Mechanisms of hypoxia-inducible factor-1αin cartilage degradation by mechanical stress

Objective: Osteoarthritis (OA) is a complex disorder. Various systemic and/or extrinsic factors including aging, obesity, genetics, epigenetics, and local mechanical stress have been pointed out as the risk factors. We previously demonstrated that hypoxia-inducible factor-1 (HIF-1) was activated specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats and induced osteoclast via suppression of osteoprotegerin. The purpose of this study was thus defined to clarify the regulatory mechanisms of hypoxia-inducible factor-1α (HIF-1α) by mechanical stress and the roles in TMJ-OA. Materials and Methods: Osteosarcoma cell line, Saos-2, was employed to develop a model for analyzing the HIF-1 signaling pathway. The cells were cultured under normoxic (21% pO2) or hypoxic (1% and 5% pO2) conditions for 24 h. Then, various pieces (1, 2, 4, 8 pieces/well) of cover glasses were directly placed on cells to simulate a loading with mechanical stress on culture cells. HIF-1 activities were evaluated by transient transfection assays with hypoxia response element (HRE)-driven luciferase reporter or real-time RT-PCR analysis for HIF-1 target genes. Results: Mechanical stress induced by the weight of cover glasses significantly activated HRE-luciferase reporter even in normoxic cells, suggesting activation of HIF-1. The lucifease activity became highest in cells with a piece of cover grass, and decreased as the loaded weight increased. Interestingly, cultures with cover glasses substantially enhanced HRE-luciferase activities induced by hypoxia, suggesting an additive effect of mechanical stress on hypoxia. HIF-1 activation by mechanical stress was also demonstrated by enhanced expression of HIF-1 target genes, BHLHB2, ADM, and CA9, from a real-time RT-PCR analysis. Conclusions: An in vitro model for analyzing the HIF-1 signaling pathway, induced by mechanical stress, was successfully developed. It is shown that excessive mechanical stimuli accelerate the expression of HIF-1, leading to a progress in articular cartilage degradation or TMJ-OA through target gene regulations. This study was funded by a Grant-in-Aid (No.22792053) from the Ministry of Education, Science, Sports and Culture of Japan.