Reciprocal Regulation of GLI1 and GLI3 Fine Tunes the Pathogenic Behavior of Synovial Fibroblasts in Rheumatoid Arthritis

OBJECTIVE: sublining subset of rheumatoid arthritis synovial fibroblasts (RASFs), in regulating their pathogenic behavior. METHODS: GLI3 protein levels were quantified in freshly isolated RASF subsets by Western blotting. Bulk RASFs were subjected to siRNA-mediated knockdown (KD) of GLI3 or GLI1, followed by RNA sequencing. The effects of GANT61 on RASF proliferation, cell-cycle progression, migration, viability, and apoptosis were assessed using EdU/PI analysis, scratch assays, CCK-8 assays, and Annexin V/PI flow cytometry. RESULTS: RASFs at both mRNA and protein levels. GLI3 KD increased GLI1 expression and upregulated genes involved in inflammation, matrix remodeling, and cell cycle regulation, including IL6, IL11, IL24, IL33, MMP3, PLAU, CCNA2, and E2F1. Pathway enrichment analysis revealed activation of ECM-receptor interaction, PI3K-Akt, and TNF signaling. Co-silencing GLI1 with GLI3 blunted the induction of IL11, IL24, IL33, CCNA2, E2F1, and PLAU observed with GLI3 KD alone, indicating that GLI1 mediates a subset of the transcriptional effects induced by GLI3 loss. GANT61 suppressed CCNA2 and E2F1 expression, inhibited RASF proliferation and migration, and did not markedly increase apoptosis. CONCLUSION: GLI3 functions as a negative regulator of GLI1 and its downstream targets that drive the pathogenic behavior of RASFs. Targeting the GLI1-GLI3 axis may represent a promising therapeutic strategy to modulate fibroblast-driven inflammation and joint destruction in RA.