The Effect And Mechanism Of KLF15 On The Expression Of MMP-3 In Human Chondrocytes

Background: Osteoarthritis(OA)is a kind of bone and joint disease characterized by degeneration of articular cartilage.It is one of the most common chronic health disorders in the world;with a higher prevalence among the aging population.It is also a huge economic burden to social,families and patients.The etiology and pathogenesis of OA is still unclear.Many studies suggest that abnormal metabolism of chondrocytes is an important cause of OA.Imbalance of metabolism on catabolic and anabolic molecules in chondrocytes has been associated with the cartilage damage in OA.Matrix metalloproteinase-3(MMP-3),one of the most important catabolic factors,acts as a cartilage degrading enzyme which is associated with the degradation of type II collagen and especially,aggrecan.Kruppel-like factor 15(KLF15)is an important member of the KLFs family and has a variety of biological functions.However,the physiological functions of the KLF15 in chondrocytes and the pathological progressions of OA are still unknown.In this study,we investigated the expression of KLF15 in human chondrocytes and its relationship with MMP-3 synthesis,which revealed its possible role in the physiological and pathological processes of chondrocytes.This research was divided into the following parts:PART1: The expressions of KLF15 in human chondrocytesObjective: To investigate whether KLF15 is expressed in human cartilage cells and the expression difference of KLF15 between OA and normal chondrocytes.Materials and methods: 1.Cell lines: Human chondrosarcoma cell SW1353 was purchased from Shanghai cell bank of Chinese Academy of Sciences;OA and healthy synovial chondrocytes were were obtained from primary culture;MC cells were preserved in the laboratory.2.Methods: The phenotype of chondrocytes was observed using AEC type II collagen staining and toluidine blue staining;RT-PCR and WB methods were used for detecting KLF15 expressions in each cell line;western-blot,q RT-PCR and immunofluorescence was used to evaluate expressions of KLF15 in OA and healthy human chondrocytes.Results: We observed typical chondrocyte morphologies using optical microscopy and typical phenotypes using immunocytochemistry staining in cultured cells.The RT-PCR analysis revealed that KLF15 was expressed in primary chondrocytes as well as SW1353 chondrogenic cell lines at the m RNA level.In addition,western blot analysis revealed the expression of KLF15 in primary chondrocytes as well as SW1353 chondrogenic cell lines at the protein level.The results showed that the m RNA level of KLF15 was significantly reduced in OA chondrocytes compared with normal chondrocytes.These results were confirmed by western-blot analysis.Consistently,typical immunofluorescence results showed that KLF15 was mainly localized in the nuclei of chondrocytes and the staining of KLF15 was significantly weaker in chondrocytes from OA patients.Conclusion: KLF15 is expressed in human chondrocytes.Expression of KLF15 is reduced in primary chondrocytes from OA patients as compared with those from normal samples.PART2: KLF15 down-regulates TNF-?-induced increased expression of MMP-3 in human chondrocytesObjective: To investigate whether TNF-? had an impact on KLF15 expression in chondrocytes and whether KLF15 affected the expression of MMP-3.Materials and methods: 1.Cell lines: Human chondrosarcoma cell SW1353 was purchased from Shanghai cell bank of Chinese Academy of Sciences;2.Methods:western blot,and q RT-PCR were used to evaluate expressions of KLF15 in SW1353 induced by TNF-?.The effect of overexpression of KLF15 on the expression of MMP-1,3,9,13,ADAMTS-4 in cells was determined by q RT-PCR and western-blot.The effect of KLF15 knockdown on the expression of MMP-1,3,9,13,ADAMTS-4 in cells was determined by q RT-PCR and also western-blot on the expression of MMP-3.Results: TNF-? significantly reduced the expression of KLF15 in a dose-dependent manner at both the m RNA level and the protein level.TNF-? induced expression of MMP-3 was inhibited by overexpression of KLF15 at both the m RNA and protein level in SW1353 cells.Knockdown of KLF15 exacerbated TNF-? induced increase in the expression of MMP-3 at both the m RNA and the protein levels.Conclusion: TNF-? reduces the expression of KLF15 in human chondrocytes.KLF15 could inhibit the expression of MMP-3 induced by TNF-? in human chondrocytes.PART3: Mechanism of KLF15 regulating MMP-3 expression in human chondrocytes Objective: To understand how KLF15 regulates the expression of MMP-3 in human chondrocytes.Materials and methods: 1.Cell lines: Human chondrosarcoma cell SW1353 was purchased from Shanghai cell bank of Chinese Academy of Sciences;2.Methods: The expression of TP53 and phosphorylated TP53 in SW1353 was determined by q RT-PCR and western-blot.We investigated effects of KLF15 on the MMP-3 promoter activity using a dual-luciferase report assay.The chromatin immunoprecipitation(Ch IP)assay was performed to access whether KLF15 could bind to the MMP-3 promoter.Results: We found that TNF-? significantly increased the phosphorylation of TP53 and the p53 specific inhibitor Pifithrin-? significantly prevented TNF-?-induced reduction of KLF15 at both the m RNA and protein level.Overexpression of KLF15 significantly inhibited the promoter activity of MMP-3.KLF15 was able to bind to the promoter region of MMP-3.In addition,TNF-? treatment attenuated the recruitment of KLF15 to MMP-3 promoter.Conclusion: TNF-?-induced reduction of KLF15 is mediated by phosphorylation level of TP53.KLF15 inhibits transcription of MMP-3 by binding to the MMP-3 promoter region.