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MOLECULAR MECHANISMS OF ACTION"
Biochemistry and Molecular Biology Program
B.S. 2002, University of Pune
M.S. 2004, University of Pune
Thesis Advisor: Sylvia Christakos, Ph.D.
Department of Biochemistry and Molecular Biology
Friday, July 8, 2011
MSB E-609, 12:00 P.M.
A new class of inflammatory CD4+T cells producing IL-17 (Th17) has been identified which play a critical role in numerous inflammatory conditions and autoimmune diseases. The active form of vitamin D, 1,25(OH)2D3, has a direct repressive effect on the expression of IL-17A in both human and mouse T cells. In vivo treatment of mice with ongoing experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) diminishes paralysis and progression of the disease and reduces IL-17A secreting CD4+T cells in the periphery and CNS. 1,25(OH)2D3 treatment of Th17 cells was also found to protect against EAE induction when adoptively transferred to na´ve mice. The mechanism of 1,25(OH)2D3 repression of IL-17A expression was found to be transcriptional repression mediated by VDR. Transcription assays, gel shift and ChIP assays indicate that 1,25(OH)2D3 mediates the suppression of hIL-17A at least in part by blocking NFAT from binding to its sites and by recruitment of the vitamin D receptor (VDR) and histone deacetylase (HDAC) to the IL-17A promoter. Runx1 mediated activation of IL-17A transcription is also inhibited by 1,25(OH)2D3. Co-immunoprecipitation and ChIP assays suggest that the mechanism involves VDR-Runx1 interaction. In addition, I found that Foxp3, a lineage-specific transcription factor for regulatory T cells which associates with NFAT as well as Runx1 for transcriptional repression, is induced by 1,25(OH)2D3 at the transcriptional level. 1,25(OH)2D3 also stimulated expression of Foxp3 in both mouse and human CD4+CD25- T cells. These findings suggest, for the first time, that the negative effect of 1,25(OH)2D3 on IL-17A involves blocking of NFAT, recruitment of HDAC, sequestration of Runx1 by 1,25(OH)2D3 /VDR as well as a direct effect of 1,25(OH)2D3 on induction of Foxp3. My results describe novel mechanisms and new concepts with regard to vitamin D and the immune system and suggest therapeutic targets for the control of autoimmune diseases.
The second part of my study describes the involvement of the SWI/SNF complex in VDR mediated gene transcription. The mammalian SWI/SNF related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. Each SWI/SNF contains two homologous ATPases, Brg-1 and hBrm. Little is known about the role of the SWI/SNF complex in vitamin D receptor (VDR) mediated transcription. Here, I demonstrate that the SWI/SNF complex contribute to transcriptional activation by VDR and cooperates with VDR and C/EBPβ in the regulation of 25-hydroxyvitamin D3 24-hydroxylase (24(OH)ase) transcription. In MC3T3-E1 osteoblastic cells and COS-7 cells, I found that the N-terminal region of Brg-1 (Brg-1-d/n) acted as a dominant negative inhibitor and significantly inhibited 1,25(OH)2D3 mediated induction of 24(OH)ase transcription. Using SW13 and C33A cells, which are deficient in Brm and Brg-1, I found that 1,25(OH)2D3 induction of 24(OH)ase transcription is markedly reduced, suggesting that the SWI/SNF complex is an important component of VDR mediated transcription. Activation of transcription was restored in these cells preferentially by Brg-1. Brg-1 d/n, inhibits 1,25(OH)2D3 induction of 24(OH)ase expression 2 fold as indicated by Western blot analysis in UMR osteoblastic cells. Brg-1-d/n also inhibits C/EBPβ enhancement of VDR mediated 24(OH)ase transcription and expression. Brg-1 enhancement of VDR mediated transcription is not observed using a 24(OH)ase promoter construct with the C/EBP site mutated. Brg-1 d/n also inhibited the C/EBP beta enhancement of VDR mediated activation of 24(OH)ase transcription in MC3T3 osteoblastic and LLCPK kidney cell lines,Brg-1- d/n at the concentrations used had no effect on basal promoter activity. Co-immunoprecipitation assays indicate that Brg-1 interacts with C/EBPβ. Chromatin immunoprecipitation assays show that C/EBPβ and Brg-1 are recruited simultaneously to the 24(OH)ase promoter, suggesting co-operatively between VDR, the SWI/SNF complex and C/EBP beta in regulating 24(OH)ase transcription.