TGFβ-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression

Abstract

Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβmediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβmediated fibroblast activation via targeted gene repression.

A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis

“…Finally, in order to confirm the proper functionality and the suitability to use 10-4A BFP cells in high throughput assays, we performed plate-reader based experiments to analyze the dose- and time-response of Acta2 expression (i.e., myofibroblast induction) in response to exposure to distinct profibrotic cytokines. The effective concentrations were in agreement with previous reports for induction of fibrosis ( Saito et al, 2003 ; Yanaba et al, 2011 ; Datta et al, 2013 ; Lee et al, 2017 ; Jones et al, 2019 ).…”

TGF-β-driven downregulation of the Wnt/β-Catenin transcription factor TCF7L2/TCF4 in PDGFRα+fibroblasts

“…RNA sequencing analysis from recent work on lung fibroblasts helped us to corroborate our results (Fig. 5G) (Jones et al, 2019). The pan HDAC inhibitor, pracinostat, attenuates TGF-β-induced repression of Tcf7l2 gene expression (Fig.…”

“…5C,D). Based on recent findings that demonstrated that TGFβ-mediated fibroblast activation requires HDAC-mediated transcriptional repression (Jones et al, 2019), we examined whether inhibiting HDACs with TSA may modify TGF-β-mediated reduction of TCF7L2 expression. we examined whether inhibiting HDACs with TSA may alter TCF7L2 expression in response to TGF-β signaling.…”

TGF-β-driven downregulation of the transcription factor TCF7L2 affects Wnt/β-catenin signaling in PDGFRα+ fibroblasts

“…S3H). Furthermore, RNA-seq analyses of TGF-β-treated idiopathic pulmonary fibrosis (IPF) lung (Jones et al, 2019) and cardiac fibroblasts (Schafer et al, 2017) show that TGF-β1 alters the expression of several validated target genes of the Wnt/β-Catenin/ TCF7L2 pathway. Dkk1, Dkk3, Lrp5, Lef1, Tcf7l1, Tgfbr2, Ctbp2, Sox9, Cxcl12 and Nfatc4 expression is reduced, whereas expression of the Fn1, Col1a1, Ctgf, Tgfb1, Foxo1, Wnt5a, Wnt5b, Wnt11, Wnt9a, Myc, and Oat TCF7L2-target genes is increased after TGF-β1 treatment (Fig.…”

“…5C,D). Based on recent findings that demonstrated that TGFβ-mediated fibroblast activation requires HDAC-mediated transcriptional repression (Jones et al, 2019), we examined whether inhibiting HDACs with TSA modified the TGF-β-mediated reduction of TCF7L2 expression. Therefore, cells were treated with TGF-β1 and/or TSA for 8 h and then TCF7L2 protein levels were evaluated by western blotting.…”