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The Role of TRPC3 Channels in Molecular and Cellular Events A Dissertation entitled The Role of TRPC3 Channels in Molecular and Cellular Events Associated to Atherogenesis by Kathryn B. Smedlund Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences __________________________________________ Dr. Guillermo Vazquez, Major Advisor __________________________________________ Dr. David Giovannucci, Committee Member __________________________________________ Dr. Sonia Najjar, Committee Member __________________________________________ Dr. Sandrine Pierre, Committee Member __________________________________________ Dr. Edwin Sanchez, Committee Member __________________________________________ Dr. Patricia Komuniecki, Dean College of Graduate Studies The University of Toledo June 2012 Copyright 2012, Kathryn B. Smedlund This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without expressed permission of the author. ii An Abstract of The Role of TRPC3 Channels in the Molecular and Cellular Events Associated to Atherogenesis by Kathryn B. Smedlund Submitted to the Graduate Faculty in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo June 2012 Atherosclerosis is a chronic inflammatory vascular disease that constitutes the major cause of death in western societies and one of the main vascular complications of diabetes, obesity and metabolic syndrome. Recruitment of circulating monocytes to the endothelium and their migration into the subintima is a fundamental event in both initiation as well as progression of the atherosclerotic lesion. Endothelial vascular cell adhesion molecule-1 (VCAM-1), which is upregulated in response to pro-atherogenic stimuli, is critical in mediating monocyte tethering and firm adhesion to the endothelium. Whereas Ca2+ signaling has been recognized to play a role in regulation of VCAM-1 expression and atherogenesis. An understanding of the molecular identity of the channels involved and/or the mechanism by which they contribute to the atherogenic process is still lacking. Transient Receptor Potential Canonical (TRPC) channels are among the most important non-voltage gated Ca2+-permeable non-selective cation channels in vascular iii endothelium where they participate in the modulation of various vascular functions. Recent observations from our laboratory have revealed new aspects within the mechanism underlying regulated expression of VCAM-1 and monocyte adhesion, two of the earliest events in atherogenesis. While studying the role of Ca2+ influx in ATP- dependent expression of VCAM-1 in human coronary artery endothelial cells (HCAECs) we found that native TRPC3 proteins form, or are part of, Ca2+-permeable channels that 2+ participate in the Ca influx that follows P2Y2 receptor activation, and that TRPC3 expression is required for ATP-induced VCAM-1 and monocyte adhesion. Notably, ATP also induces TRPC3 expression and this is paralleled by augmented constitutive, non- regulated TRPC3-mediated cation influx. Interestingly, tumor necrosis factor-alpha (TNFα), a cytokine involved in the pathogenesis of atherosclerosis but not associated with regulated Ca2+ influx, induces VCAM-1 expression in HCAECs through a mechanism that also requires, at least in part, Ca2+ entry into the cell. Based on these findings and considering that high constitutive, non-regulated activity is an intrinsic property of TRPC3 channels, we hypothesized that upregulated expression of constitutively active TRPC3 in coronary endothelium might represent a novel contributing factor to the molecular/cellular events associated to initiation and progression of atherosclerotic lesions. Additionally, the balance between apoptosis and survival is crucial in maintaining endothelial cell integrity during vascular damage and alterations in these processes has been associated to atherosclerosis. It has recently been shown that stimulation of non- neuronal nicotinic acetylcholine receptors (nAChR) is involved in a cholinergic anti- inflammatory reflex signaling circuit, with anti-inflammatory actions at the macrophage iv level that are suggestive of potential anti-atherogenic role for nAChRs. Furthermore, many studies on endothelial cells from different vascular beds have revealed anti- apoptotic actions of nAChR stimulation; however, whether this action involves participation of downstream survival signaling has not been explored. Our lab has revealed that in HCAECs cholinergic receptors promote activation of three typical survival signaling pathways: the phosphatidyl-inositol-3 kinase (PI3K)/AKT axis, activated downstream muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively); the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both mAChR and nAChR. In addition, we have identified nAChR-dependent regulation of JAK2/STAT3 and ERK1/2 downstream α7- nAChR. Altogether, these findings suggest that stimulation of cholinergic receptors in HCAECs may act in a concerted way to induce an efficient survival signaling response when endothelial cells are exposed to pro-apoptotic stimuli. v This work is dedicated to my wonderful parents Eugene and Wavalynne Smedlund. Thank you for all your love, encouragement and support. I would not have been able to accomplish such an achievement without your loving support. vi Acknowledgments First and foremost, I would like to thank my major advisor Dr. Guillermo Vazquez for allowing me the opportunity to work in his laboratory and being a great mentor and giving me exceptional training for my future career. Secondly, I would like to thank my advisory committee members: Dr. David Giovannucci, Dr. Sonia Najjar, Dr. Sandrine Pierre and Dr. Edwin Sanchez for the time and advice they have provided to me during my graduate studies. Lastly, I would like to thank my lab members, Jean-Yves Tano and Bobby Lee, for all their help and friendships throughout the years. vii Table of Contents Abstract…………………………………………………………………………………...iii Dedication………………………………………………………………………………...vi Acknowledgments……………………………………………………………………….vii Table of Contents………………………………………………………………………..viii List of Figures…………………………………………………………………………...xiv Chapter 1: Literature Review ……………………………………………………………..1 1.1. Atherosclerosis………………………………………………………………..1 1.1.1. Endothelial Dysfunction…………………………………………..4 1.1.2. Cytokines………………………………………………………….6 1.1.3. Adhesion Molecules……………………………………………….7 1.1.4. VCAM-1 Expression in atherosclerotic lesions…………………...8 1.1.5. VCAM-1 Structure and Function………………………………….9 1.1.6. Role of calcium signaling in VCAM-1 expression………………10 1.2. Transient Receptor Potential Canonical (TRPC) channels: structure and function……………………………………………………………………...12 1.2.1. TRPC channels in Cardiovascular Disease………………………14 1.3. The Cholinergic Anti-inflammatory Reflex Pathway……………………….16 Chapter 2: Mouse Models of Atherosclerosis…………………………………………...19 viii Introduction………………………………………………………………………19 2.1. Current Mouse Models of Atherosclerosis………………………………….20 2.1.1. C57BL/6…………………………………………………………...20 2.1.2. Apolipoprotein E deficient (ApoE-/-) mice………………………..21 2.1.3. Low Density Lipoprotein Receptor deficient (LDLR-/-) mice…….22 2.2. Development of novel mouse models to study the role of TRPC3 in atherosclerosis………………………………………………………………23 2.2.1. Generation of endothelial specific TRPC3 transgenic mice………24 2.2.2. Generation of endothelial-specific TRPC3-/- mice………………...27 Chapter 3: Involvement of Native TRPC3 Protein in ATP-dependent VCAM-1 Expression and Monocyte Adherence in Coronary Artery Endothelial Cells…………...29 3.1. Abstract……………………………………………………………………...30 3.2. Introduction………………………………………………………………….32 3.3. Materials and Methods………………………………………………………33 3.4. Results……………………………………………………………………….35 3.5. Discussion…………………………………………………………………...39 3.6. Figure Legends………………………………………………………………44 3.7. References…………………………………………………………………...46 3.8. Figures……………………………………………………………………….53 Chapter 4: The Constitutive Function of Native TRPC3 Channels Modulates VCAM-1 Expression in Coronary Endothelial Cells through NFkB Signaling…………………....58 4.1. Abstract...........................................................................................................59 4.2. Introduction.....................................................................................................61 ix 4.3. Materials and Methods....................................................................................62 4.4. Results.............................................................................................................62 4.5. Discussion.......................................................................................................69 4.6. References.......................................................................................................76 4.7. Figure Legends................................................................................................82 4.8. Novelty and Significance................................................................................86 4.9. Figures.............................................................................................................88 4.10. Supplementary Information..........................................................................95 Chapter 5: Evidence for Operation of Nicotinic and Muscarinic Acetylcholine Receptor- Dependent Survival Pathways in Human Coronary Artery Endothelial Cells................126
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