IHH-GLI-1-HIF-2α signalling influences hypertrophic chondrocytes to exacerbate osteoarthritis progression

Background: Chondrocyte hypertrophy is a potential target for osteoarthritis (OA) treatment, with Indian hedgehog (IHH), glioma-associated oncogene homolog (GLI), and hypoxia-inducible factor-2α (HIF-2α) being closely associated with chondrocyte hypertrophy during OA progression. Whereas IHH can modulate chondrocyte hypertrophy, interference with IHH signalling has not achieved the anticipated therapeutic effects and poses safety concerns, necessitating further clarification of the specific mechanisms by which IHH affects articular cartilage degeneration. Inhibition of the HIF-2α overexpression in cartilage slows the progression of early OA, but the mechanisms underlying HIF-2α accumulation in OA cartilage remain unclear. The aim of this study was to determine the function of Ihh, as well as its downstream factors, in chondrocytes, based on an early osteoarthritis (OA) mouse model and in vitro chondrocyte model.

Methods: Investigated the expression levels and locations of IHH-GLI-1 pathway in normal and early degenerated human cartilage, comparing them with HIF-2α and its downstream factors. RT-qPCR, Western blotting, Crystal violet staining, and EdU assays were used to evaluate the pecific regulatory mechanisms of the IHH-GLI-1-HIF-2α signalling axis in normal chondrocytes and in chondrocytes under inflammatory conditions. Validated the impact of IHH on early cartilage degeneration and the relationship between the IHH-GLI-1 pathway and the expression levels and expression locations of HIF-2α and its downstream factors in Col2a1-Cre^ERT2;Ihh^fl/fl mice.

Results: In early-stage degenerative joint cartilage, the GLI-1 pathway in hypertrophic chondrocytes exhibited similar changes in location and levels to HIF-2α and its downstream factor vascular endothelial growth factor (VEGF). In vitro, IHH-GLI-1-HIF-2α signalling activation in chondrocytes under physiological hypoxic conditions inhibited chondrocyte proliferation. In chondrocytes stimulated by inflammatory environments, IHH inhibited the degradation of HIF-2α via the GLI-1 pathway, thereby promoting HIF-2α protein expression. Elevated HIF-2α expression further enhanced intracellular IHH-GLI-1 levels, generating a positive feedback loop to collectively regulate the expression of downstream hypertrophic factors and matrix-degradation factors. In vivo, conditional Ihh knockout in mouse chondrocytes downregulated Hif-2α protein expression in early degenerative cartilage tissue and affected the expression of downstream Vegf and hypertrophic factors.

Conclusions: During OA progression, the IHH-GLI-1-HIF-2α axis mainly operates within hypertrophic chondrocytes, exacerbating cartilage degeneration by regulating hypertrophic chondrocyte functions, cartilage matrix degradation, and microvascular invasion.

The translational potential of this article: This study identifies the IHH-GLI-1-HIF-2α signalling axis and reveals its potential as a therapeutic target for OA.