The nuclear receptor TR4 orchestrates cytoskeletal organization in a Gα12/ROCK-dependent manner to promote myofibroblast differentiation and tissue fibrosis in systemic sclerosis.

Abstract

Members of the superfamily of nuclear receptors have been implicated in inflammatory processes and pathologic tissue remodeling and have emerged as attractive targets for pharmaceutical intervention. However, the role of the testicular receptor 4 (TR4, or Nr2c2) in fibroblast activation and rheumatologic diseases has not yet been investigated. TR4 expression in human skin and experimental fibrosis were determined by immunofluorescence staining and western blot. RNA sequencing (RNAseq) was performed in TGFβ-stimulated fibroblasts with or without TR4 knockdown by siRNA. Myofibroblasts differentiation were assessed by α smooth muscle actin (αSMA) and extracellular matrix staining, and ROCK activity by ELISA. Fibroblast-specific Tr4 knockout mice were subjected to TBRIact-, bleomycin-, and cGvHD-induced dermal fibrosis, and bleomycin-induced pulmonary fibrosis. Precision-cut skin slices (PCSS) from systemic sclerosis (SSc) patients were used for TR4 knockdown studies. TR4 was upregulated in fibroblasts in the skin of SSc patients and in murine models of SSc. TGFβ induced TR4 in fibroblasts in a SMAD3-dependent manner. Knockdown of TR4 prevented fibroblast-to-myofibroblast-transition, and fibroblast-specific-knockout of Tr4 ameliorated experimental murine skin and pulmonary fibrosis. RNAseq and functional experiments identified the profibrotic effects of TR4 were dependent on Gα12- and ROCK-associated cytoskeletal remodeling. PCSS confirmed that TR4 regulates ROCK signaling and the expression of profibrotic genes in SSc skin. TR4 is upregulated in SSc in a TGFβ-dependent manner and drives fibroblast activation. Inhibition of TR4 prevents ROCK activation, fibroblast-to-myofibroblast transition and ameliorates dermal and pulmonary fibrosis, highlighting TR4 as a potential therapeutic target in SSc and related diseases.