Background/Purpose: The Retinoic-acid related Orphan Receptor-alpha (RORα) is a member of the nuclear receptor superfamily and a ligand-dependent transcription factor implicated in a wide range of physiological and pathological processes. Although individual key mediators of fibroblast activation such as TGFβ and WNT pathways have been identified, the factors that coordinate the activation and interaction of those pathways remain largely enigmatic. Here, we aim to evaluate the role of RORα in the pathogenesis of fibrotic diseases.

Methods: Mice carrying Rorα^fl/flxCol1a2-CreER were generated to specifically knockout Rorα in fibroblasts in an inducible manner. RORα was inactivated using the selective inhibitor SR3335. The role of RORα was investigated in different mouse models: Bleomycin- and TBRI^CA -induced dermal fibrosis, bleomycin-induced pulmonary fibrosis, carbon tetrachloride (CCL4)-induced liver fibrosis and in Wnt-10b transgenic mice. The activity of TGFβ/SMAD signaling pathway was analyzed by reporter assays. RNA sequencing was performed to determine target genes of RORα in fibroblasts.

Results: The expression of RORα was upregulated in fibroblasts in both human and murine fibrotic lung and liver. Activation of canonical Wnt/β-catenin signaling mimicked the increase of RORα in fibrosis and potently induced RORα expression. Reporter assays showed that canonical Wnt ligands induced the transcriptional activity of RORα, which could be prevented by Wnt signaling antagonists Dickkopf. Inhibition of RORα signaling by incubation with SR3335 inhibited WNT- and TGFβ-dependent myofibroblast differentiation and effectively reduced the release of collagen. Reporter assay demonstrated that SR3335 dramatically inhibited TGFβ/SMAD signaling pathway activity. Knockout of RORα reduced the stimulatory effects of TGFβ and WNT on fibroblast activation and collagen release. In contrast, fibroblasts overexpressing RORα are more susceptible to the profibrotic effects of TGFβ and WNT. In addition, fibroblast-specific inactivation of RORα in vivo protected from experimental fibrosis induced by bleomycin injection. Furthermore, treatment with SR3335 effectively reduced Wnt-induced fibrosis in vivo. Dermal thickening, myofibroblast differentiation and hydroxyproline content were all significantly reduced in Wnt10b-tg mice treated with SR3335 as compared to vehicle-treated littermates. Inhibition of RORα also ameliorated TGFβ-dependent fibrosis with potent antifibrotic effects in mice overexpressing TBRI^CA. Treatment with SR3335 also reduced bleomycin-induced skin and lung fibrosis and CCL4-induced liver fibrosis.

Conclusion: The present study characterizes RORα as a key checkpoint of TGFβ- and WNT- induced fibroblast activation. RORα is induced in fibrotic diseases in a WNT-dependent manner to promote TGFβ- and WNT-induced fibroblast activation and tissue fibrosis. Targeting of RORα simultaneously interferes with TGFβ- and WNT-signaling as two core pathways in the pathogenesis of fibrotic diseases. The inhibition of those core pathways translates into potent antifibrotic effects across different models and organ systems.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/orphan-nuclear-receptor-ror%ce%b1-is-a-key-regulator-of-tgf%ce%b2-and-wnt-signaling-in-fibrotic-diseases/