JNK Activation and Shear Stress: Implications for Adaptive and Maladaptive Signaling

Atherosclerosis is a leading cause of medical complications and death in the developed world. While its progression is influenced by many systemic risk factors, atherosclerosis initiates at vessel branch points and curvatures, where shear stress is low and exhibits complex flow patterns. Endothelial cells in these areas show increased inflammatory gene expression, cytoskeletal rearrangement and apoptosis. In vivo, the extracellular matrix in areas of disturbed flow changes from a basement membrane of collagen and laminin, to one with increased fibronectin deposition. These matrix changes correlate with inflammatory gene expression, suggesting that certain shear stress responses are matrix-specific. The c-Jun NH 2 -terminal kinase (JNK) is activated in response to shear stress. It is implicated in inflammation, cell migration and apoptosis, and feeding atheroprone mice the JNK inhibitor SP600125 inhibits atherosclerotic plaque development, making JNK an interesting candidate for mediating disease progression. However, both the mechanisms and role of shear-induced JNK activation, aside from its effects on inflammatory gene expression, remain elusive. This first part of this study focuses on the pathway of JNK activation by shear stress. In arteries from wild-type and atheroprone ApoE -/- mice, JNK activation localized to endothelial cells in areas of early atherogenesis that showed increased deposition of fibronectin. JNK activation by shear stress was matrix-specific, with enhanced activation on fibronectin compared to collagen I or matrigel, and involved new integrin binding to matrix. MKK4 and PAK were key upstream elements in the pathway. The second part of this study identifies a novel role for JNK in endothelial cell alignment in response to shear stress. Phosphorylated JNK localized to both the nucleus and focal adhesions in iii response to shear stress, and knockdown of JNK2 inhibited actin stress fiber alignment with shear. Cells on collagen I showed a late activation of JNK compared to cells on fibronectin, and in accordance with this observation, alignment was less efficient than in cells on fibronectin. Thus, JNK promotes alignment in response to high laminar shear, but under disturbed shear, where matrix remodeling occurs, its sustained activation contributes to the inflammatory processes associated with atherosclerotic development and progression. Dedication iv To Dr. Martin Schwartz, for giving me the opportunity to pursue this work, for always (heroically) having his office door open for questions, no matter how small, and for understanding that sometimes the best way to learn is by finding the answers for yourself. While this is not always the easiest way to proceed for mentor and student, the professional and intellectual growth that has resulted will stand me in good stead throughout my life. For that, I will always be grateful. I would like to thank the members of my graduate committee, Dr. Tom Parsons, Dr. Rick Horwitz, Dr. Sally Parsons and Dr. Brett Blackman for their advice and expertise. If mentors are people who allow you to learn from their experiences, then I could not have asked for better guides during this learning process. Members of the Schwartz Lab, both past and present, deserve thanks for their advice and unfailing support. Even in the most uncertain of times, a kind word and the chance to discuss science and life with sympathetic colleagues makes the day brighter. Notably, I would like to thank A. Wayne Orr for being both mentor and friend from the very beginning. In addition, Kostas Moissoglu, Elizabeth Coleman, Alexandra Pacheco and Brenton Hoffman were there when I needed encouragement the most. Finally, my deepest appreciation goes to my family and friends. Without their ever-present love, optimism and support, I would not be the person I am today. To my parents, Eli and Chin-lan, and my sister, Suzanna, for being only a phone call away from happiness. To Sandra Wong, for always being willing to fly across the country to keep me company. To David Scott, for the gift of laughter in the bleakest of times and love and joy in the happiest of times.

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