0961: Retinoic acid-related orphan receptor-α: A novel upstream regulator of Hippo signaling and potential therapeutic target in fibrosis

Background/Purpose: Retinoic acid-related orphan receptor-α (RORα) is a nuclear transcription factor implicated in immune regulation, circadian rhythm, and metabolism. However, its role in fibrotic diseases such as systemic sclerosis (SSc) remains undefined. Given that aberrant fibroblast activation and extracellular matrix deposition are hallmarks of fibrotic tissue remodeling, we investigated the mechanistic involvement of RORα in fibrogenesis and evaluated its therapeutic potential as a modulator of the Hippo signaling pathway.

Methods: In human samples (SSc and other fibrotic conditions) and in mouse fibrosis models, RORα expression was measured by quantitative PCR, Western blot, and immunofluorescence. RORα was silenced in cultured fibroblasts (siRNA) and in RORα-deficient mice to evaluate its function in fibrosis. The selective RORα inverse agonist SR3335 was used to pharmacologically inhibit RORα in three fibrosis models: bleomycin-induced skin and lung fibrosis, and CCl4-induced liver fibrosis. Transcriptomic profiling (RNA-Seq) identified RORα-dependent genes and signaling pathways.

Results: RORα expression was elevated in fibroblasts from various fibrotic diseases and organs, including hypertrophic scars, fibrotic skin, lungs and liver, compared to non-fibrotic controls. Increased RORα expression was also observed in murine SSc models. RORα knockdown in fibroblasts attenuated TGFβ-induced myofibroblast differentiation and collagen production. Similarly, pharmacologic inhibition of RORα with SR3335 reduced TGFβ-driven myofibroblast activation and extracellular matrix production (as indicated by decreased α-smooth muscle actin, type I collagen, and fibronectin) in human fibroblasts. In vivo, SR3335 treatment significantly ameliorated fibrosis in all three mouse models. RNA-Seq showed that RORα regulates a profibrotic gene network, including COL1A1, ACTA2 (αSMA), CTGF, and PAI-1. Mechanistically, RORα modulates the Hippo signaling pathway, a key regulator of fibroblast activation. SR3335 abrogated TGFβ-induced Hippo reporter activity and prevented YAP/TAZ upregulation and nuclear translocation in fibroblasts.

Conclusion: RORα expression was upregulated in fibroblasts from fibrotic human tissues (skin, lung, and liver, including samples from SSc patients) and in fibrotic mouse models, compared to controls. RORα knockdown in fibroblasts attenuated TGFβ-induced myofibroblast differentiation and collagen production. Similarly, pharmacologic inhibition of RORα with SR3335 reduced TGFβ-driven myofibroblast activation and extracellular matrix production (as indicated by decreased α-smooth muscle actin, type I collagen, and fibronectin) in human fibroblasts. In vivo, SR3335 treatment significantly ameliorated fibrosis in all three mouse models. RNA-Seq showed that RORα regulates a profibrotic gene network, including COL1A1, ACTA2 (αSMA), CTGF, and PAI-1. Mechanistically, RORα modulates the Hippo signaling pathway, a key regulator of fibroblast activation. SR3335 abrogated TGFβ-induced Hippo reporter activity and prevented YAP/TAZ upregulation and nuclear translocation in fibroblasts.