Background/Purpose: 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, also known as Nr2c2) in fibroblast activation and rheumatologic diseases has not yet been investigated.

Methods: TR4 expression in fibroblasts in the skin of systemic sclerosis (SSc) patients and in murine models of SSc were analyzed by immunofluorescence staining and Western blot. We performed bulk RNA sequencing (RNAseq) of TGFβ-stimulated fibroblasts with or without knockdown of TR4 by siRNA. The effect of TR4 on fibroblasts activation was studied using extracellular matrix staining. ROCK activity was quantified by ELISA in vitro. To confirm the findings, we applied TBRIact-, bleomycin-, cGvHD-induced dermal fibrotic as well as bleomycin-induced pulmonary fibrotic model in mice with fibroblast-specific-knockout of Tr4.

Results: Here we find that the expression of TR4 was upregulated in fibroblasts in the skin of systemic sclerosis (SSc) patients and in murine models of SSc. TGFβ stimulation induced the expression of TR4 in fibroblasts in a SMAD3-dependent manner. TR4 regulated numerous genes and functional terms associated with cytoskeletal remodeling. Knockdown of TR4 by siRNA prevented fibroblast-to-myofibroblast-transition, whereas plasmid-driven-overexpression of TR4 fostered TGFβ-induced fibroblast activation. Fibroblast-specific-knockout of Tr4 ameliorated skin fibrosis induced by overexpression of TBRIact, by bleomycin, associated with sclGvHD as well as bleomycin-induced pulmonary fibrosis. By RNA sequencing of TR4 target genes in fibroblasts and by analyzingthe functional role of ROCK and Gα12 using small molecule inhibitors and siRNA,we identified the profibrotic effects of TR4 were dependent on Gα12- and ROCK-associated cytoskeletal remodeling.

Conclusion: Our data indicate that TR4 is upregulated in SSc in a TGFβ-dependent manner to promote fibroblast activation. Inhibition of TR4 interferes with activation of ROCK, prevents cytoskeletal remodeling and fibroblast-to-myofibroblast transition and ameliorates dermal and pulmonary fibrosis. Inactivation of TR4 might thus offer therapeutic potential for the treatment of fibrosis in SSc and related diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-nuclear-receptor-tr4-orchestrates-cytoskeletal-organization-in-a-g%ce%b112-rock-dependent-manner-to-promote-myofibroblast-differentiation-and-tissue-fibrosis-in-systemic-sclerosis/