UNIVERSITY OF CINCINNATI Date: 23-Jul-2010 I, Michael C Tranter , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Molecular, Cellular & Biochemical Pharmacology It is entitled: Investigation of NF-kappaB-Dependent Transcriptional and Post-Transcriptional Regulatory Networks in Late Ischemic Preconditioning Student Signature: Michael C Tranter This work and its defense approved by: Committee Chair: Walter Keith Jones, PhD Walter Keith Jones, PhD 8/18/2010 927 Investigation of NF-κB-Dependent Transcriptional and Post- Transcriptional Regulatory Networks in Late Ischemic Preconditioning A dissertation submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In the Department of Pharmacology and Cell Biophysics of the College of Medicine 2010 by Michael Tranter B.S. Rose-Hulman Institute of Technology, 2004 Dr. W. Keith Jones, Committee Chairperson Abstract Ischemic preconditioning (IPC) is a cardioprotective phenomenon initiated in response to short bouts of sublethal ischemia and reperfusion that serves as an endogenous defense mechanism to protect the mammalian heart against a subsequent prolonged ischemia/reperfusion (I/R) injury. The late phase of IPC provides protection for approximately 12-72 hours after the IPC stimulus and requires de novo gene expression. Activation of the transcription factor NF-κB has been shown to be necessary for the development of late phase IPC-induced cardioprotection. However, the specific mechanisms and downstream gene expression profiles that underlie the cardioprotection of NF-κB in late IPC remain unknown. To investigate NF-κB-dependent gene expression in IPC, we conducted a directed microarray analysis to compare the gene expression patterns of wild-type and NF-κB dominant negative mice after an IPC stimulus. Microarray results indicated that NF-κB- dependent gene expression changes after late IPC were enriched for genes involved in angiogenesis, programmed cell death, and the heat shock response. We further demonstrated that the expression of two inducible heat shock protein 70 genes, Hsp70.1 and Hsp70.3, are induced by NF-κB after late IPC. However, results of infarct studies show that only the Hsp70.3 gene contributes to cardioprotection in the late phase of IPC, while the Hsp70.1 gene, encoding a nearly identical protein, does not contribute to IPC- induced cardioprotection and is pro-cell death after ischemia/reperfusion injury. iii Interestingly, inhibition of NF-κB in late IPC only partially (50%) reduced IPC- induced expression of Hsp70.3 mRNA, while it completely blocked the increase of Hsp70.3 protein. Upon further investigation of the Hsp70.3 3’-untranslated region (3’- UTR) of the mRNA transcript, we found evidence for miRNA and alternative polyadenylation mediated post-transcriptional regulation of Hsp70.3. To assess potential miRNA regulation of Hsp70.3, comparative miRNA expression arrays were performed in wild-type and NF-κB dominant-negative mice after IPC. Two Hsp70.3 targeting miRNAs, miR-378* and miR-711, were found to be downregulated by IPC, coinciding with increased Hsp70 protein expression. However, only miR-711 was suppressed in an NF-κB-dependent manner after late IPC. Our results demonstrate that both of these miRNAs exert a 3’-UTR-mediated post-transciptional suppression on Hsp70.3 expression. In addition to regulation by miRNAs, it was found that four distinct size populations of Hsp70.3 transcripts exist as a result of alternative polyadenylation (polyA) within the 3’-UTR. Two predominant populations of alternatively polyadenylated Hsp70.3 transcripts exist that represent the result of polyadenylation at one of two sites on either side of the binding site for miR-378*. Quantitative analysis of polyA site utilization after late IPC using QRT-PCR revealed a significant increase in Hsp70.3 mRNA transcripts with shortened 3’-UTRs leading to the removal of the miR-378* binding site. These results indicate for the first time that alternative polyadenylation plays a role in post-transcriptional regulation of cardioprotective gene programs following a late IPC stimulus. In addition, it appears that these processes may be coordinated with miRNA-mediated post-transcriptional regulation. iv Analysis of predicted transcription factor binding sites within the Hsp70.3 depicted binding sites for the IPC-dependent transcription factors HSF-1, STAT3, and AP-1, in addition to NF-κB. Hsp70.3-luciferase reporter constructs were used to assess the role of these transcription factors on the regulation of Hsp70.3 gene expression in H9c2 cells and murine embryonic fibroblasts (MEFs) after stimulus by either heat shock (HS) or simulated I/R. Results indicate that HSF-1 is absolutely obligatory to Hsp70.3 expression following both HS and simulated I/R. Inhibition of either NF-κB, STAT3, or AP-1 blunted Hsp70.3 reporter expression following HS and simulated I/R. This work demonstrates that mRNA expression of Hsp70.3 is transcriptionally co-regulated through the obligatory coordinated actions of NF-κB, STAT3, AP-1 and HSF-1. As a future means to acutely inhibit activation of multiple transcription factors in IPC and examine the resulting effects on gene expression, we sought to demonstrate the efficacious application of non-viral PGAA glycopolymers (T4) for the functional delivery of transcription factor decoys and transcriptional blockade in the in vivo heart. Results show that T4-mediated pericardial delivery of NF-κB decoys resulted in efficient transfection of the myocardium and functional blockade of NF-κB as evidenced by inhibition of NF-κB induced COX-2 gene expression and a decrease in infarct size after I/R injury. Successful application of this system for the blockade of NF-κB, STAT-3, or AP-1 transcriptional activation in late IPC demonstrated that the activation of these factors are all critically important to late phase IPC cardioprotection. This is in agreement with our in vitro results indicating coordinated gene expression by these three factors. The use of PGAA glycopolymers makes it possible to specifically inhibit transcription v factor activation in vivo for the semi-high-throughput study of transcriptional networks in myocardial pathophysiology. The results presented herein delineate the immediate NF-κB-dependent transcriptome after late IPC and contribute to the global understanding of the cardioprotective mechanisms of late IPC. In addition, the regulation of cardioprotective Hsp70.3 requires coordinated regulation of transcription as well as post-transcriptional modulation by polyadeylation site selection and miRNA binding/translational regulation. Our studies of the pre- and post-transcriptional regulation of Hsp70.3 expression elucidate a complex multi-factorial regulation of Hsp70.3 and contribute to the understanding of the regulatory networks controlling gene expression in late IPC. vi vii Acknowledgements I would like to extend my sincere appreciation and thanks to the many people who have provided both the personal and professional guidance that have made possible my completion of this dissertation. Special thanks to my committee members, Dr. Keith Jones, Dr. Theresa Reineke, Dr. Scott Belcher, Dr. JoEl Schultz, and Dr. Hong-Sheng Wang, for their time spent discussing and reviewing my research and the invaluable expertise and feedback they have provided throughout my graduate career. I am very grateful for the genuine interest that each has shown in not only my current research, but also in my future success and development as a scientist. I respect and appreciate the advice and mentoring I have received from each. I owe much of my development as a scientist to Dr. Keith Jones, who gave me the opportunity to perform my graduate work in his laboratory. Keith has been an excellent mentor and friend, and his dedication and passion for science is contagious and evident throughout the lab. I feel very luck to have had the privilege of performing my graduate research and training in Keith’s lab. I considered it a great compliment when Keith once said that I reminded him of a younger scientific version of himself. It is my hope and goal that I may someday achieve the same level of professional success in my career as Dr. Keith Jones. I would also like to thank Dr. Theresa Reineke for the opportunity for me to perform a large chapter of my graduate work as a collaborative effort with her lab. I am viii grateful for her continuing collaboration and support of my work even following her move from the University of Cincinnati to Virginia Tech. I would like to thank all the members of the Jones lab, both past and present, for their help, teaching, and camaraderie during my graduate career. Dr. Maria Brown and Dr. Suiwen He both deserve credit for providing additional teaching and mentoring during my early time in the lab. I am very indebted to Jackie Belew for her time and effort in maintaining the Jones lab mouse colonies, which were very critical to my research. I am also thankful for the friendship of my lab mates, whose conversational company helped make coming to the lab everyday an enjoyable experience. Many great scientific ideas and experimental plans were also born out of casual conversations with labmates. I would also like to thank other teachers and mentors who have helped in my scientific development through the years. Rose-Hulman Institute of Technology did an outstanding job of preparing me academically for the rigors of graduate school. Dr. Richard Anthony and Dr. Christine Buckley, along with the rest of the RHIT department of Applied Biology and Biomedical Engineering, deserve special thanks for their time and advice. Dr. Leland Sudlow, and Dr. Ron Millard were patient and kind enough to mentor me as an NSF REU summer student when I just beginning to learn experimental procedures. Thanks to Dr. Ron Millard for the continued and often needed support and advice from the time I joined his lab as a summer student in 2003 throughout my graduate career.