The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

Abstract

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition.Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

AdipoRon reduces TGFβ1‐mediated collagen deposition in vitro and alleviates knee stiffness in vivo

“…Western blot analysis was performed using standard western blot analysis technology as previously described (Bayram et al, 2022; Bayram, Owen, et al, 2020; Dudakovic et al, 2015; Dudakovic et al, 2023) or using Jess (Bio‐Techne), an automated western blot system, according to instructions provided by the manufacturer. Cells were lysed with radio‐immunoprecipitation buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific).…”

“…Gene expression analysis by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was performed as previously described (Dudakovic et al, 2023; Paradise et al, 2022). Briefly, cell lysis and RNA isolation were completed with TRI‐Reagent (Zymo Research) and Direct‐zol RNA isolation kit (Zymo Research), respectively.…”