Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

Abstract

AbstractBackgroundTissue fibrosis is a common feature of many organ dysfunctions, such as heart failure and chronic kidney disease. However, no fundamental treatment has been developed. This study aims to identify novel molecular mechanisms for antifibrotic intervention, focusing on fibroblast activation.MethodsWe performed a forward genetic screen using a genome-wide CRISPR library in the context of transforming growth factor β (TGF-β)-mediated connective tissue growth factor (CTGF) expression, and used unbiased techniques such as Cleavage Under Targets and Tagmentation (CUT&Tag) and proximity-dependent biotin labeling by TurboID to reveal the detailed molecular mechanisms.ResultsCRISPR library screening identified a number of players in both the canonical Smad pathway and the non-canonical pathway. In addition to the known factors, the Keap1-Nrf2 pathway was identified as a predominant regulator of TGF-β-mediated CTGF expression.Keap1deletion and consequent Nrf2 activation broadly suppressed profibrotic gene expression, independently of conventional antioxidant effects. CUT&Tag revealed that Nrf2 bound to the proximity of fibrosis-related genes includingCtgfandFn1.Subsequent individual analysis revealed Smad3 and RNA polymerase II binding to the Nrf2 peak site, which was attenuated byKeap1deletion. TurboID experiments further discovered that Nrf2 interacts with Ddx54, which acts as a corepressor. Consistently,Keap1deletion-mediated repression of profibrotic gene expression was reversed by additionalDdx54deletion. The impact of the Keap1-Nrf2 pathway on pathological fibrosis was examined using tamoxifen-inducible fibroblast-specificKeap1knockout mice. Pressure overload for 4 weeks robustly induced cardiac hypertrophy, fibrosis and contractile dysfunction. However, deletion ofKeap1in thePostnlineage attenuated these cardiac pathologies. The anti-fibrotic effects of Keap1 deletion were also confirmed in renal fibrosis in the unilateral ureteral obstruction (UUO) model.ConclusionsFibroblast Nrf2 transcriptionally represses fibrosis-related genes in cooperation with the corepressor Ddx54. Fibroblast-specific deletion ofKeap1attenuated pathological fibrosis in pressure overload heart failure and renal fibrosis.