible sGC activity and express

CD47 at the cell surface. They also respond to E3CaG1. In the process of defining our experimental system, I discovered that while CD47 is necessary for TSP-1 inhibition of sGC, it might not be sufficient. The baculoviral vector pAcGP67.coco encoding E3CaG1 was a kind gift from Dr. Deane Mosher (University of Wisconsin). We purified E3CaG1 using a protocol given by Doug Annis (Mosher Lab). Dr. Stacy Mazzalupo isolated and cultured aortic smooth muscle cells from mice and performed immuno-cytochemistry on

MCF-7 cells and HEK 293T cells to measure endogenous sGC expression. Some of the work in this chapter has been published (1).

2.2. Materials and Methods

2.2.1. Materials

FITC-conjugated monoclonal anti-human CD47 antibody (B6H12) and an isotype control antibody were obtained from BD Biosciences (San Jose, CA). Anti-integrin antibodies to αV (P2W7 and 272-17E6) were obtained from Abcam. Mouse monoclonal anti-FLAG antibody was from Sigma Aldrich. 2-(N,N-Diethylamino)-diazenolate-2- oxide (DEA/NO) was a kind gift from Dr. Katrina Miranda (University of Arizona).

DEA/NO is a NO donor. It spontaneously dissociates in a pH dependent, first-order process with a half-life of 2 min and 16 min at 37 °C and 22-25 °C, pH 7.5 respectively, to liberate 1.5 moles of NO per mole of parent compound (70). Phosphate-buffered saline (PBS) was prepared as 10 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM

KCl, pH 7.4. Tris-buffered saline (TBS) was prepared as 10 mM Tris.HCl, 150 mM

NaCl, pH 7.4. Krebs buffer was prepared as 25 mM HEPES, 120 mM NaCl, 4.75 mM

KCl, 1.44 mM MgSO4, 11 mM glucose, pH 7.4. All other reagents were obtained from

Sigma unless otherwise noted.

2.2.2. Cell culture

Sf9 cells were maintained in Grace’s Insect Media (Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biologicals), gentamicin (10 mg/ml) and fungizone

(0.25 µg/ml). Jurkat T cells (TIB-152™) were purchased from ATCC. Jurkat T cells lacking CD47 (JinB8) (71) were the kind gift of Dr. David Roberts (NIH). All Jurkat cell lines were maintained in RPMI 1640 (Invitrogen) supplemented with 5-10% fetal bovine

38 serum (FBS), penicillin (5 mg/ml) and streptomycin (1 mg/ml). Jurkat T cells were maintained below 2 x 106 cells/ml unless otherwise noted, and were weaned from 5%

FBS to serum-free conditions starting 48 h prior to all experiments. MCF-7 cells were obtained from ATCC (HTB-22™) and maintained in DMEM supplemented with 10%

FBS, penicillin (5 mg/ml) and streptomycin (1 mg/ml), and used for experiments within

10 passages after thawing. HT-1080-28 cells that were stably transfected with sGC alpha and beta subunits (cell line made by Susan Kunz and Dr. Roger Miesfeld) were maintained in DMEM supplemented with 5% FBS and penicillin (5 mg/ml) and streptomycin (1 mg/ml). HEK 293T cells were a kind gift from Dr. Roger Miesfeld and were maintained in DMEM supplemented with 5% FBS and penicillin (5 mg/ml) and streptomycin (1 mg/ml).

2.2.3. Expression and purification of E3CaG1

Expression and purification were carried out as described (72). Briefly, Sf9 cells were grown at 27 °C and were maintained in Grace’s Insect Media supplemented with

10% fetal bovine serum and gentamicin and fungizone. When the cells reached a density of 1 x 106 cells/ml, they were transferred to SF900II media (Invitrogen) and were infected with high titer virus at a multiplicity of infection of 5. Media was collected 65 h post infection. After the His-tagged E3CaG1 was purified by immobilized metal ion affinity chromatography, it was stored at -80 °C in TBS supplemented with 2 mM CaCl2.

Protein concentrations were determined by the BCA assay (Thermo Scientific, Rockford,

IL) using bovine serum albumin as the standard.

2.2.4. Cloning and transient transfection of human sGC

Primers 5'-ctcagtctcgagatctattcctgatgc-3' and 5'-cagtcaggatccgatgttctgcacgaagc-3' were used to amplify human sGC α1 cDNA (ATCC clone MGC-33150) for cloning into pCMV-3Tag-9 (Clontech, Mountain View, CA) between BamHI and HindIII sites, yielding a C-terminal myc-tagged protein (vector WM397). Human sGC β1 was cloned into pCMV-3Tag-3A (Clontech) between SacI and XhoI sites, yielding a C-terminal

FLAG-tagged protein (vector WM434). Primers 5'-gcactcgaggtcatcatcctgctttg-3' and 5'- cactgtgagctcatgtacggatttgtg-3' were used to amplify the cDNA from plasmid pSTBlue1-

Huβ1 bearing the human sGC β1 cDNA – a gift from Dr. Alan Nighorn (University of

Arizona). The Stratagene QuikChange® Lightning Site-Directed Mutagenesis Kit

(Agilent, La Jolla, CA) was used to correct all errors in both plasmids to match

CCDS34085.1 (GUCY1A3) and CCDS47154.1 (GUCY1B3) (this was done by Dr. Stacy

Mazzalupo, a postdoctoral researcher in our lab) (73). Transfection reagent TurboFect™

(Fermentas, Glen Burnie, MD) was used at a ratio of 20 µg plasmid DNA (1:1 ratio of sGCα: sGCβ) to 25 µl reagent per 10-cm dish of cells at 50% confluency. Cells were harvested by trypsinization 12 h after transfection and the cell pellet was quickly frozen in liquid nitrogen. Transfection efficiency was analyzed by western blot. Briefly, lysates of transiently transfected (mock and sGC) MCF-7 cells were separated by SDS-PAGE and transferred onto nitrocellulose membrane. sGC β subunit was detected by probing membranes with mouse monoclonal anti-FLAG antibody (1:2000 dilution) and signal was detected by goat anti-mouse antibody conjugated to either HRP or IR-dye. We do

40 not have a good sGC alpha antibody that can be used for western blotting. Presence of heterodimeric functional protein was confirmed by NO-inducible activity.

2.2.5. sGC activity and cGMP accumulation in intact cells

MCF-7 cells, HEK 293T (transiently transfected) and HT1080-28 were trypsinized and resuspended in media (1 x 106 cells per assay condition). Jurkat T cells

(1 x 106 per assay condition) were resuspended in Krebs buffer. Cells were incubated with 10 mM NaOH (buffer control) or 50 µM DEA/NO. Reactions were stopped after 2 min by placing the cell suspensions on ice, pelleting and quickly freezing. For cGMP measurements, the cell pellets were thawed and resuspended with 100 µL cell lysis buffer. The basal and NO-induced sGC activities of intact cells were expressed in terms of picomoles cGMP produced per 2 minutes per milligram of total protein content (pmol cGMP/mg protein/2 min), using the CatchPoint cGMP ELISA kit (Molecular Devices,

Sunnyvale, CA). Protein concentrations were determined by the BCA assay (Thermo

Scientific) using bovine serum albumin as the standard.

To examine E3CaG1 inhibition, cells were incubated with indicated concentrations of E3CaG1 in Krebs buffer for 15 min at room temperature, followed by the addition of buffer control or 10 µM DEA/NO. Reactions were stopped after 2 min by quickly pelleting cells. Pellets were lysed in lysis buffer (MD kit) and cGMP accumulation measured.

2.2.6. Flow cytometry

To measure relative expression of CD47 on the cell surface of different cell lines,

1 x 106 cells (HEK 293T, MCF-7, HT-1080-28 and Jurkat T cells) were resuspended in stain/wash buffer (PBS supplemented with 0.1% BSA, 0.01% NaN3). Except for Jurkat

T cells, all of the cells used were adherent. The adherent cells were lifted off the tissue culture dishes with EDTA and not trypsin. Once in suspension, the cells were incubated with FITC-conjugated monoclonal anti-human CD47 antibody for 1 hour at 4 °C. Cells were washed three times with stain/wash buffer to remove unbound antibody and fixed with 4% paraformaldehyde prior to analysis by flow cytometry.

To measure E3CaG1 binding to CD47 by competition with the CD47 antibody,

1 x 106 Jurkat T cells resuspended in stain/wash buffer (PBS supplemented with 0.1%

BSA, 0.01% NaN3) were used per assay condition. Cells were incubated with stain/wash buffer or stain/wash buffer supplemented with E3CaG1 (22 nM) for 1 h at 4 °C and were fixed with 4% paraformaldehyde prior to incubation with FITC-conjugated monoclonal anti-human CD47 antibody. Cells were washed with stain/wash buffer to remove unbound antibody followed by addition of 4% paraformaldehyde.

One-color flow cytometric analysis was performed at 488 nm using a FACScan flow cytometer (BD Biosciences). The emission fluorescence of FITC-conjugated CD47 antibody was detected using a 530/30 bandpass filter and recorded at a rate of 200-400 events per second for 10,000 events gated on FSC (forward scatter) vs. SSC (side scatter). Data were analyzed using CellQuest PRO software (BD Biosciences) or FlowJo

(v.7.6.4). Appropriate electronic compensation was adjusted by acquiring cell

42 populations stained with each dye/fluorophore individually, as well as an unstained control.

2.2.7. Expression and purification of the G1 domain of human TSP-1

The gene encoding the G1 domain of TSP-1 was synthesized between BamHI and

HindIII sites in the pGS-21a vector from Genscript. We expressed this domain as a fusion protein consisting of an N terminal His tag fused to glutathione-s-transferase

(GST) to increase the solubility of the protein. A TEV protease cleavage site was engineered in between the 6xHis-GST and the G1 domain. Pilot expression studies (data not shown) showed that a 3 h induction time (with 0.4 mM IPTG) at 37 °C yielded ~30% of the protein in the soluble fraction. His-GST-G1 was first purified from the rest of the bacterial protein using Ni2+-affinity column. Following elution and dialysis to remove imidazole, the protein was concentrated and used for activity assays along with GST controls. Preliminary efforts to remove GST from the G1 domain by TEV protease cleavage resulted in unstable protein that could not be used for activity measurements.

2.3. Results

2.3.1. Expression of sGC and measurement of NO-inducible sGC activity

Four cells lines were examined for sGC activity. MCF-7 cells and HEK 293T cells show no detectable endogenous expression of sGC (immuno-staining data not shown), but they can be transiently transfected to yield active protein (Figure 2.1 and

2.2). The HT-1080-28 cell line (made by Susan Kunz and Dr. Roger Miesfeld, that stably expresses the myc-tagged α1 and FLAG-tagged β1 subunit of sGC) exhibit NO-

43 inducible sGC activity, but these cells are very difficult to grow and obtain in large numbers. Jurkat T cells are reported to express 540 fold higher α1 mRNA and 14 fold higher β1 mRNA than a control prostate cancer cell line DU-145 (74), therefore we obtained Jurkat T cells and tested their NO-inducible sGC activity. They exhibit robust sGC activity as shown in Figure 2.2. In addition, cultured aortic smooth muscle cells obtained from mice displayed very little activity (performed by Dr. Stacy Mazzalupo).

2.3.2. CD47 expression on cell surface

CD47 expression on the cell surface was measured by flow cytometry. Mouse monoclonal anti-CD47 antibody (B6H12) conjugated to FITC was used to label CD47 on the cell membrane. All cell types exhibited very low autofluorescence and only a small increase in fluorescence upon treatment with a FITC-conjugated isotype control antibody, indicating very little non-specific binding occurs. All the cells under study expressed

CD47 at the membrane; however, Jurkat T and MCF-7 cells have relatively higher expression than HEK 293T and HT-1080 cells (Table 2.1). Due to the higher expression of both sGC and CD47 in Jurkat T cells, we decided to use these cells as our model system.

2.3.4. E3CaG1

Due to difficulties in obtaining TSP-1 from our collaborators and to the general instability of the protein, we decided to express and purify a functional fragment of TSP-

1, E3CaG1.

Figure 2.1. Western blot showing transient transfection of MCF-7 cells. Equimolar ratio of sGC α-myc and β-FLAG results in expression of β-FLAG. Lysates from mock and sGC transfected cells were separated on SDS-PAGE gel, followed by transfer to nitrocellulose membrane. Blot was probed with mouse monoclonal anti-

FLAG antibody. Goat-anti mouse secondary conjugated to HRP was used to detect signal. Since we do not have a good sGC α antibody, expression of active protein is determined by measuring NO-inducible activity.

Figure 2.2. NO-induced sGC activity in different cell types. HT-1080-28, Jurkat T cells and aortic smooth muscle cells (aortic SMC) and transiently transfected HEK

293T and MCF-7 cells were treated with either 10 mM NaOH or 50 µM DEA/NO for 2 minutes. Cell pellets were frozen in liquid nitrogen and cGMP accumulation was measured. Error bars represent the range from mean of replicates

Cell line Cells only FITC-Isotype CD47-FITC

HEK 293T 9.7 11.9 45.2

MCF-7 10.2 21.1 1763.0

Jurkat T cells 9.4 11.0 409.0

Table 2.1. Summarizes relative cell surface expression of CD47 on HEK 293T, MCF-

7 and Jurkat T cells. Cells were resuspended in stain/wash buffer and incubated with buffer, FITC-conjugated isotype control antibody or FITC-conjugated anti-human

CD47 antibody for 1 hour at 4 °C. Cells were washed three times with stain/wash buffer to remove unbound antibody. One-color flow cytometric analysis was performed at 488 nm using a FACScan flow cytometer. The emission fluorescence of

FITC-conjugated antibody was detected using a 530/30 bandpass filter. The numbers represent the corresponding mean values of FITC fluorescence.

Figure 2.3. Schematic diagrams of full length TSP-1 and the recombinant C-terminal fragment E3CaG1. E3CaG1 consists of the third EGFβ-like type 2 repeat, the seven calcium binding type 3 repeats and the globular C-terminal CD47 binding domain.

The protein has a N-terminal 6xHis tag.

Figure 2.4. Gel showing purification of E3CaG1. 1 liter of Sf9 cells were infected with baculovirus and 65 hours later cells were harvested and supernatant containing

E3CaG1 was purified to apparent homogeneity using Ni2+-affinity chromatography.

As shown in in Figure 2.3 E3CaG1 consists of the last EFGβ-like type II repeat, all of the calcium binding type III repeats, and the C-terminal cell binding domain required for

CD47-dependent activity. Expression of the protein in Sf9 cells, using baculovirus and purification via batch Ni2+-affinity based isolation led to ~10 mg of protein per liter of cell culture. Representative gel for purification is shown in Figure 2.4.

We observed strong inhibition of NO-stimulated sGC activity for E3CaG1 concentrations as low as 0.22 nM (42%) and found inhibition to be maximal for concentrations above ～ 20 nM (～67%), similar to previous reports for E3CaG1 and full length TSP-1 (Figure 2.5) (55). Subsequent experiments were performed with 22 nM

E3CaG1.

2.3.5. CD47 is necessary but insufficient for E3CaG1 binding to Jurkat T cells and inhibition of sGC

Surprisingly, Jurkat T cells that had been in culture for less than 3 weeks and those that were grown at low density (0.5 x 106 cells/mL) responded to E3CaG1, while those cells that had been in culture for more than 4 weeks or those grown at high densities (3 x106 cells/mL) did not respond. This could be due to one of two reasons, a loss of CD47 on cell surface as a function of time or growth conditions, or a loss of

E3CaG1 binding to CD47. As shown in Figure 2.6, CD47 expression did not change over time or growth densities, as assessed by CD47 antibody. I then measured binding of

E3CaG1 to Jurkat T cells through its ability to compete with a FITC-conjugated monoclonal anti-human CD47 antibody using flow cytometry (Figure 2.7). When cells

50 were incubated with FITC-conjugated CD47 antibody, there was an ～100 fold increase in fluorescence, indicating the presence of CD47 on the membrane. When E3CaG1 (22 nM) was added to cells prior to the addition of CD47 antibody, the mean fluorescence decreased significantly, indicating that E3CaG1 competes with the monoclonal antibody and binds to CD47 on the Jurkat T cell surface (Figure 2.7A). Older cells (> 6 weeks), or cells that had been grown at higher density (3 x106 cells/mL), could still bind antibody

(Figure 2.6 and Figure 2.7B), but E3CaG1 was no longer able to compete with antibody binding. Consistent with the binding studies, E3CaG1 (22 nM) inhibited NO-stimulated sGC activity in younger cells but no inhibition was seen in older cells (Figure 2.8)

From these experiments, I concluded that although CD47 remains on the cell surface, CD47 or a complex that includes it has changed and can no longer interact with the TSP-1 fragment. All subsequent experiments were therefore performed on cells that were within 3 weeks of growth and kept well below 1 x 106 cells/mL.

2.3.6. Role of integrin αv in E3CaG1 signaling via CD47

CD47 is known to associate with αv integrin (61, 75). To investigate the role of

αv integrin in E3CaG1 signaling, I examined E3CaG1 activity in the presence of anti-αv antibodies. Blocking of αv integrin with antibodies (PW27 or 272-17E6) did not alter

E3CaG1 inhibition of sGC (Figure 2.9). In contrast, and as expected from previous studies (55), antibody B6H12 (monoclonal antihuman CD47 antibody) abolished

E3CaG1-dependent inhibition of sGC, showing that CD47 is necessary.

Figure 2.5. Inhibition of sGC activity in Jurkat T cells as a function of E3CaG1 concentration. Error bars represent the range in values of means from two independent experiments. Jurkat T cells were incubated with E3CaG1 (0-220 nM) for 15 min prior to the addition of 10 µM DEA/NO. Cell pellets were lysed in lysis buffer (MD kit) and cGMP accumulation measured. Reprinted with permission from (1). Copyright 2011

American Chemical Society.

Figure 2.6. CD47 expression on young and old Jurkat T cells. Cells were stained with

FITC-conjugated isotype control antibody or FITC-conjugated anti-CD47 antibody

(B6H12). 1 x 106 cells were used per assay condition.

Figure 2.7. E3CaG1 binding as measured by flow cytometry. Flow cytometry histogram of young Jurkat T cells (A) and old Jurkat cells (B) labeled with FITC- conjugated anti-human CD47 antibody in the presence or absence of E3CaG1, or with isotype or vehicle control, as indicated. 1 x106 cells were used per condition.

Figure 2.8. E3CaG1 inhibits sGC in young cells but not in older cells. (A) Young

Jurkat T cells were examined for cGMP production (1 x106 cells per assay condition).

Where indicated, cells were incubated with E3CaG1 at room temperature (15 min), followed by the addition of 10 µM DEA/NO. Error bars represent the standard deviation from mean of independent experiments (n = 5), and * denotes p < 0.001.

(B) is the same as above, except that older cells (> 6 weeks in culture) were used.

Figure 2.9. Role of αv integrin in E3CaG1 signaling via CD47. Addition of anti-

CD47 antibody B6H12 prevents E3CaG1 induced inhibition of sGC, whereas addition of anti-integrin αv antibodies P2W7 or 272-17E6 prior to the addition of E3CaG1 does not. Jurkat T cells were incubated with the respective antibodies for 30 min at 4

°C prior to the addition of E3CaG1 (22 nM). After 15 min, 10 mM NaOH (buffer control) or 10 µM DEA/NO was added as appropriate, followed by incubation for 2 min. Error bars represent the standard deviation from mean of independent experiments (n = 5) and * denotes p < 0.001. Reprinted with permission from (1).

2.3.7. G1 domain of TSP-1

E3CaG1 must be expressed using baculovirus, which makes mutational analysis time consuming and to investigate if the integrin binding domain of E3CaG1 (type 3 repeats) is necessary for inhibition of sGC, we expressed the G1 domain of TSP-1 in a bacterial expression system. When the G1 domain was fused to 6xHis-GST the fusion protein was stable and inhibited NO-stimulated cGMP accumulation (Figure 2.10). The

G1 domain alone (post TEV cleavage) was unstable and prone to precipitation. Further optimization of purification strategies is required to yield stable and robust protein.

2.4. Conclusions and Discussion

I surveyed several cell lines to establish our model system, analyzing NO- inducible sGC activity, surface expression of CD47 and the ability of cells to respond to

E3CaG1 (C terminal fragment of TSP-1). In our initial survey, we found that sGC was not expressed in MCF-7 and HEK 293T cells; we were successful in transiently transfecting these cells to make active protein. In primary cell lines such as aortic smooth muscle cells and HMVEC (human microvascular endothelial cells) sGC expression decreased over time in culture (data not shown). Based on this and the fact that Jurkat T cells expressed abundant CD47 at the cell surface, we chose the Jurkat T cells for most subsequent experiments. I also found that while CD47 is necessary for E3CaG1 mediated inhibition of sGC, it might not be sufficient. I showed that integrin αv is not

Figure 2.10. GST-G1 activity assay. Jurkat T cells (0.5 x 106 cells per assay condition) were incubated with buffer, GST or GST-G1 at room temperature (15 min), followed by the addition of 10 µM DEA/NO. cGMP accumulation was measured for each experimental condition. Error bars represent the range from means of two independent experiments.

58 necessary for E3CaG1 signaling via CD47. For all further experiments with Jurkat T cells, I used younger/resting T cells that were capable of responding to E3CaG1.

Further exploration into whether the loss of E3CaG1 binding to CD47 in old or activated Jurkat T cells is due to change in expression or location of signaling partners of

CD47 or post-translational modification of CD47 itself was beyond the scope of my project and is under investigation by others in our group.

CHAPTER 3

E3CaG1 AND ANGIOTENSIN II INHIBIT sGC VIA INCREASE IN

INTRACELULAR CALCIUM

3.1. Hypothesis

As described previously (56) and shown in Figure 2.5 and 2.8A, incubation of cells with TSP-1/E3CaG1 for 15 minutes is sufficient to inhibit sGC and therefore NO signaling. This short response time rules out transcriptional or translational regulation of sGC as the mechanism responsible for inhibition and implicates post-translational modification as likely responsible. Those data and two key reports in the literature led to my central hypothesis. TSP-1 and TSP-1 derived peptides 4N1 and 4N1K (modified

4N1) binding to CD47 have each been shown to increase intracellular calcium

2+ concentration ([Ca ]i) in mast cells and fibroblasts (68, 76). In mast cells, increase in

2+ [Ca ]i by TSP-1/CD47 interaction is thought to be mediated via G-protein coupled signaling where CD47 and integrins function as non-canonical GPCRs. In fibroblasts, increase in intracellular calcium by TSP-1/CD47 interaction occurs via opening of a plasma membrane channel, again thought to be mediated by CD47 interaction with

2+ integrins. The second important finding in the literature was that high [Ca ]i levels lead to attenuation of NO-induced cGMP production in transformed HEK 293 cells (33), in primary astrocytes (34) in primary pituitary gland cells (35), and that micromolar calcium concentrations can directly inhibit isolated sGC (33, 36, 37). On the basis of the foregoing, I hypothesized that TSP-1 inhibition of sGC was mediated through Ca2+

60 signaling. In this chapter, I describe experiments that showing that E3CaG1 binding to

2+ CD47 in Jurkat T cells results in an increase in [Ca ]i and that this increase is necessary for inhibition of sGC. I also show that Angiotensin II, a potent vasoconstrictor that

2+ induces an increase in [Ca ]i through GPCR AT1, also inhibits sGC, providing a new link between signals for vasodilation (NO) and vasoconstriction (Ang II). Most of the work in this chapter has been published (1). Calcium imaging and data analysis was performed in the laboratory of Dr. Scott Boitano, with his assistance.

3.2. Materials and Methods

3.2.1. Materials

Ionomycin, thapsigargin, PHA and BAPTA-AM were obtained from Invitrogen

(Carlsbad, CA). Fura 2-AM and Fluo-3AM were obtained from Calbiochem/EMD

Biosciences (San Diego, CA). All other reagents were from Sigma unless otherwise noted.

3.2.2. Cell culture

3T3-L1 fibroblasts were the kind gift of Dr. Tsu-Shuen Tsao (University of

Arizona) and were maintained in DMEM (Invitrogen) supplemented with 10% FBS, penicillin (5 mg/mL), and streptomycin (1 mg/mL). Dr. Betsy Dokken provided the

Human microvascular endothelial (HMVE) cells and the maintenance media with necessary supplements. All other cells used were cultured as described in Chapter 2.

3.2.3. Calcium imaging

2+ In order to assay [Ca ]i in Jurkat T cells, which normally grow in suspension, 3T3 L1 fibroblasts were grown on glass coverslips for 3-4 days before the experiment. On the day of the experiment, fibroblasts were hypotonically lysed, and cellular debris was mechanically removed with a cell scraper. Jurkat T cells were then allowed to adhere to the matrix-coated coverslips. The cells were left undisturbed for a minimum of 1 hour before use and they remained attached to the coverslips under these conditions for up to 4 hours. Attached cells were loaded with Fura-2AM for 30 min at room temperature.

Fura-2 fluorescence was observed on an Olympus (Center Valley, PA) IX70 microscope equipped with a 75 W Xenon lamp while alternating between excitation wavelengths of

340 and 380 nm. Images of emitted fluorescence above 505 nm were captured by an

ICCD camera (Photon Technology International, Birmingham, NJ) under ImageMaster

2+ software control (PTI). Effective [Ca ]i was calculated from equations published in (77).

2+ Initial [Ca ]i was assessed over 20-60 s to establish a consistent baseline, and changes in

2+ [Ca ]i were monitored over time for each experimental condition. Depending on the experiment, measurements were taken every 0.6 s (for 3-5 min experiments) up to 5 s (for experiments >5 min). Cell morphology within the time period of measurement was assessed by differential interference contrast microscopy and found not to vary.

3.2.4. Flow cytometry

2+ To examine change in [Ca ]i by flow cytometry, Jurkat T cells or CD47 deficient

Jurkat T cells (JinB8 cells) (71) were loaded with 5 µM Fluo-3AM in Krebs buffer for 30

62 min at room temperature with gentle mixing every 10 min. HMVE cells were detached from tissue culture dish by treatment with EDTA for 10 min at room temperature. They were loaded with 10 µM Fluo-3 AM in Krebs buffer for 1 hour with gentle mixing every

10 min. The green fluorescence emission of calcium binding dye Fluo-3 was then analyzed following 488 nm laser excitation on a BD LSRII flow cytometer (Becton

Dickinson, Inc.). Buffer or E3CaG1 (2.2-220 nM) was added to cell suspension and data were collected after 10 min. Where indicated, cells were incubated with anti-CD47 antibody (B6H12) or antibodies to αv integrin (PW27 and 272-17E6) for 20-30 min at 4

°C prior to the addition of E3CaG1. Data were analyzed using FlowJo (v.7.6.4).

3.2.5. sGC activity and cGMP accumulation in intact cells

2+ To manipulate [Ca ]i, cells were incubated with ionomycin (1 µg/mL) and thapsigargin (400 nM), 20 mM EGTA or vehicle control, and 0-10 mM CaCl2 for 15 min, followed immediately by the addition of 10 µM DEA/NO. For experiments examining intracellular calcium chelation, cells were incubated with BAPTA-AM (10 µM, added from a 2 mM stock solution in DMSO) or vehicle control for 15 min prior to the addition of E3CaG1 (16 nM) or buffer for 15 min, and then DEA/NO (10 µM, for 2 min).

BAPTA-AM is a membrane permeable Ca2+ chelator that is converted to BAPTA in the cytosol, where it becomes trapped.

2+ To examine if E3CaG1 mediates an increase in [Ca ]i via activation of GPCRs and IP3 generation, Jurkat T cells were incubated with a cell permeable inhibitor of the

IP3-receptor, xestospongin-C (10 µM), for 30 min prior to the addition of E3CaG1 (22 nM). This was followed by the addition of DEA/NO (10 µM) for 2 min.

To examine the effect of PHA or Ang II on sGC activity, Jurkat T cells were grown in serum free media 12 h prior to the experiment at a density of less than 1 x 106 cells/mL. Where indicated, cells were incubated with 5 µM BAPTA-AM or vehicle control (DMSO) for 15 min, followed by the addition of indicated concentrations of PHA or 1 µM Ang II for an additional 2 min, and then DEA/NO (10 µM) for 2 min.

To examine the effect of E3CaG1 on sGC activity in HMVE cells, the cells were detached from tissue culture dish by treatment with EDTA and resuspended in Krebs buffer. Where indicated, E3CaG1 (22 nM) was added to cells for 15 min prior to the addition of 10 µM DEA/NO.

In all cases, reactions were stopped by quick pelleting of cells, followed by freezing in liquid nitrogen. Cell pellets were lysed in lysis buffer (Molecular Devices,

Catch-Point cGMP assay kit) or conjugate/lysis buffer (HTRF assay, Cisbio, Bedford,

MA). cGMP concentrations were measured by competitive ELISA (Catch-Point assay) or homogeneous time-resolved fluorescence (HTRF assay) according to the manufacturer’s instructions and expressed as pmoles of cGMP produced per mg of protein per 2 min (pmol/mg protein/2 min).

3.3. Results

2+ 3.3.1. E3CaG1 induces an increase in [Ca ]i

2+ TSP-1 and peptide 4N1K are known to increase [Ca ]i in fibroblasts and mast cells through a mechanism thought to require direct binding to CD47 (68, 76). We hypothesized that E3CaG1 inhibition of sGC in Jurkat T cells also involved changes in

2+ [Ca ]i and used digital imaging microscopy to examine this possibility. Jurkat T cells were transferred to matrix-coated coverslips for these experiments and allowed to attach for at least 1 hour, well beyond the time where attachment-associated Ca2+ spikes have been previously described, which persist for ～ 8 min post-attachment (78). At rest,

2+ Jurkat T cells displayed [Ca ]i of 10-25 nM. Addition of E3CaG1 (22 nM final

2+ concentration) induced an increase in [Ca ]i to 150-300 nM (Figures 3.1 and 3.2).

Similar increases were not observed after washing with Hank’s basal salt solution

(HBSS, pH 7.4) alone (Figure 3.2, black trace). Calcium concentrations could be experimentally controlled within Jurkat cells using the calcium chelator BAPTA (Figure

3.3) or the Ca2+ ionophore ionomycin and sarco/endoplasmic reticulum Ca2+ ATPase

(SERCA) pump inhibitor thapsigargin (Figure 3.4). None of the treatment conditions altered cell morphology within the period of measurement as assessed by differential interference contrast microscopy.

2+ 3.3.2. E3CaG1-dependent increases in [Ca ]i requires CD47

We examined the requirement for CD47 using flow cytometry and the fluorescent

Ca2+ indicator Fluo-3. Binding of 2.2 nM or 22 nM E3CaG1 to Jurkat T cells in

65 suspension led to an ~100-fold increase in average fluorescence over addition of buffer alone (Figure 3.5A). Thus, Jurkat T cells in suspension behaved similarly to those attached to coverslips. Addition of anti-CD47 antibody B6H12 completely blocked

E3CaG1-dependent calcium mobilization (Figure 3.5A and 3.6). Likewise, cell line

JinB8, which is a modified Jurkat T cell lacking CD47 (71), is not sensitive to E3CaG1

(Fig. 3.5B). We conclude that E3CaG1 signaling requires CD47, as expected from previous studies (55). In contrast, antibodies to integrin αV, which is known to associate

2+ with CD47 (61), have no effect on E3CaG1-dependent increases in [Ca ]i (Figure 3.6) and subsequent inhibition of sGC (Figure 2.9).

2+ 3.3.3. E3CaG1 mediates increase in [Ca ]i and inhibits sGC in human microvascular endothelial cells

To test if our signaling pathway is intact in a primary cell line, we tested E3CaG1

2+ mediated increase in [Ca ]i in human microvascular endothelial cells. Addition of 22 nM E3CaG1 to these cells in suspension led to an ~25 fold increase in average fluorescence over addition of buffer alone (Figure 3.7A). Consistent with increase in

2+ [Ca ]i, cells incubated with E3CaG1 prior to the addition of 10 µM DEA/NO showed decreased cGMP accumulation, over buffer control (Figure 3.7B). We observed that sGC expression/activity in these cells decreases over time in culture (data not shown).

Figure 3.1. E3CaG1 induces an increase in intracellular calcium. Snapshot of cells in the imaging field at individual time points over a 10 min experiment. The coloration

2+ indicates [Ca ]i after addition of E3CaG1 (22 nM). Six Jurkat T cells of the 40 in he

2+ field of view are circled for emphasis. E3CaG1 induced [Ca ]i peaks in this and similar experiments range from 75 nM up to 300 nM. Reprinted with permission from

2+ Figure 3.2. Calcium ion fluctuations with time. The red and blue lines are [Ca ]i traces over time for two representative cells upon addition of E3CaG1 (22 nM). The black trace represents a typical cell response following addition of the vehicle control

American Chemical Society.

2+ Figure 3.3. [Ca ]i over time of a typical Jurkat T cell following the addition of

E3CaG1 (22 nM) after preincubation with intracellular calcium chelator BAPTA-AM

Society.

2+ Figure 3.4. [Ca ]i over time of a typical Jurkat T cell following addition of 2 mM extracellular CaCl2 in the presence of ionomycin (1 µg/mL) and thapsigargin (400 nM).

2+ [Ca ]i rises to 800 nM under these conditions. Reprinted with permission from (1).

2+ Figure 3.5. E3CaG1-induced increase in [Ca ]i are cell adhesion independent but require CD47. A. Flow cytometry histograms of green fluorescence emission by calcium binding dye Fluo-3. Addition of E3CaG1 (2.2 or 22 nM) to Jurkat T cells results in a 90-100-fold increase in Fluo-3 fluorescence over addition of buffer. Prior incubation with anti-CD47 antibody B6H12 abolishes E3CaG1-dependent increases in

American Chemical Society.

2+ Figure 3.6. CD47 is necessary for E3CaG1-dependent increase in [Ca ]i. Flow cytometry histograms showing incubation of Jurkat T cells with E3CaG1 increases

2+ [Ca ]i and this is abolished with prior incubation of cells with anti-CD47 antibody

(B6H12). In contrast prior incubation with αv integrin antibodies has no effect,

2+ showing that αv integrin is not involved in E3CaG1-mediated increase in [Ca ]i..

Reprinted with permission from (1). Coyright 2011 American Chemical Society.

2+ Figure 3.7. E3CaG1 induces increase in [Ca ]i in human microvascular endothelial cells and inhibits sGC. A. Flow cytometry histogram showing E3CaG1 (22 nM)

2+ increases [Ca ]i, in primary human microvascular endothelial cells. B. Consistent

2+ with increase in [Ca ]i,, these cells show inhibited sGC activity when treated with

E3CaG1, prior to the addition of DEA/NO (10 µM). Error bars represent the range between means of two independent experiments.

3.3.4. Calcium inhibits NO-inducible sGC activity in Jurkat T cells

On the basis of previous reports showing that calcium can inhibit sGC activity in

HEK 293 cells and also with purified protein (33, 36, 37), we examined whether this was also the case for Jurkat T cells. Jurkat cells were resuspended in Krebs buffer containing

1 µg/mL ionomycin and 400 nM thapsigargin and varying concentrations of extracellular

2+ 2+ 2+ calcium ([Ca ]e). Under these conditions, [Ca ]i is effectively set by [Ca ]e. NO-

2+ inducible cGMP accumulation was inversely proportional to [Ca ]e, and complete

2+ inhibition occurred at [Ca ]e = 4 mM (Figure 3.8). Chelating of extracellular calcium with EGTA abolished inhibition. Approximately 99% of cells were viable under each experimental condition, as indicated by trypan blue dye exclusion. Chelating intracellular Ca2+ with compound BAPTA also overcame inhibition of sGC by E3CaG1,

2+ indicating that E3CaG1 inhibits sGC through a mechanism requiring increased [Ca ]i.

In the absence of BAPTA, E3CaG1 reduced NO-stimulated cGMP production by 50%.

However, after preloading cells with BAPTA, E3CaG1 had no effect on cGMP

2+ production (Figure 3.9), consistent with no increase in [Ca ]i (Figure 3.3).

3.3.5. Angiotensin II and Phytohemoagglutinin inhibit NO-driven cGMP accumulation

Angiotensin II (Ang II) is a hormone that induces vasoconstriction through

2+ binding GPCR AT1 and inducing a sustained increase in [Ca ]i in target cells (79, 80).

2+ Figure 3.8. Increased [Ca ]i leads to sGC inhibition in Jurkat T cells. Jurkat T cells

(1 x 106) were incubated with Ca2+ ionophore ionomycin (1 µg/mL) and the SERCA- inhibitor thapsigargin (400 nM) for 15 min at room temperature, followed by the addition of EGTA (20 mM) or vehicle control, 0-10 mM CaCl2 as indicated, and 10

µM DEA/NO. The reaction was stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 5). * denotes p <

American Chemical Society.

Figure 3.9. Chelating free cytosolic Ca2+ with cell permeable chelator BAPTA-AM reverses sGC inhibition by E3CaG1. Jurkat T cells (0.5 x106) were incubated with 10

µM BAPTA-AM or vehicle control (DMSO) 15 min prior to the addition of E3CaG1

(16 nM) or buffer for an additional 15 min. This was followed by addition of

DEA/NO (10 µM); the reaction was stopped after 2 min. cGMP accumulation was measured and expressed in terms of percentage control (10 µM DEA/NO). Error bars represent the standard deviation from the mean of independent experiments (n = 4), and * denotes p < 0.001. Reprinted with permission from (1). Copyright 2011

American Chemical Society.

Phytohemoagglutinin (PHA) is a natural agonist of the T-cell receptor that transiently

2+ increases [Ca ]i (81). Since Jurkat T cells have Ang II and T-cell receptors (81, 82), we asked whether Ang II and PHA would inhibit cGMP production by sGC. Addition of

PHA inhibited NO-stimulated sGC activity in a dose-dependent manner to 60% at the highest concentration examined (50 µg/mL, Figure 3.10). Addition of Ang II increased

2+ [Ca ]i to a similar level as did E3CaG1 and inhibited NO-stimulated sGC activity by

40% (Figures 3.11 and Figure 3.12). As with E3CaG1, chelating intracellular calcium with BAPTA reversed this inhibition (Figure 3.12). Pretreatment with pertussis toxin has no effect in either case, showing lack of involvement of G protein, Gi (Figure 3.11).

2+ 3.3.6. E3CaG1 does not increase [Ca ]i via an IP3- dependent mechanism

2+ To elucidate whether E3CaG1 causes an increase in [Ca ]i via release of IP3- sensitive intracellular calcium stores, we used xestospongin C, a membrane permeable inhibitor of IP3 receptor. Incubation of cells with xestospongin C (10 µM) for 30 min prior to the addition of E3CaG1 does not reverse sGC inhibition. In contrast, addition of xestospongin C to cells prior to the addition of Ang II abolished the Ang II-dependent

2+ increase in [Ca ]i, as expected since Ang II is known to signal via IP3 generation. This

2+ shows that E3CaG1-dependent increase in [Ca ]i does not involve IP3 (Figure 3.13).

Attempts to measure IP3 in Jurkat T cell lysates were unsuccessful. We did not see any appreciable change in IP3 production across all conditions we used, even for our positive control (Ang II).

Figure 3.10. Stimulation of calcium release by PHA results in sGC inhibition. Jurkat cells (0.5 x106) were incubated with BAPTA-AM (5 µM) for 15 min prior to the addition of the indicated concentrations of PHA or buffer, followed immediately by the addition of DEA/NO (10 µM). Reactions were stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 5), and * denotes p < 0.01. Reprinted with permission from (1). Copyright 2011

American Chemical Society.

2+ Figure 3.11. Ang II increases [Ca ]i. to the same levels as does E3CaG1. Flow cytometry histograms of green fluorescence emission of calcium binding dye Fluo-3

2+ 2+ as a function of changing [Ca ]i. Ang II (1 µM) increases [Ca ]i to the same levels as does E3CaG1. Pretreatment with pertussis toxin has no effect in either case.

Figure 3.12. Stimulation of calcium release by Ang II results in sGC inhibition.

Jurkat cells (0.5 x106) were incubated with 5 µM BAPTA-AM or vehicle control

(DMSO) 15 min prior to the addition of Ang II (1 µM) or buffer and additional 2 min incubation, followed by the addition of DEA/NO (10 µM). The reaction was stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 5), and * denotes p < 0.01. Reprinted with permission from (1).

2+ Figure 3.13. E3CaG1 does not mediate changes in [Ca ]i in an IP3-dependent mechanism. Jurkat T cells (0.5 x 106) were incubated with xestospongin C (10 µM) for 30 min prior to the addition of E3CaG1 or Ang II. This was followed immediately by addition of DEA/NO (10 µM). Reactions were stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 3).

3.4. Conclusions and Discussion

A major finding from the experiments described in this chapter is that TSP-1 and

Ang II inhibit NO signaling via sGC by increasing intracellular calcium levels. Binding of the TSP-1 derived fragment E3CaG1 to CD47 in Jurkat cells and HMVE cells causes

2+ free [Ca ]i to increases from resting levels of 5-10 nM to peak levels of 300 nM, leading to strong inhibition of sGC (Figures 3.2, 3.7 and 3.9). In Jurkat T cells, blocking this increase with chelator BAPTA reverses the sGC inhibition (Figure 3.9). Inclusion of an

IP3 receptor inhibitor has no effect on E3CaG1 inhibition of sGC (Figure 3.13). Inducing

2+ an increase in [Ca ]i with PHA (Figure 3.10), Ang II (Figure 3.12), or with calcium ionophore ionomycin and SERCA inhibitor thapsigargin (Figure 3.8), also leads to sGC inhibition. These data make clear that Ca2+ regulates sGC activity.

The link between Ang II and sGC is particularly interesting. Ang II is part of the renin-angiotensin-aldosterone system for controlling blood pressure through the sensing of blood volume and the linking of kidney function to blood flow (83). The Ang II receptors are G-protein coupled receptors, the most common of which is angiotensin

2+ receptor type 1 (AT1). Binding leads to an increase in [Ca ]i through the production of inositol triphosphate (IP3) and subsequent binding to the IP3-sensitive calcium channel of the sarco/endoplasmic reticulum. In vascular smooth muscle, Ca2+ stimulates myosin light chain kinase, which phosphorylates myosin, leading to vasoconstriction.

Angiotensin converting enzyme inhibitions (84), and AT1 inhibitors, are commonly used to block this pathway and vasoconstriction in the treatment of hypertension (85). NO-

2+ stimulated sGC produces cGMP, which lowers [Ca ]i through multiple mechanisms, but

82 in particular via phosphorylation of regulatory protein phospholamban by cGMP- dependent protein kinase G (PKG), which leads to stimulation of SERCA and the pumping of calcium from the cytosol into cellular stores (86). Interestingly, TSP-1 can also inhibit PKG, further attenuating NO signaling (87). Thus, our Ang II data suggest a feedback mechanism that serves to balance vasodilation through NO and vasoconstriction through Ang II by directly raising and lowering calcium levels. While it is clear that calcium regulates sGC activity, the nature of this regulation is explored in the following chapter.

CHAPTER 4

MECHANISM OF CALCIUM REGULATION OF sGC

4.1. Calcium regulation of sGC

Subcellular localization, dimerization status, phosphorylation, protein-protein interaction and S-nitrosation have all been implicated in sGC regulation (13, 24, 27, 29,

31, 32, 88-96). The studies in this chapter explore how calcium regulates sGC in the

TSP-1/CD47 pathway. Possible mechanisms we considered were:

1. Calcium was directly inhibiting sGC through binding to an allosteric site or

displacing Mg2+. These possibilities have been previously raised (36, 37).

2. Calcium stimulated calcium-dependent kinases/phosphatases leading to covalent

modification of sGC through phosphorylation or dephosphorylation.

3. Calcium stimulated a phosphodiesterase (PDE) leading to lower cGMP

accumulation in cells.

We explored these possibilities and found that Ca2+-dependent inhibition of sGC requires a tyrosine kinase and that sGC was inhibited through phosphorylation, leading to an increase in Km for GTP. We also found that YC-1 family of compounds, in clinical trial for treating hypertension, overcomes inhibition. Most of the work in this chapter has been published (1).

4.2. Materials and Methods

4.2.1. Materials

PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and 8-methoxymethyl

IBMX were obtained from Calbiochem. Compounds YC-1 and BAY 41-2272 were obtained from Cayman Chemicals. All other materials were obtained from Sigma unless otherwise noted.

4.2.2. Cell culture

Jurkat T cells and MCF-7 cells were cultured as described in chapter 2.

4.2.3. Phosphodiesterase activity

4.2.3.1. Jurkat T cells

25 x 106 Jurkat T cells were used for each assay condition and were incubated with buffer or E3CaG1. Cell pellets were lysed into 600 µL homogenization buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, 1 mM PMSF, protease inhibitor cocktail (10 µL/mL cell lysate)) using a homogenizer. Lysate was spun at 13000 x g and supernatant was incubated with or without IBMX (0.5 mM) and 8- methoxymethyl IBMX (0.4 mM) for 10 min. This was followed by the addition of Mg2+-

GTP reaction buffer and DEA/NO (10 µM). Reactions were stopped after 2 min by the addition of 250 µL of cell lysis buffer (Molecular Devices, Sunnyvale, CA). cGMP

85 concentrations were determined by competitive ELISA using the CatchPoint cGMP

(Molecular Devices), following manufacturers instructions.

4.2.3.2. MCF-7 cells

To examine the effect of phosphodiesterases on cGMP accumulation in intact cells, MCF-7 cells transiently transfected with sGC were used. 14 hours post- transfection, cells were trypsinized and resuspended in Krebs buffer. Cells were incubated with vehicle/DMSO, IBMX (0.5 mM), or 8-methoxymethyl IBMX (0.4 mM) for 30 min, followed by the addition of ionomycin (1 µg/mL), thapsigargin (400 nM) and calcium chloride (0.1 mM) to appropriate samples, followed immediately by the addition of DEA/NO (10 µM). After 2 min, cells were spun down and cell pellets frozen and cGMP accumulation measured by ELISA (Catchpoint-cGMP kit, Molecular Devices).

4.2.4. Effect of compounds YC-1 and BAY 41-2272

4.2.4.1. Jurkat T cells

To examine the effect of compounds YC-1 and BAY 41-2272 on E3CaG1 inhibition of sGC, cells were incubated with E3CaG1 (22 nM) for 15 min prior to the addition of 10 µM YC-1 or 10 µM BAY 41-2272, or vehicle control (DMSO), followed immediately by the addition of DEA/NO. After 2 min, cells were spun down and cell pellets frozen and cGMP accumulation measured.

4.2.4.2. Immuno-precipitated sGC

sGC immunoprecipitated from transiently-transfected MCF-7 cells was treated with 0 µM or 100 µM CaCl2 prior to the addition of Mg-GTP reaction buffer and 10 µM

YC-1 or 10 µM BAY 41-2272. This was followed immediately by the addition of 10 µM

DEA/NO. Reactions were carried out at 37 °C for 5 min and cGMP accumulation was measured.

4.2.5. Kinetics

To study kinetic properties of calcium-inhibited sGC, transiently transfected

MCF-7 cells were either treated with DMSO (vehicle control) or ionomycin, thapsigargin, and 2 mM CaCl2 for 5 min. Cell pellets were lysed as described above and incubated with FLAG agarose beads for 1.5 hours at 4 °C. Following this, the beads were washed three times and resuspended in an appropriate volume of Tris buffered saline (pH 7.5). Aliquots of this slurry were then used for activity measurements.

Reactions were carried out at 37 °C in a final volume of 150 µL and initiated by the addition of reaction buffer (5-2000 µM GTP, 8 mM MgCl2, 50 mM HEPES, pH 7.5, prepared at 10X concentration just prior to use). Where NO-induced activities were measured DEA/NO (50 µM) was added immediately after the addition of reaction buffer.

Reactions were quenched by the addition of cell lysis buffer from the cGMP kit, generally after 10 min (−NO) or 3 min (+ NO). Catalytic rates were linear over these time periods for all GTP concentrations used. Inclusion of equal quantities of immunoprecipitated sGC was confirmed by western blot analyses. For each experiment,

87 cGMP accumulation was measured in duplicate using the cGMP-ELISA kit from

Molecular Devices; higher concentrations (upto 4 mM GTP) did not interfere with the measurements. Kinetic parameters were obtained by non-linear fitting of the Michealis-

Menten equation, using Sigma Plot (SPSS Inc., Chicago, IL). Km and Vmax are presented as the average and standard deviation of three independent experiments.

4.2.6. Cell-free inhibition of sGC

MCF-7 cells were transiently transfected with sGC; 14 h post-transfection, cells were trypsinized and pellets were lysed in homogenization buffer. Immunoprecipitation of sGC was performed as described above. Jurkat cell lysate was then incubated with sGC on the beads for 15 min at 37 °C with or without 250 nM Ca2+ and/or staurosporine

(1 µM) and genistein (10 µM). Where indicated, sGC was treated with alkaline phosphatase for 30 min at room temperature. Following this, beads were washed five times with TBS and resuspended in an appropriate volume for the activity assay. Where indicated, 10 µM DEA/NO and 10 µM YC-1 were included in the reactions.

4.3. Results

4.3.1. Phosphodiesterases have minimal effect

Previous studies have indicated that TSP-1 dependent inhibition of cGMP accumulation was through inhibition of sGC and not through stimulation of phosphodiesterases (45, 46). To confirm that this was also true under the conditions of our experiments, we examined cGMP accumulation when PDE was inhibited. We first

88 sought to directly inhibit PDE proteins in live Jurkat T cells using 3-isobutyl-1- methylxanthine (IBMX), a general PDE inhibitor, or 8-methoxymethyl IBMX, a specific inhibitor of calcium/calmodulin-dependent PDE1. Unfortunately, these compounds activate T cells, possibly through a cAMP-dependent mechanism (97), which interferes with the measurement of E3CaG1 activity. We therefore measured NO-dependent cGMP accumulation in Jurkat T cell lysate obtained from cells that were previously treated with

E3CaG1 or buffer control; measurements were made in the presence or absence of IBMX and 8-methoxymethyl IBMX. Under these conditions, calcium is diluted and PDEs inhibited. Inhibition of NO-driven sGC activity persisted in the cell free lysate, consistent with the lack of involvement of PDEs in this pathway (Figure 4.1).

We further examined the role of PDEs using transiently expressed human sGC in

2+ MCF-7 cells, which do not normally express sGC. Raising [Ca ]i in these cells led to pronounced inhibition of NO-stimulated sGC activity (Figure 4.2). Addition of IBMX or

8-methoxymethyl IBMX led to small increases in basal and NO-stimulated and calcium- inhibited cGMP levels; however, the 60% reduction in NO-stimulated cGMP

2+ accumulation due to increased [Ca ]i was unchanged in the presence of these compounds, indicating PDEs have at most a minor role in the observed loss of cGMP under the conditions of our experiments.

4.3.2. YC-1 and BAY 41-2272 overcome Ca2+ inhibition of sGC in live cells.

YC-1 and BAY 41-2272 are small molecule activators of sGC that act synergistically with NO and CO (98); the related compound BAY 63-2521 (Riociguat) is

89 in clinical trial for pulmonary hypertension (99). Cells were incubated with E3CaG1 for

15 min, prior to the addition of YC-1 or BAY 41-2272. When included at 10 µM,

E3CaG1 inhibiton of sGC was completely overcome by either YC-1 or BAY 41-2272

(Figures 4.3 and 4.4), suggesting that Ca2+ inhibition of sGC is through an allosteric mechanism that can be overcome by allosteric stimulators.

That sGC in diluted extracts remains inhibited (Figure 4.1) suggested that inhibition was through covalent modification. To further examine this possibility, we transiently expressed sGC in MCF-7 cells and isolated the protein through immunoprecipitation using a FLAG purification tag. The immunoprecipitated protein displays NO-stimulated activity (Figure 4.5). However, when cells were first treated with

Ca2+/ionomycin, the isolated protein was substantially inhibited (Figure 4.5). As with cellular sGC, addition of YC-1 or BAY 41-2272 reversed the inhibition. Thus, higher

2+ [Ca ]i leads to a modified sGC with reduced activity, and this activity can be overcome by allosteric stimulators.

2+ Two reports indicate that Ca can directly inhibit sGC isolated from bovine lung, with Ki values ranging between 0.15 and 98.5 µM depending on conditions and laboratory (36,

37). We examined direct inhibition of immunoprecipitated human sGC and found that addition of 100 µM Ca2+ led to 50 ± 2% inhibition of the NO-stimulated protein. Neither

YC-1 nor BAY 41-2272 was able to overcome direct inhibition by 100 µM Ca2+, in contrast to the inhibition of sGC by E3CaG1 in whole cells (Figure 4.6).

Figure 4.1. Phosphodiesterases are minimally involved in Ca2+-dependent lowering of cGMP in Jurkat cells. Jurkat T cells (25 x 106) were incubated with 22 nM E3CaG1 or vehicle control (buffer) for 15 min prior to lysis. Lysates were incubated with

IBMX (0.5 mM) and 8-methoxymethyl IBMX (0.4 mM) or vehicle control (DMSO) for 10 min. Following this, Mg2+-GTP reaction buffer and DEA/NO (10 µM) were added, and the reaction was stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 3), and * denotes p < 0.001.

Figure 4.2. Phosphodiesterases are minimally involved in Ca2+-dependent lowering of cGMP in MCF-7 cells. Transiently transfected MCF-7 cells were incubated with

IBMX (0.5 mM) or 8-methoxymethyl IBMX (0.4 mM), or DMSO (vehicle control) for 30 min prior to the addition of ionomycin (1 µg/mL), thapsigargin (400 nM), and calcium chloride (0.1 mM) to appropriate samples, followed immediately by the addition of DEA/NO (10 µM). After 2 min, cells were spun down and cell pellets frozen. cGMP was measured and expressed in terms of percentage control (10 µM

DEA/NO) to account for transfection efficiencies. Error bars represent the standard deviation from the mean of independent experiments (n = 3); and * denotes p < 0.001 and # denotes p < 0.01. Reprinted with permission from (1). Copyright 2011

American Chemical Society

Figure 4.3. YC-1 overcomes E3CaG1-dependent inhibition of sGC in Jurkat T cells.

Jurkat T cells (0.5 x 106 cells) were incubated with 22 nM E3CaG1 or buffer for 15 min prior to the addition of 10 µM YC-1 or vehicle control DMSO. This was followed immediately by the addition of 10 µM DEA/NO. Reactions were stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 5), and * represents p < 0.001. Reprinted with permission from (1).

Figure 4.4. BAY 41-2272 overcomes E3CaG1-dependent inhibition of sGC in Jurkat

T cells. Jurkat T cells (0.5 x 106 cells) were incubated with 22 nM E3CaG1 or buffer for 15 min prior to the addition of 10 µM YC-1 or vehicle control DMSO. This was followed immediately by the addition of 10 µM DEA/NO. Reactions were stopped after 2 min. Error bars represent the standard deviation from the mean of independent experiments (n = 5), and * represents p < 0.001. Reprinted with permission from (1).

Figure 4.5. Immunoprecipitated sGC remains inhibited, but YC-1 and BAY 41-2272 overcome inhibition. Transiently transfected MCF-7 cells treated with DMSO or 0.1 mM CaCl2 in the presence of ionomycin/thapsigargin. Immunoprecipitated sGC was treated with 10 µM YC-1 or 10 µM BAY 41-2272, followed immediately by the addition of 10 µM DEA/NO. Reactions were carried out at 37 °C for 5 min, and cGMP accumulations was measured. Error bars represent the standard deviation from mean of independent experiments (n = 5). Reprinted with permission from (1).

Figure 4.6. YC-1 and BAY 41-2272 do not overcome direct Ca2+ -inhibition of sGC. sGC immunoprecipitated from transiently-transfected MCF-7 cells was treated with 0

µM or 100 µM CaCl2 prior to the addition of 10 µM YC-1 (A) or 10 µM BAY 41-

2272 (B). This was followed immediately by the addition of 10 µM DEA/NO.

Reactions were carried out at 37 °C for 5 min and cGMP accumulation was measured.

Error bars represent the standard deviation from the mean of independent experiments

(n = 5), and * denotes p < 0.001. Reprinted with permission from (1). Copyright

2011 American Chemical Society.

The fact that a high concentration of Ca2+ is needed to achieve substantial direct inhibition in vitro (micromolar vs nanomolar in the cell) and that YC-1 or BAY 41-2272 does not overcome this inhibition indicates that direct binding of Ca2+ to sGC does not contribute to the observed intracellular sGC inhibition.

4.3.3. Inhibited sGC exhibits an increase in Km

To further characterize inhibited sGC, we measured steady-state kinetic parameters for the protein after immunoprecipitation (Figure 4.7 and Table 4.1). The uninhibited protein displayed typical values for Km and Vmax (36, 37, 100, 101) and the expected decrease in Km and increase in Vmax upon stimulation by NO. In contrast, Km values for sGC isolated from calcium treated cells were dramatically increased and unaltered by NO stimulation (Table 4.1). The inhibited value (Km ～870 µM) is about twice the value for cellular GTP concentrations which are estimated to be ～470 µM in mammalian tissues (102). At this GTP concentration, the inhibited protein would operate in the cell well below Vmax, while the uninhibited protein, with Km = 67 µM when bound to NO, would be nearly saturated with GTP and operating at near maximal velocity.

4.3.4. Inhibition of sGC requires a calcium-dependent tyrosine kinase

We developed a cell-free assay for evaluating calcium-dependent inhibition of sGC. Jurkat T cell lysate led to inhibition of sGC upon addition of 250 nM Ca2+ but had no effect in its absence (Figure 4.8).

Figure 4.7. Representative kinetic plots for immunoprecipitated sGC obtained from

MCF-7 cells. Cells were lysed after treatment for 5 min with ionomycin, thapsigargin, and 2 mM CaCl2, or vehicle control. Reactions were carried out at 37 °C for 10 min

(− NO) or 3 min (+ NO). Where included DEA/NO (50 µM) was added just prior to measurement. Shown are the averages of duplicate measurements ± range in measured values. The solid curves represent the nonlinear fit to the Michaelis-Menten equation. Reprinted with permission from (1). Copyright 2011 American Chemical

Society.

KmGTP (µM) Vmax (pmol cGMP Fold-increase min-1)b (+Ca2+/–Ca2+)c

2+ 2+ 2+ 2+ –Ca +Ca –Ca +Ca Km Vmax sGC 234 ± 16 857 ± 31d 6.3 ± 1.4 5.1 ± 0.3d 3.7 0.8

+NO 69 ± 6 887 ± 71 41 ± 9 51 ± 16 12.9 1.2

Table 4.1. Cellular Calcium-Induced Changes in Kinetic Parameters of sGCa aValues obtained for immunoprecipitated sGC from MCF-7 cells. Ionomycin, thapsigargin and 2 mM CaCl2, or vehicle control, were added to the cells 5 min prior to lysis. bPresented as pmol min-1 since the quantity of sGC protein attached to the anti-FLAG agarose beads is unknown. Total lysate protein was ~65 µg per sample. cPresented as the ratio of +Ca2+/–Ca2+ values. dThis value is the average ± range of two independent experiments performed in duplicate. All other values are the average

± standard deviation of three independent experiments performed in duplicate.

Addition of a pan-kinase inhibitor, staurosporine, prevented inhibition indicating that a calcium-activated kinase is required for inhibition. Obvious candidates for this role are the multifunctional Ca2+/calmodulin-dependent protein kinases I, II and IV. However, addition of compounds KN-62 or KN-93, which inhibit these proteins (103, 104), had no effect on E3CaG1 inhibition of sGC (Figure 4.9).

Addition of genistein, a specific inhibitor of tyrosine kinases (105), also reversed

Ca2+-induced inhibition of sGC. Consistent with a phosphorylation event, treatment of sGC, with alkaline phosphatase after addition of Ca2+, restored activity (Figure 4.10).

Decrease in phosphorylation upon treatment with alkaline phosphatase was confirmed by western blot (Figure 4.11).

4.4. Conclusion and discussion

Phosphodiesterase inhibitors did not reverse E3CaG1/Ca2+-mediated inhibition of sGC, therefore we ruled out the involvement of phosphodiesterases in the pathway. Allosteric activators BAY 41-2272 and YC-1 overcome Ca2+-inhibition of sGC, suggesting that inhibition is through an allosteric mechanism. Moreover, we can isolate inhibited sGC and kinetic analysis shows that inhibited sGC exhibits an increase in Km for GTP.

We developed a cell free assay, wherein, sGC isolated from transiently transfected cells when incubated with cell lysate (Jurkat T, MCF-7 or HEK 293T cells) and 250 nM Ca2+ is less active. Calcium-inhibition of sGC requires a tyrosine kinase and treatment of inhibited sGC with alkaline phosphatase reverses the inhibition.

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Figure 4.8. Cell free assay. Immunoprecipitated sGC treated with Jurkat cell lysate and 250 nM Ca2+ is inhibited; inhibition is reversed by broad-range kinase inhibitor staurosporine. sGC was immunoprecipitated from transiently transfected MCF-7 cells and incubated with Jurkat T cell lysate with or without 250 nM Ca2+ and/or staurosporine (1 µM). Calcium and staurosporine were washed away and cGMP activity was measured. Where indicated, DEA/NO and YC-1 were added to a final concentration of 10 µM. Error bars represent the standard deviation from the mean of independent experiments (n = 3). Reprinted with permission from (1). Copyright

2011 American Chemical Society.

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Figure 4.9. Calcium/Calmodulin-dependent kinases are not involved in Ca2+- dependent inhibition of sGC. Jurkat T cells (0.5 x106) were incubated with or without

KN-93 or KN-62 (10 µM) for 30 min prior to the addition of E3CaG1 or buffer for 15 min. This was followed by the addition of DEA/NO (10 µM) for 2 min. Cells were spun down and cGMP accumulation was measured. Error bars represent range from means from two independent experiments.

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Figure 4.10. Genistein and alkaline phosphatase reverse inhibition of sGC.

Immunoprecipitated sGC treated with Jurkat lysate with or without 250 nM Ca2+ is inhibited; inhibition is reversed by broad-range tyrosine kinase inhibitor genistein (10

µM). Consistent with this, treatment of sGC with alkaline phosphatase after treatment with calcium, results in restoration of activity. sGC was immunoprecipitated from transiently transfected MCF-7 cells and incubated with Jurkat T cell lysate with or without 250 nM Ca2+ and/or genistein. Calcium was washed away before incubation with alkaline phosphatase where indicated, for 30 min at room temperature. Where indicated, DEA/NO (10 µM) was added and cGMP activity measured. Error bars represent standard deviation from the mean of independent experiments (n = 3).

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Figure 4.11. Alkaline phosphatase reverses phosphorylation of sGC. Western blot showing alkaline phosphatase treatment leads to loss of phosphorylation of sGC. sGC immunoprecipitated from transiently transfected MCF-7 cells was incubated with

Jurkat cell lysate with or without Ca2+ (250 nM). After washing calcium away, sGC was incubated with or without alkaline phosphatase for 30 min at room temperature.

Samples that were used for measuring cGMP were later used for western blotting.

Samples were separated on SDS-PAGE gel and transferred onto nitrocellulose membrane. The blot was probed with anti-pTyr antibody (Santa Cruz Biotechnology) and anti-FLAG-antibody (Sigma-Aldrich Inc.) and the heavy chain of IgG was used as

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This suggests that sGC is phosphorylated and that the phosphorylation is inhibitory. We do not know how many steps there are between release of calcium and phosphorylation of sGC. That sGC is also regulated by phosphorylation comes as no surprise.

Phosphorylation is a post-translational modification that regulates most of the proteome.

The net phosphorylation status of proteins may never change, but change in phosphorylation at key residues can lead to significant changes in activity. In order to pursue the role of phosphorylation in regulation of activity of sGC and towards understanding the complete mechanism by which calcium inhibits sGC, we needed a more robust source of human sGC. Other members of our lab have tried to purify human sGC from other sources such as E.coli and SF9 insect cells. However the protein is either insoluble or inactive. Therefore we decided to use lentiviral expression system. We reasoned that production of human sGC from transformed human cell lines such as HEK

293T or MCF-7 cells might be more fruitful.

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CHAPTER 5

IDENTIFICATION OF KINASE(S) REQUIRED FOR CALCIUM INHIBITION OF

SOLUBLE GUANYLATE CYCLASE

5.1. Human kinome

Kinases are broadly divided into three categories depending on the preference for the residue they phosphorylate in a target protein, serine/threonine kinases, tyrosine kinases and dual specificity kinases. The human genome has 518 protein kinases, constituting about 1.7% of the human genome. Out of these, 90 are tyrosine kinases

(106). The tyrosine kinases can be subdivided into receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases. RTKs are type I transmembrane proteins possessing an N- terminal extracellular domain, which can bind activating ligands, a single transmembrane domain and a C-terminal catalytic domain. Non-receptor tyrosine kinases are mostly soluble cytosolic proteins. A subset of these cytosolic tyrosine kinases can be targeted to the membrane (107).

This chapter describes efforts made to uncover which of the tyrosine kinases of the human kinome are required for calcium-inhibition of sGC. In order to establish a more robust source of sGC, I developed a lentiviral expression system. The advantage of using a lentivirus-based expression system is that they can infect both dividing and non- dividing cells, so even primary cell lines can be used for experiments. In addition, lentivirus encoding the gene of interest can be stored for years without significant loss in titer. This chapter outlines expression and purification of sGC produced by lentiviral

106 expression. I also describe experiments I did to uncover which kinase(s) are required for calcium-dependent inhibition of sGC. We used a pharmacological approach with some broad-range inhibitors to rule out major classes of kinases, such as PKA and PKC. I used fractionation of cellular lysate to rule out RTKs. I then used some specific inhibitors of the non-receptor tyrosine kinases to narrow down the possibilities. In addition, I performed protein-knockdown via antibody depletion of certain candidate kinases that we hypothesized were involved, such as Pyk2 and c-src kinase. I also performed bioinformatics analysis of the expression of tyrosine kinases in the three cell lines that showed calcium-dependent inhibition of sGC. Dr. Stacy Mazzalupo, a former post doctoral researcher in our lab had cloned rat and bovine sGC subunits into various carrier vectors. I subcloned rat sGC α subunit from pGEMT-Easy (Promega Inc.) into pCMV-

3Tag-3A (Agilent Technologies) and cow sGC β subunit into pCMV-3Tag-9. All of the work in this chapter and future RNAi experiments will be described in a manuscript currently under preparation.

5.2. Materials and Methods

5.2.1. Materials

The lentiviral expression system (Lenti-X™ Tet-Off® Advanced Inducible expression system) was purchased from Clontech Laboratories, Inc. (Mountain View,

CA). Restriction enzymes and other routine molecular biology grade reagents were from

Fermentas Inc. Inhibitors PP1, PP2, GF109203X, Sunitinib were a kind gift from Dr.

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Benjamin Jester and Dr. Indraneel Ghosh. Gleevec® was a kind gift from Dr. Richard

Vallaincourt. All other reagents were from Sigma Aldrich, unless otherwise noted.

5.2.2. Methods

5.2.2.1. Cloning of human sGC alpha and beta subunit into lentiviral vector

Primers 5’-cccggatccatgttctgcacgaagctcaaggat-3’ and 5’- gggcccgaattcttacagatcttcctcagagat-3’ were used to amplify human sGC α1 subunit for cloning into pLVX-Tight-Puro (Clontech Laboratories, Inc., Mountain View, CA) between BamHI and EcoRI sites, while keeping the c-terminal myc tag on the protein intact. Primers 5’-accgcggccgcatgtacggatttgtgaatcacgccctg-3’ and 5’- gccgaattcctatttatcgtcatcatctttgtagtccttgtcatcatc-3’ were used to amplify human sGC β1 subunit for cloning into pLVX-Tight-Puro (Clontech Laboratories Inc., Moutain View,

CA) between NotI and EcoRI sites, while keeping the c-terminal FLAG tag on the protein intact.

5.2.2.2. Production of Lentivirus

Approximately 24 hours prior to transfection, 4.5 x 106 Lenti-X HEK 293T cells were seeded into one 10 cm tissue culture plate and incubated overnight at 37 °C, 5 %

CO2. Three such plates were made, one for each of the viruses to be made (Tet-activator, sGC alpha and sGC beta). On the day of transfection, the cells were 80-90% confluent.

Transfection was performed as per manufacturers instructions. 48 h after the addition of transfection complexes, lentiviral supernatants were collected and filtered through a 0.45

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µm cellulose acetate filter to remove cellular debris. Lentivirus produced was aliquoted and stored at -80 °C. The virus titer was determined using an ELISA for p24 (Clontech,

Mountain View, CA) and expressed as IFU/ml.

5.2.2.3. Transduction of target cells

We transduced MCF-7, HEK 293T and HEK 293S cells. Multiplicity of infection

(MOI) was determined as follows:

MOI = (Virus titer * ml of virus)/ number of cells

We initially chose an MOI of 0.5 and 2 to test the efficacy of transduction. 12-18 hours prior to transduction, cells were seeded so as to yield 15-20% confluence. Appropriate volume of each of the three viruses was added to cells to yield an MOI of 0.5 or 2.

Polybrene (4 µg/mL) was added to the cells to increase efficiency of virus transduction.

Doxycycline (1 µg/mL) was also added to inhibit sGC transcription. Transduced cells were then maintained in media containing G418 (500 µg/mL) and puromycin (3 µg/mL) to select for cells that had integrated the Tet-transactivator gene (along with the gene for

G418 resistance) and human sGC alpha and beta subunit genes (along with the gene for puromycin resistance) into their genome. Selection of cells that had all three genes stably integrated took 4 weeks, after which the antibiotic concentrations were decreased (G418-

200 µg/mL and puromycin-1 µg/mL), doxycycline concentration was maintained at 1

µg/mL. To ascertain inducibility of expression, both transiently transduced (cells that were not subject to selection with antibiotics for 4 weeks) and stably transduced cells

109 were placed in media containing no doxycycline/tetracycline (DMEM supplemented with

Tc-free FBS) for a period of 48 hours. Cells were harvested at this time and cell lysates used for western analysis to check for expression of both alpha and beta subunit of sGC.

NO-inducible activity was also measured.

5.2.2.4. Inhibitor screening

HEK 293T-hsGC cells (cells that were stably transduced) were placed in doxycycline-free media for 48 hours. Cells were harvested and lysed in homogenization buffer and sGC was immunoprecipitated with FLAG beads. Jurkat T cell lysate was then incubated with sGC (on FLAG beads) for 15 min at 37 °C with or without 250 nM Ca2+ and/or genistein, PP1, PP2, Gleevec, GF109203X, or Sunitinib (0-1 mM). Following this, beads were washed five times with TBS and resuspended in an appropriate volume measuring NO-inducible activity.

5.2.2.5. Jurkat T cell lysate fractionation

Jurkat T cell lysate was first centrifuged at 14000 rpm on a tabletop centrifuge and then ultracentrifuged at 54000 rpm for 20 min at 4 °C. The ultracentrifuged supernatant containing only cytosolic proteins was used for cell free assay. sGC immunoprecipitated from HEK 293T-hsGC cells was incubated with ultracentrifuge supernatant for 15 min at 37 °C, with or without 300 nM Ca2+. Following this, beads were washed five times with TBS and resuspended in appropriate volume for activity measurement. Where indicated, 10 µM DEA/NO was included in the reactions.

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5.2.2.6. Depletion of candidate kinases

Jurkat T cell lysate was depleted of certain candidate kinases such as Pyk2 and c- src, to see if the depleted lysate was still capable of inhibiting sGC in the presence of calcium. Lysate was incubated with appropriate antibody-protein A/G beads overnight at

4 °C. Lysate was checked for depletion by western blot and used for cell-free sGC inhibition as described above.

5.2.2.7. Transient transfection of rat sGC

MCF-7 cells were plated in 10-cm dishes so as to yield 50-60% confluence on the day or transfection. Transfection reagent TurboFect™ (Fermentas, Glen Burnie, MD) was used at a ratio of 20 µg plasmid DNA (1:1 ratio of sGCα: sGCβ) to 25 µl reagent per

10-cm dish of cells. 14 hours post-transfection, cells were harvested by trypsinization and cell pellets were quickly frozen in liquid nitrogen. Expression of sGC subunits was detected by presence of NO-inducible activity and western blotting.

5.2.2.8. Cell free assay with rat sGC

The purpose of this experiment was to see if rat sGC was regulated by calcium.

Rat sGC on FLAG beads, were incubated with Jurkat T cell lysate for 15 min at 37 °C with or without 250 nM Ca2+. Following this, beads were washed five times with TBS and resuspended in appropriate volume for activity measurement.

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5.2.2.9. Bioinformatics

I performed bioinformatics analysis on already published gene expression studies in the three cell lines that I have used to study our signaling pathway: HEK 293T cells,

MCF-7 and Jurkat T cells. NCBI has assembled gene expression studies in a central place called the Gene Expression Omnibus (GEO) Data sets. An initial search for each of the three different cell lines produced 2, 7 and 29 data sets for HEK 293T cells, Jurkat T cells and MCF-7 cells, respectively. The description for each of these data sets outlines the study; from this I determined whether the study included gene expression data for untreated control and only those data sets were downloaded. Fully annotated excel spreadsheets that contained gene ID, gene name and expression levels were used. The expression of genes such as GAPDH, CD47, ubiquitin and actin were used for internal controls and if expression for these genes were not consistent among data sets for the same cell line, then the data set was disregarded and not used for further analysis. A list of tyrosine kinases expressed in the three cell lines was generated as outlined in Figure

5.2.

5.3. Results

5.3.1. Lentivirus production, titer determination and production of stable cell line HEK

293T-hsGC

We chose Lenti-X™ Tet-Off® Advanced Inducible expression system from Clontech

Laboratories, Inc. We wanted an inducible expression system since we had determined, with both transient transfections and the stable HT-1080-28 cell line that expresses sGC,

112 that constitutive expression of sGC is toxic to cells. In E.coli, the Tet repressor protein

(TetR) negatively regulates the genes of the tetracycline-resistance operon. TetR blocks the transcription of these genes by binding to the tet operator sequences (tetO) in the absence of tetracycline (Tc). In the presence of Tc, TetR can no longer bind to the operator sequences and transcription of resistance-mediating genes begin (108). This forms the basis for the Lenti-X™ Tet-Off® expression system from Clontech (Figure

5.1). I cloned sGC alpha and beta subunit into the lentiviral vector. Transfection of

Lenti-X HEK293T cells produced lentiviral particles 48 hours post-transfection. The titer of all three viruses (Tet, sGC alpha and sGC beta) was determined to be between 106-108

IFU/ml using p24 ELISA kit (Clontech, Moutain View, CA). We transduced cells

(MCF-7, HEK 293T and HEK-S) with an MOI of 0.5 or 2 for all three viruses. Of the three cell lines tested, the transduction efficiency was highest in HEK 293T cells, as determined by the number of cells that survived the antibiotic selection. Table 5.1 describes our findings. We have since made permanent cell line HEK 293T-hsGC that, upon induction by withdrawal of doxycycline, expresses active human sGC. Expression is analyzed by western blotting with anti-FLAG antibody for the beta subunit. We do not have a good antibody for the alpha subunit; therefore NO-inducible activity is a measure of functional protein. Frozen aliquots of these cells survive freezing and thawing. We have used these cells as our source for sGC for all experiments described in this chapter.

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Figure 5.1. Illustration of the Tet-Off lentiviral expression system. In the absence of tetracycline/doxycycline the tet activator protein (tTA) binds to the operator and drives transcription of the gene of interest via a CMV promoter. In the presence of tetracycline/doxycycline, tTA is unable to bind to the tet operator and transcription is maintained in a basal inactive state.

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Figure 5.2. Flowchart illustrating bioinformatics analyses.

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MCF-7 cells Expression NO-inducible Survival activity Transient Yes 29 fold Yes transduction

Stable Yes Not determined Did not survive transduction HEK-293T cells Transient Yes 24 fold Yes transduction

Stable Yes 45 ± 10 fold Yes transduction HEK 293-S cells Transient Yes 23 fold Yes transduction

Stable Not determined Not determined Did not survive transduction

Table 5.1. Summary of lentiviral expression studies. MCF-7 cells can be transiently

transduced to express sGC, however stable transduction led to cells with an

unfavorable phenotype: 50% of cells would still attach to tissue culture dishes and

look very healthy; however, after a few days in culture there were increasing amounts

of cell debris/floating populations of cells. HEK 293T cells were successfully

transduced both transiently and stably to yield active protein. HEK 293-S cells could

only be transiently transduced to yield active protein. Stable transduction did not yield

many surviving cells.

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5.3.2. Jurkat T cell lysate fractionation

I had previously shown that staurosporine, a broad range protein kinase inhibitor, and genistein, a broad range tyrosine kinase inhibitor (105, 109), were able to reverse calcium-mediated inhibition of sGC (Figure 4.8 and 4.10). This led us to conclude that a tyrosine kinase is central to our pathway. To rule out the involvement of trans-membrane receptor tyrosine kinases, I fractionated Jurkat T cell lysate into membrane fraction and cytosolic fraction by ultracentrifugation. I used the cytosolic fraction of the lysate for the cell free assay. Incubation of sGC on beads with the ultracentrifuge supernatant, with

250 nM Ca2+, led to an inhibition in activity, indicating that the tyrosine kinase involved in our pathway is a cytosolic protein and not a receptor tyrosine kinase.

5.3.3. Inhibitor screening

To further narrow the possible tyrosine kinases involved, more specific inhibitors were examined. IC50 values for these were determined so as to match the published values for specific kinases. Table 5.2 summarizes these findings. Genistein, a tyrosine kinase inhibitor, inhibited the kinase in the calcium-sGC pathway with an IC50 of 56 ± 7 nM. Gleevec® (Imatinib), a selective inhibitor of bcr-abl kinase and to a lesser extent platelet-derived growth factor receptor (PDGF-R) and c-kit (110), had no effect on reversing calcium induced sGC inhibition, even at 0.1 mM. Similarly, potent PKC inhibitor GF109203X (111) and PKA inhibitor H89 (112) had no effect.

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Figure 5.3. Cell free inhibition of sGC by calcium using fractionated Jurkat lysate.

Immunoprecipitated sGC is inhibited when treated with ultracentrifuged Jurkat T cell lysate (depleted of membrane fractions) and 250 nM Ca2+. Where indicated, 10 µM

DEA/NO was added. cGMP accumulation was measured and expressed in terms of percentage control (10 µM DEA/NO).

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PP1 and PP2, potent inhibitors of the src family of kinases reversed calcium mediated inhibition of sGC with IC50 values 70 ± 20 nM for PP1 and 92 ± 4 nM for PP2. The reported IC50 values for fyn, lyk and src kinases are, 5, 6 and 170 nM respectively (113).

Sunitinib, an inhibitor of receptor tyrosine kinases and VEGF receptors, did not reverse calcium-mediated inhibition of sGC. These findings suggest the involvement of src family kinases.

Pyk2, a kinase related to focal adhesion tyrosine kinase (FAK), is regulated by calcium in several cell types (114). Although it is known that Pyk2 can be regulated by calcium, the mechanism by which this occurs is not fully understood and is thought to involve the N-terminal FERM (4.1/ezrin/radixin/moesin) domain of Pyk2. The current model for activation of Pyk2 involves autophosphorylation at Tyr 402 followed by recruitment of src family kinase (115). Based on the foregoing, we obtained antibodies to

Pyk2, c-src kinase antibody and a pan-antibody that would bind to all proteins in the src kinase family. Depletion of lysate for the protein of interest was analyzed by western blotting. In the case of pyk2 (Figure 5.4) and c-src (data not shown) the antibody- mediated depletion worked, but there was no reversal in inhibition (Figures 5.4 and 5.5), suggesting that these two proteins are perhaps not involved in the pathway. In the case of the pan-antibody that binds to all src family kinases, the antibody depletion was incomplete and there was no reversal. This experiment therefore proved to be inconclusive.

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2+ Inhibitor Class of kinase Ca -sGC IC50 Reference

Genistein Tyrosine kinases Yes 56± 2 nM* (105)

Staurosporine All kinases Yes ND (109)

H89 PKA No ND (112)

GF 109203X PKC No ND (111)

PP1 Src kinase family Yes 70 ± 20 nM* (113)

PP2 Src kinase family Yes 92 ± 4 nM # (113)

Gleevec c-abl No ND (110)

Sunitinib VEGFR2, PDGFR, No ND (116)

c-Kit

KN-62 CAMK No ND (103)

KN-93 CAMK No ND (117)

Table 5.2. Summary of inhibitor screen. Inhibitors to several different kinases were

used to test the involvement of kinases in calcium dependent inhibition of sGC.

Where applicable, IC50 values were calculated from the dose-response. * denotes n =

3 experiments, whereas # denotes n = 2 experiments. ND denotes not-determined and

in these cases the highest dose of inhibitor used was 1 mM and even at that

concentration, calcium-dependent inhibition of sGC was not reversed.

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Figure 5.4. Depletion of pyk2 from MCF-7 lysate does not reverse calcium-mediated inhibition of sGC. MCF-7 cell lysate was incubated mouse monoclonal Pyk2 antibody or buffer control for 16 h at 4 °C. Protein A/G agarose beads were added at this time to capture the antibody-pyk2 complex. An aliquot of the lysates were analysed for depletion by western blotting. GAPDH was used for loading control and

Jurkat T cell lysate was used as a positive control. The appropriate lysates were then incubated with sGC on FLAG beads with or without 250 nM Ca2+. Following this beads were washed five times with TBS and resuspended in appropriate volume for activity measurement. Where indicated, 10 µM DEA/NO was added.

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Figure 5.5. Depletion of c-src kinase from Jurkat T cell lysate does not reverse calcium- mediated inhibition of sGC. Jurkat T cell lysate was depleted of c-src kinase

(as described in Figure 5.4). Cell lysates were analysed for depletion along with loading controls by western blotting (data not shown). Appropriate lysates were then incubated with sGC on FLAG beads with or without 250 nM Ca2+. Following this beads are washed five times with TBS and resuspended in appropriate volumes for activity measurement. Where indicated, 10 µM DEA/NO was added.

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5.3.4. Cell free assay with rat sGC

Transient transfection of rat sGC (FLAG-tagged alpha and myc-tagged beta) yielded active NO-inducible protein. Inclusion of Jurkat T cell lysate with 300 nM Ca2+ resulted in inhibition (Figure 5.6). This implies that rat isoform of sGC is also regulated by calcium and that the phosphorylation site on sGC is conserved.

5.3.5. Bioinformatics

Cell lines MCF-7, Jurkat and HEK 293T have 38 tyrosine kinases in common, based on published expression profiles. Table 5.3. lists these kinases along with their

NCBI gene ID and gene symbol. Among these kinases, c-abl, c-src kinase, Pyk2, Lck and Syk are all inhibited by PP1 and PP2. We have ruled out receptor tyrosine kinases, therefore c-abl is not likely to be involved. siRNA/shRNA-mediated knockdown of c- src, pyk2, Lck and Syk are planned to ascertain if any of these are involved.

5.4. Conclusions and Discussion

I successfully established a lentiviral expression system for human sGC. Lenti-X™ HEK

293T cells consistently produce high titer virus for all three genes. Transduction of HEK

293T cells with MOI of 2 for all three viruses results in maximum transduction efficiency. Selection with antibiotics, puromycin and G418, results in an apparently homogenous population of cells with all three genes (Tet activator, sGC alpha and sGA beta) stably integrated into the genome of HEK 293T cells. Western blot analysis of

FLAG-tagged beta subunit shows that β-FLAG is expressed only upon induction by

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Figure 5.6. Rat sGC is inhibited by calcium. MCF-7 cells were transiently transfected with rat sGC alpha and beta subunits. Incubation of rat sGC on FLAG beads with

Jurkat lysate with Ca2+, led to inhibition of NO-induced activity.

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Table 5.3. List of tyrosine kinases expressed in HEK 293T, Jurkat T and MCF-7 cells.

NCBI Gene NCBI Protein Symbol Gene ID ABL1 25 c-abl oncogene 1, receptor tyrosine kinase AXL 558 AXL receptor tyrosine kinase BLK 640 B lymphoid tyrosine kinase BMX 660 BMX non-receptor tyrosine kinase BTK 695 Bruton agammaglobulinemia tyrosine kinase DDR1 780 discoidin domain receptor tyrosine kinase 1 CSK 1445 c-src tyrosine kinase PTK2B 2185 PTK2B protein tyrosine kinase 2 beta FER 2241 fer (fps/fes related) tyrosine kinase FLT1 2321 fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) FLT3 2322 fms-related tyrosine kinase 3 FLT4 2324 fms-related tyrosine kinase 4 KDR 3791 kinase insert domain receptor (a type III receptor tyrosine kinase) LCK 3932 lymphocyte-specific protein tyrosine kinase LTK 4058 leukocyte receptor tyrosine kinase MATK 4145 megakaryocyte-associated tyrosine kinase MST1R 4486 macrophage stimulating 1 receptor (c-met-related tyrosine kinase) MUSK 4593 muscle, skeletal, receptor tyrosine kinase NTRK2 4915 neurotrophic tyrosine kinase, receptor, type 1 NTRK3 4916 neurotrophic tyrosine kinase, receptor, type 3 ROR1 4919 receptor tyrosine kinase-like orphan receptor 1 ROR2 4920 receptor tyrosine kinase-like orphan receptor 2 DDR2 4921 discoidin domain receptor tyrosine kinase 2 PTK2 5747 PTK2 protein tyrosine kinase 2 PTK6 5753 PTK6 protein tyrosine kinase 6 PTK7 5754 PTK7 protein tyrosine kinase 7 ROS1 6098 c-ros oncogene 1 , receptor tyrosine kinase RYK 6259 RYK receptor-like tyrosine kinase SYK 6850 spleen tyrosine kinase TEC 7006 tec protein tyrosine kinase TEK 7010 TEK tyrosine kinase, endothelial TIE1 7075 tyrosine kinase with immunoglobulin-like and EGF-like domains 1 TXK 7294 TXK tyrosine kinase TYK2 7297 tyrosine kinase 2 TYRO3 7301 TYRO3 protein tyrosine kinase TNK1 8711 tyrosine kinase, non-receptor, 1 TNK2 10188 tyrosine kinase, non-receptor, 2 MERTK 10461 c-mer proto-oncogene tyrosine kinase

125 withdrawal of doxycycline. We could not analyze sGC alpha subunit expression due to the lack of a good antibody. However, sGC immunoprecipitated from these cells remains active and exhibits similar calcium-dependent inhibition as seen in Jurkat T cells.

Repetition of the transduction protocol consistently yields cells that have inducible sGC expression, indicating that the lentivirus is efficient and our protocol stream-lined.

Genistein, a tyrosine kinase specific inhibitor, reverses calcium-dependent inhibition of sGC implying that tyrosine kinase (s) are central to calcium-mediated inhibition of sGC. PP1 and PP2, both potent inhibitors of the src family kinases reversed calcium mediated inhibition of sGC. Pyk2, a tyrosine kinase activated by calcium recruits c-src kinase for downstream signaling (115). Therefore, we depleted Jurkat T cell and MCF-7 cell lysate of these two proteins. However, the depleted lysates still inhibit sGC when calcium was included. Bioinformatics analyses using published expression profiles reveal 38 tyrosine kinases in common between the three cell lines under investigation. Of these, 5 are potently inhibited by PP1 and PP2. Therefore, siRNA/shRNA mediated knockdown of these kinases is the next step. We have also considered the possibility that the kinase that phosphorylates sGC is distinct from the kinase that is activated upon increase in intracellular calcium. The site of modification on sGC is also unknown. Data from rat sGC imply that the site of phosphorylation is a conserved site. Efforts are underway to scale up purification of sGC from HEK 293T- hsGC cells in order to be able to obtain enough material for proteomics.

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CHAPTER 6

SUMMARY AND FUTURE DIRECTIONS

6.1. Summary

TSP-1 via CD47 inhibits NO signaling by regulating sGC activity. This provided us with an opportunity to study the regulation of sGC in the context of a signaling pathway as well as to complement the structure and function studies on sGC currently going on in the lab. In chapter 2, I discussed the development of Jurkat T cells as a model system for uncovering the mechanism behind TSP-1 regulation of NO signaling. After surveying a few different cell immortalized cell lines, we chose Jurkat T cells, they show robust NO- inducible sGC activity, express abundant CD47 at the cell surface and are responsive to

E3CaG1, a C-terminal fragment of TSP-1. In addition, I discovered that while CD47 is necessary for TSP-1 inhibition of sGC, it is not sufficient. Jurkat T cells lose the ability to bind to E3CaG1 after they have been 3 weeks in culture or if they are grown at higher densities. This loss of binding is not due to the loss of CD47, but rather to a loss of a binding partner of CD47 or a post-translational modification of CD47 itself. I also expressed and purified from E. coli a GST fusion construct of the G1 domain of human

TSP-1. This protein binds to CD47 and inhibits sGC. The activity of this protein is not as robust as E3CaG1, perhaps due to instability.

I describe in Chapter 3 my discovery that calcium is required for E3CaG1

2+ mediated inhibition of sGC. I have shown that E3CaG1 increases [Ca ]i from basal levels of 5-10 nM to 300 nM in Jurkat T cells. When this increase in calcium

127 concentration is chelated with intracellular calcium chelator BAPTA, E3CaG1 inhibition of sGC is reversed. I have also shown that blocking CD47 with a CD47 antibody

2+ prevents E3CaG1 mediated changes in [Ca ]i, showing that CD47 is necessary. In addition, JinB8 cells, that lack CD47 (71), also do not respond to E3CaG1. Angiotensin

II, a potent vasoconstrictor that also increases intracellular calcium, via an increase in IP3, also inhibits sGC. This establishes calcium ion as a regulator of sGC activity and functioning in two independent pathways.

Chapter 4 focuses on the nature of calcium inhibition of sGC in this signaling pathway. While there is evidence in the literature that calcium can inhibit sGC by directly binding to sGC and competing with Mg2+ ion required for catalysis, in the TSP-1 pathway, calcium inhibits sGC via a post-translational modification. We developed a cell free assay, where incubation of sGC with Jurkat T cell, HEK 293T or MCF-7 lysate along with 250-300 nM Ca2+, leads to inhibited sGC. Allosteric stimulators, compounds

YC-1 and BAY 41-2272, restore the activity of this inhibited protein, so does broad range kinase inhibitor staurosporine. Further, treatment of the inhibited protein with alkaline phosphatase also restores activity. Therefore, calcium inhibits sGC via phosphorylation.

Chapter 5 delves into establishing a robust source of sGC. For uncovering the phosphorylation site, and for use in other structure/function studies underway in our group, I established a lentiviral expression system that should yield sufficient material once expanded to higher scale tissue culture. In this chapter, I have also used a preliminary inhibitor screen, to elucidate which kinase is responsible to phosphorylation

128 of sGC. The inhibitor screen indicates that the src kinase family is likely responsible for calcium regulation of sGC. The major drawback of using inhibitors is the promiscuity with which they inhibit kinases and the false positives that this might generate. The alternative approach underway is to introduce point mutations of sGC at several consensus phosphorylation sites as predicted in silico. Unfortunately, all of the mutants generated so far were inhibited by calcium, although some did not express (Data not shown, all experiments performed by Dr. Stacy Mazaluppo). In addition, there is a possibility that calcium induces multiple phosphorylation events on sGC, the net effect of which is inhibitory. Rat sGC is also inhibited by calcium, suggesting that the phosphorylation site(s) is conserved evolutionarily. The lentiviral expression system provides a stable platform where multiple mutants can be generated and screened in a high throughput manner. Figure 6.1 summarizes my findings in this dissertation.

6.2. Future directions

6.2.1. Identification of kinase(s) regulating sGC

A genetic approach with gene knockdown studies could be used. This will begin with shRNA-mediated knockdown of src family kinases, and is underway. Should this not work, whole kinome knockdown or even whole genome knockdown is now possible, but still challenging to perform. Alternatively, a fractionation approach may yield the answer. Lysate from tissue culture could be fractionated by standard approaches such as column chromatography and ammonium sulphate precipitation. The fractions retaining activity will be analyzed by mass spectrometry to identify candidate proteins. We are

129 examining bovine tissue, beginning with lung, which is rich in sGC, as a larger source of material.

6.2.2. Identifying the site of sGC modification

Calcium regulation of sGC in this pathway is via phosphorylation of sGC. To elucidate which site is modified, we need large quantities of modified sGC for use with mass-spectrometry. Since we already know that sGC is phosphorylated in its basal state, we would look for changes in phosphorylation upon addition of calcium. Upon identifying residues that show changed phosphorylation status, site-directed mutagenesis will be used to ascertain validity of the role of these sites in sGC regulation. I have established a lentiviral expression system for human full-length sGC that should provide sufficient material for mass spectrometry. Efforts are underway to increase the scale of tissue culture for obtaining a large number of cells. Our lab has acquired the WAVE™ bioreactor and experiments with micro-carriers (to increase surface area for growth of cells) are being conducted. Currently, HEK 293T-hsGC cells (cells that are stably transduced with human sGC) are being used for this purpose. In the future, we could identify another cell line that produces protein more efficiently, such as HEK 293S cells.

6.2.3. Other pieces of the puzzle.

In the TSP-1/CD47 signaling pathway, the other piece of the puzzle is calcium itself, where is it coming from? Some preliminary experiments have ruled out involvement of IP3-mediated increase in calcium, but this leaves exploration into the involvement of membrane channels and other mechanisms that could release calcium.

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Preliminary data with the GST-G1 domain suggests that in the presence of extracellular

EDTA, GST-G1 no longer inhibits sGC. Further investigation into the source of calcium could include using membrane channel blockers in conjunction with E3CaG1.

Elucidation of CD47 binding partners is also key; it is not at all known how CD47 relays information from TSP-1 in the extracellular region, to release of calcium in the intracellular region. Integrin αv and αvβ3 have been ruled out as potential CD47 binding partners in this pathway, but other integrins and potentially any membrane protein are possible candidates. Similarly, post-translational modification of CD47 could account for lack of E3CaG1 binding in older Jurkat T cells. The possible approach to attack this question is both gene and protein microarray analysis (gene microarray data from Chris

Giron are available) to look for change in gene and protein expression between cells that respond to E3CaG1 and those that do not. Additionally, MudPIT analyses of cell lysates might be able to identify any post-translational modification on CD47.

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Figure 6.1. Research summary. TSP-1 mediates inhibition of NO signaling via sGC by increasing calcium concentration in the cell. Interestingly, Ang II a potent vasoconstrictor also inhibits sGC via an increase in calcium. Thus, two branches of signaling that antagonize NO signaling use calcium for their regulation. Calcium- mediated inhibition of sGC requires a tyrosine kinase. Phosphorylation of sGC leads to inhibition. Allosteric activators of sGC, BAY-41-2272 and YC-1, reverse inhibition. Rat and cow sGC are also inhibited by calcium, suggesting that the site of modification is evolutionarily conserved. E3CaG1 binding to CD47 requires an additional modification of CD47 or another binding partner (indicated by X). E3CaG1

2+ binding to CD47 leads to increase in [Ca ]i. However, the mechanism for this is not known (indicated by Y), although it does not involve IP3-receptor.

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