The contribution of methyltransferases : demethylases to renal fibrosis

Abstract

TGF β-1 signalling regulates many cellular processes, including proliferation, differentiation, apoptosis, immune responses, and fibrogenesis. The essential role of TGF β-1/SMAD signalling in stimulating fibrogenic cells to produce extra cellular matrix proteins and promoting proliferation of myofibroblasts is widely recognised. SMAD3 is known as a mediator in TGF β- 1-induced fibrosis in the kidney. Upon activation of TGF-β receptors, SMAD2 and SMAD3 are phosphorylated and form cytoplasmic heteromeric complexes with SMAD4. These complexes translocate to the nucleus where they regulate expression of TGF β-1 target genes. Tissues undergoing fibrosis exhibit markedly increased expression of a-SMA and interstitial matrix components, such as collagen and fibronectin which are TGF β-1/SMAD3-responsive genes. The mechanism by which SMADs mediate transcriptional regulation of these genes is incompletely understood, however SMAD3-null mice are protected against renal tubulointerstitial fibrosis, glomerular sclerosis, and also fibrosis in other organs. Data from this thesis has demonstrated for the first time that methylation might be important in the regulation of SMAD3 transcriptional activity; indeed the introduction of siRNAs targeting demethylases/methyltransferases led to changes in SMAD3 transcriptional activity. The introduction of siRNAs targeting both methyltransferases and demethylases resulted in changes in α-SMA and fibronectin expression at the protein level. The work in this dissertation again confirms that TGF β-1 signalling is a SMAD3- dependent pathway. SET9 is a methyltransferase enzyme that can methylate non-histone protein substrates including the transcription factors p53, Stat3, Rb, TAF10, E2F1, ERα, NF-κB, and DNMT1. I show that SET9 plays a central role in regulating SMAD3 activity, as shown by SET9 knockdown. I also show that wild type SET9 overexpression results in increased SMAD3 activity, in the presence of TGF β-1. Conversely, expression of a mutant SET9, which lacks methyltransferase activity, 15 failed to increase SMAD3 activity, even in the presence of TGF β-1. Furthermore, SET9 gene silencing with siRNAs significantly attenuated TGF β-1–induced ECM gene expression. These novel effects of SET9 warrant further evaluation of SET9 as a target in the treatment of fibrotic diseases such as CKD. Screening a demethylase siRNA library showed that the putative demethylase HSPBAP-1 is also involved in TGF β/SMAD signalling. Interestingly, I show that HSPBAP-1 interacts with SMAD3, and suppresses the transcriptional activity of SMAD3-driven reporter-genes. This is the first report of such an interaction, and the first data implicating a potential demethylation event in TGF β-1/SMAD3 signalling. Taken together, the work in this study defines novel roles of SET9 and HSPBAP-1 in fibrosis by mediating TGF β-1/SMAD3 signalling. On the basis of my work, future examination of SET9/HSPBAP-1 in whole organism models of renal fibrosis should be considered.