Hypoxic Regulation Of Matrix Metalloproteinase-1 Expression In Cartilage Stem/Progenitor Cells Of The Rat Knee Articular Cartilage

Objective:To select HIF-1?-binding sites in rat CSPCs by the hypoxia condition;To illuminate the regulatory mechanism between HIF-1?and MMP-1 in cartilage-derived stem/progenitor cells(CSPCs)from knee joint of rats.;To further explore the hypoxia response mechanism of CSPCs'migration,apoptosis,and chondrogenesis in OA model in vitro.Methods:CSPCs were isolated from knee joint of 7-14d rats,cultured in 10%mesenchymal stem cell medium in a humidifed atmosphere containing 1%O₂,5%CO₂,94%N₂ at 37°C.We utilized Ch IP coupled to next-generation high-throughput sequencing to define HIF-1?-binding sites across the whole genome of rat CSPCs.The pc DNA3.1-based HIF-1?over-expression vectors and commercially purchased si HIF-1?were transient transfected into CSPCs by using liposomes.Then the expression levels of MMP-1 and HIF-1?were detected by RT-PCR,Western Blot,flow cytometry,and immunofluorescence.We further vertified the chromatin-proteins interaction mechanism between HIF-1?and MMP-1 by using Ch IP-q PCR and dual luciferase reporter assay.IL-1?was applied to intervene CSPCs to mimic an OA model in vitro.After that,RT-PCR and Western Blot were respectively applied to monitor the expression of HIF-1?and MMP-1 genes and proteins.Transwell migration assay and FITC-PI staining were conducted following by the transient transfection of si HIF-1?.YC-1 was applied to inhibit the expression of HIF-1?,then the chondrogenic activity was measured by Alcian blue staining.Results:We demonstrated the existence of a large number of HIF-1?binding sites distributed in the whole genome of rat CSPCs(details below).Among them,MMP-1 gene is highly expressed in OA articular cartilage,which is highly related to chondrocyte migration,apoptosis and metabolic processes.Also,there is a HIF-1?-recognition sequence in MMP-1 promoter beyond the core RCGTG motif(chr8:5702223-5702476).Transient transfection of the pc DNA3.1-based HIF-1?over-expression vectors could effectively inhibited the gene and protein expression of MMP-1 in a hypoxia-independent manner.In the meanwhile,the inhibition of HIF-1?by si HIF-1?led to a hypoxia-independent increase in MMP-1 gene and protein expression.We further verified that HIF-1?functions as a transcriptional repressor of MMP-1 through binding to the HRE sequence by using Ch IP-q PCR,dual luciferase report experiments.The enhanced migration capacity,and limited chondrogenesis induced by IL-1?,which was consistent with the characterstic of CSPCs in OA cartilage.Hypoxic environment could effectively reduce cell apoptosis and limit the enhancement of cell migration ability,but the chondrogenic differentiation ability had no obvious effect.However,under the OA environment induced by IL-1?,hypoxia could inhibit cell migration and apoptosis,and promote chondrogenic differentiation,and the above effects were mediated by HIF-1?.When the expression of HIF-1?was silenced,the protective effect would be weakened.Conclusion:There is a direct molecule mechanism of interaction between HIF-1?and MMP-1 in rat CSPCs.HIF-1?inhibits MMP-1 transcription activity by directly binding to the HRE region of the MMP-1 gene promoter,thereby inhibiting MMP-1 gene and protein expression levels.It is clearly known that hypoxia is a protective factor in IL-1?-induced OA model in vitro.The hypoxic regulation of migration,apoptosis,and chondrogenesis was operated in a HIF-1?-dependent manner.