Cyclin-Dependent Kinases and their role in Inflammation, Endothelial Cell Migration and Autocrine Activity Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shruthi Ratnakar Shetty Graduate Program in Pharmaceutical Sciences The Ohio State University 2020 Dissertation Committee Dale Hoyt, Advisor Liva Rakotondraibe Moray Campbell Keli Hu Copyrighted by Shruthi Ratnakar Shetty 2020 Abstract Inflammation is the body’s response to infection or injury. Endothelial cells are among the different players involved in an inflammatory cascade. In response to an inflammatory stimuli such as bacterial lipopolysaccharide (LPS), endothelial cells get activated which is characterized by the production of important mediators, such as inducible nitric oxide synthase (iNOS) which, catalyzes the production of nitric oxide (NO) and reactive nitrogen species and cyclooxygenase-2 (COX-2) that catalyzes the production of prostaglandins. Though the production of these mediators is required for an inflammatory response, it is important that their levels are regulated. Continued production of iNOS results in increased accumulation of reactive nitrogen species (RNS) that might lead to cytotoxicity, whereas lack of/suppression results in endothelial and vascular dysfunction. On the other hand, severe cardiovascular, intestinal and renal side effects are observed with significant suppression of COX-2. Thus, studying factors that could regulate the levels of iNOS and COX-2 could provide useful insights for developing novel therapeutic targets. Regulation of protein levels involves control of protein induction or turnover. Since protein induction requires transcription, in this dissertation we studied the role of a promoter of transcription “Cyclin- dependent kinase 7 (CDK7)” in iNOS and COX-2 protein induction. We first depleted CDK7 in mouse aortic endothelial cells (MAEC) using siRNA, followed by stimulus with ii lipopolysaccharide/interferon gamma (LPS/IFN) to induce iNOS and COX-2 protein. Interestingly, CDK7 depletion resulted in excessive induction of both iNOS and COX-2 protein levels compared to control. This observation was recapitulated using a covalent inhibitor of CDK7, THZ1. Further investigation showed the protein induction due to lack of CDK7 was not due to increased mRNA levels. CDK7 knockdown however decreased the turnover of iNOS, but not COX-2 protein. We identified a novel mechanism by which CDK7 could regulate the stability of iNOS protein. We demonstrated how in the absence of CDK7, iNOS was resistant to degradation by protease, calpain, due to increased association of iNOS with its binding partner calmodulin. CDK7 depletion was also found to exert a post-transcriptional effect on COX-2, by activating upstream signaling cascades from mTOR and MAPK pathways that might promote translation. Activation of the components of the translation pre- initiation complex was also observed in the absence of CDK7, which contributed to increased COX-2 protein levels. This dissertation thus identifies CDK7 as a repressor of both iNOS and COX-2 proteins in endothelial cells by affecting their turnover and translation, respectively. The research provides novel insights, for the therapeutic manipulation of iNOS and COX-2 expression in endothelial cells via the less explored post-transcriptional effects of CDK7. We also investigated the roles of different transcriptional and atypical CDKs in angiogenesis and autocrine activity in endothelial cells. CDK5 and CDK9 were found to be potential regulators of endothelial cell migration whereas CDK7 and CDK9 were found iii to regulate autocrine activity in endothelial cells. Although these studies are highly preliminary, they pave the way to understand and further explore these CDKs as potential angiogenic or autocrine targets in various therapeutic conditions. iv Dedication This Dissertation is dedicated to my family, my father Ratnakar Shetty, my mother Mamatha Shetty and my sister Reshma Shetty, who have been incredibly supportive and have always encouraged me to do my best. v Acknowledgement I would like to thank my family especially my parents for their incredible support. The last five years of my PhD journey would not have been possible without the training and guidance from my advisor Dr. Dale Hoyt. I would also like to thank my committee members Dr. Liva, Dr. Hu and Dr. Campbell for their useful suggestions and insights on how to improve my research project. Finally, my doctoral training has not only enabled me to be a better scientist but also has helped improve my scientific communication skills for which I am extremely grateful to the department of Pharmacology, including all faculty and students for their insightful comments during seminars and weekly journal clubs. Lastly, I would like to thank all my friends and those lasting relationships I have built here in Columbus, Ohio. Go Bucks! vi Vita 2008-2012…………………....Bachelor’s in Pharmacy, University of Mumbai, Mumbai, India 2012-2014…………………....Master’s in Pharmacology, Toxicology and Therapeutics, University of Mumbai, India 2014-2015……………………Drug Safety Associate, TATA Consultancy Services, Mumbai, India 2015-2020……………………PhD. Pharmaceutical Sciences, College of Pharmacy, The Ohio State University, Columbus, Ohio 2015-2019……………………Graduate Teaching Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio 2018-2020…………………....Delegate, Council of Graduate Students, The Ohio State University, Columbus, Ohio 2019-2020…………………....Graduate Minor Applied Clinical and Preclinical Research, The Ohio State University, Columbus, Ohio vii 2019-2020……………………Graduate Fellow, College of Pharmacy, The Ohio State University, Columbus, Ohio 2019-2020……………………Graduate Representative, University Conduct Board, The Ohio State University, Columbus, Ohio Publications 1. Shetty S, Spaulding AM, Baker C, Baine SH, Hoyt DG. Translational Regulation of Cyclooxygenase-2 (COX2) by Cyclin-Dependent Kinase 7 (CDK7) in Endothelial Cells. The FASEB Journal. 2020;34(S1):1-1. doi:10.1096/fasebj.2020.34.s1.04431 2. Shetty S, Spaulding AM, Baker C, Baine SH, Hoyt DG. Regulation of Inducible Nitric Oxide Synthase (iNOS) by Cyclin-Dependent Kinase 7 (CDK7) in Endothelial Cells. The FASEB Journal. 2020;34(S1):1-1. doi:10.1096/fasebj.2020.34.s1.04533 3. Karmahapatra, S., Kientz, C., Shetty, S., Yalowich, J. C., & Rakotondraibe, L. H. (2018). Capsicodendrin from Cinnamosma fragrans Exhibits Antiproliferative and Cytotoxic Activity in Human Leukemia Cells: Modulation by Glutathione. Journal of Natural Products, 81(3), 625–629. https://doi.org/10.1021/acs.jnatprod.7b00887 4. Shetty, S., et al. (2015). Neuroprotective potential of escitalopram against behavioral, mitochondrial and oxidative dysfunction induced by 3-nitropropionic acid. Annals of Neurosciences, 22(1), 11–18. https://doi.org/10.5214/ans.0972.7531.220104 viii 5. Hariharan, A., Shetty, S., Shirole, T., & Jagtap, A. G. (2014). Potential of protease inhibitor in 3-nitropropionic acid induced Huntington’s disease like symptoms: Mitochondrial dysfunction and neurodegeneration. Neurotoxicology, 45, 139–148. https://doi.org/10.1016/j.neuro.2014.10.004 Fields of Study Major Field: Pharmaceutical Sciences Minor Field: Applied Clinical and Preclinical Research ix Table of Contents Abstract ...................................................................................................................... ii Dedication ...................................................................................................................v Acknowledgement ..................................................................................................... vi Vita ........................................................................................................................... vii Publications ............................................................................................................. viii Table of Contents ........................................................................................................x List of Tables............................................................................................................xvi List of Figures ........................................................................................................ xvii List of Symbols and Abbreviations .......................................................................... xx Chapter 1. Introduction and Literature Review .......................................................1 1.1 Inflammation: ....................................................................................................1 1.1.1 Activation of Inflammation: What prompts inflammatory cells to produce Cytokines? ................................................................................................................1 1.1.2 Cells and Cytokines Involved in Inflammatory Response: .................................2 1.2 Endothelial cells in Inflammation: ....................................................................8 x 1.3 Inducible nitric oxide synthase: ....................................................................... 11 1.3.1 Introduction: .................................................................................................. 11 1.3.2 Structure: ....................................................................................................... 11 1.3.3 Production of NO: ........................................................................................