Zhang Chengming, Zhao Ruipeng, Dong Zhengquan, Liu Yang, Liu Mengrou, Li Haoqian, Yin Yukun, Che Xianda, Wu Gaige, Li Pengcui, Wei Xiaochun, Yang Ziquan
Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair, Department of Orthopedics, Second Hospital of Shanxi Medical University, Taiyuan, 030001, PR China.
Department of Laboratory Medicine, Handan Second Hospital, Hebei University of Engineering, Handan, 056000, PR China.
J Orthop Translat. 2024 Oct 22;49:207-217. doi: 10.1016/j.jot.2024.09.008. eCollection 2024 Nov.
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.
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;Ihh mice.
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. , conditional knockout in mouse chondrocytes downregulated Hif-2α protein expression in early degenerative cartilage tissue and affected the expression of downstream Vegf and hypertrophic factors.
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.
This study identifies the IHH-GLI-1-HIF-2α signalling axis and reveals its potential as a therapeutic target for OA.
软骨细胞肥大是骨关节炎（OA）治疗的一个潜在靶点，在OA进展过程中，印度刺猬因子（IHH）、胶质瘤相关癌基因同源物（GLI）和缺氧诱导因子-2α（HIF-2α）与软骨细胞肥大密切相关。虽然IHH可以调节软骨细胞肥大，但干扰IHH信号传导并未达到预期的治疗效果，且存在安全问题，因此有必要进一步阐明IHH影响关节软骨退变的具体机制。抑制软骨中HIF-2α的过表达可减缓早期OA的进展，但OA软骨中HIF-2α积累的潜在机制仍不清楚。本研究旨在基于早期骨关节炎（OA）小鼠模型和体外软骨细胞模型，确定Ihh及其下游因子在软骨细胞中的功能。
研究正常和早期退变人软骨中IHH-GLI-1通路的表达水平和定位，并与HIF-2α及其下游因子进行比较。采用RT-qPCR、蛋白质免疫印迹法、结晶紫染色和EdU检测，评估IHH-GLI-1-HIF-2α信号轴在正常软骨细胞和炎症条件下软骨细胞中的具体调控机制。验证IHH对早期软骨退变的影响，以及IHH-GLI-1通路与Col2a1-Cre;Ihh小鼠中HIF-2α及其下游因子的表达水平和表达定位之间的关系。
在早期退变的关节软骨中，肥大软骨细胞中的GLI-1通路在定位和水平上表现出与HIF-2α及其下游因子血管内皮生长因子（VEGF）相似的变化。在体外，生理缺氧条件下软骨细胞中IHH-GLI-1-HIF-2α信号激活抑制软骨细胞增殖。在炎症环境刺激的软骨细胞中，IHH通过GLI-1通路抑制HIF-2α的降解，从而促进HIF-2α蛋白表达。HIF-2α表达升高进一步增强细胞内IHH-GLI-1水平，形成正反馈环，共同调节下游肥大因子和基质降解因子的表达。在小鼠软骨细胞中进行条件性敲除，可下调早期退变软骨组织中Hif-2α蛋白表达，并影响下游Vegf和肥大因子的表达。
在OA进展过程中，IHH-GLI-1-HIF-2α轴主要在肥大软骨细胞内发挥作用，通过调节肥大软骨细胞功能、软骨基质降解和微血管侵入，加剧软骨退变。
本研究确定了IHH-GLI-1-HIF-2α信号轴，并揭示了其作为OA治疗靶点的潜力。
