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In Transcriptional Regulation in Human Podocytes

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In Transcriptional Regulation in Human Podocytes THE ROLE OF ALPHA-ACTININ4 (ACTN4) IN TRANSCRIPTIONAL REGULATION IN HUMAN PODOCYTES (HPC) AND IN NEPHROTIC SYNDROME BY XUAN ZHAO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis advisor: Dr. Hung-Ying Kao Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Xuan Zhao Candidate for the Doctor of Philosophy degree* (Signed) Dr. David Samols (Chair of the committee) Dr. Hung-Ying Kao Dr. William Merrick Dr. Leslie Bruggeman Dr. David Buchner (date) June 21th, 2017 *We also certify that written approval has been obtained for any propriety material contained therein Table of contents LIST OF TABLES ............................................................................................................. iii LIST OF FIGURES ........................................................................................................... iv ACKNOWLEDGEMENTS .............................................................................................. vii LIST OF ABBREVIATIONS .......................................................................................... viii ABSTRACT ........................................................................................................................ 1 CHAPTER 1: INTRODUCTION ....................................................................................... 3 Podocytes And Nephrotic Syndrome ........................................................................ 3 Alpha Actinin 4 (ACTN4) ....................................................................................... 12 NF-κB Signaling ...................................................................................................... 18 Glucocorticoid And Glucocorticoid Receptors (GRs) ............................................. 25 The Effects Of Glucocorticoid Therapy On Nephrotic Syndrome .......................... 33 The Role Of Glucocorticoids In Podocyte .............................................................. 35 CHAPTER 2: ACTN4 POTENTIATES NF-κB ACTIVITY IN PODOCYTES INDEPENDENT OF ITS CYTOPLASMIC ACTIN BINDING FUNCION ....................................................................................................... 41 Abstract .................................................................................................................... 41 Introduction ............................................................................................................. 41 Materials And Methods ........................................................................................... 44 Results ..................................................................................................................... 48 Discussion ................................................................................................................ 74 i CHAPTER3: ACTN4 REGULATES GLUCOCORTICOID RECEPTOR- MEDIATED TRANSACTIVATION AND TRANSREPRESSION IN PODOCYTES ................................................................................................................... 79 Abstract .................................................................................................................... 79 Introduction ............................................................................................................. 80 Results ..................................................................................................................... 88 Discussion .............................................................................................................. 124 CHAPTER4: CHARACTERIZE THE NOVEL FAMILIAL FOCAL SEGMENTAL GLOMERULOSCLEROSIS-LINKED ACTN4 PROTEIN MUTATIONS ................................................................................................................. 128 Abstract .................................................................................................................. 128 Introduction ........................................................................................................... 131 Materials And Methods ......................................................................................... 131 Results ................................................................................................................... 134 Discussion .............................................................................................................. 144 CHAPTER5: FUTURE DIRECTIONS .......................................................................... 146 References ....................................................................................................................... 155 ii LIST OF TABLES Table 1. Genes associated with nephrotic syndrome .......................................................... 9 Table 2. A summary of biochemical properties of selectvie ACTN4 variants. .............. 144 iii LIST OF FIGURES Figure 1. A diagram showing the structure and components of renal glomerular filtration system, from kidney to podocyte. ..................................................... 10 Figure 2. A schematic diagram depicting components of The podocyte SD and foot processes and slit diaphragm proteins. ............................................... 11 Figure 3. A Schematic representation of the domain architect of ACTN4 and disease-associated mutations. ........................................................................... 16 Figure 4. Canonical and alternative pathway of NF-κB signaling.................................... 22 Figure 5. Domain structure of NF-κB and IκB family proteins.. ...................................... 23 Figure 6. The GR family proteins ..................................................................................... 29 Figure 7. Molecular mechanism of GR signaling pathways ............................................ 30 Figure 8. The mechanism of the Podocyte injury and the protective effect of glucocorticoids. .................................................................................. 37 Figure 9. The effect of ACTN4 knockdown on NF-κB-mediated transcriptional activation. ......................................................................................................... 51 Figure 10. The effect of ACTN4 knockdown on TNFα-induced p65 nuclear translocation in HPCs. .................................................................................... 54 Figure 11. GRα-mediated transrepression of NF-κB target genes in podocytes. ............. 57 Figure 12. ACTN4 and p65 interact in glomeruli and in mammalian kidney cells. ......... 60 Figure 13. IκBα blocks ACTN4 from interacting with p65 in the cytosol. ...................... 63 Figure 14. The association of ACTN4 variants with p65 and p50. .................................. 66 Figure 15. The role of HDAC7 in ACTN4-mediated potentiation in NF-kB transactivation activity. ................................................................................... 68 iv Figure 16. Association of p65 and ACTN4 with IL1-β and IL-8 promoters in HPCs. .... 72 Figure 17. A model in which ACTN4 binds and co-activates NF-κB transactivation activity in the nucleus. ..................................................................................... 73 Figure 18. The effect of Dex on nuclear translocation of GRα in podocytes. .................. 90 Figure 19. The effect of ACTN4 on GR-mediated transcriptional activation. ................. 94 Figure 20. Mapping of ACTN4 and GR interacting domains. ......................................... 98 Figure 21. The effect of ACTN4 LXXAA mutation on potentiating GR-mediated transcriptional activity. ........................................................... 101 Figure 22. The effect of FSGS-linked ACTN4 on GR-mediated transcriptional activation. ..................................................................................................... 104 Figure 23. The effect of ACTN4 knockdown on Dex-induced GR nuclear translocation in HPCs. .................................................................................. 107 Figure 24. The interaction between ACTN4 and GR only occurred in the nucleus in HPCs. ......................................................................................................... 109 Figure 25. The effect of knocking down HSP90 on the association between ACTN4 and GRα. ......................................................................................... 110 Figure 26. The recruitment of ACTN4 and GR with GR transactivated promoters in HPCs. ......................................................................................................... 116 Figure 27. The effects of Dex on the recruitment of GR, ACTN4, and p65 to NF-κB targeted promoters. ....................................................................... 122 Figure 28. A model depicting the mechanism by which ACTN4 regulates Dex- and TNFα-mediated transcriptional regulation in HPCs. .................... 123 v Figure 29. Characterization of wild-type and disease-associated ACTN4 protein stability ............................................................................................. 136 Figure 30. The F-actin filaments binding affinity of disease-linked ACTN4 mutations. ..................................................................................................... 138 Figure 31. Subcellular distribution of the newly identified disease-associated ACTN4 mutants.
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