NFAT5 Mediation Of MMP13 Expression Induced By Hypertonicity And Mechanical Stress In Cartilage Chondrocytes

Objective: This article imitates the articular cartilage survival microenvironment,including hyperosmotic stress and mechanical loading,in order to investigate their simulation to cartilage injury,and the molecular mechanism of cartilage degeneration or osteoarthritis.Methods: Different osmotic pressure mediums,ranging from 350 m OsM to 550 mOsM,were made by adding NaCl to provide hyperosmotic conditions to cartilage chondrocytes;Meanwhile,12% cyclic tensile strain was applied to chondrocytes.Cartilage chondrocytes cultured in vitro were identified by toluidine blue staining to proteoglycans and immunofluorescence to collagen II,which are the markers of chondrocytes extracellular matrix.The cellular viability was analyzed using formanzan to measure the absorbance of chondrocytes,which is an inhibitor of cell viability.MTT assay was used after an incubation period under the above-described stress conditions.Western blot,RT-qPCR and immunofluorescence were applied to detect nuclear factor of activated T cells,metal regulatory transcription factor 1 and matrix metalloproteinase 13 expressions.Appropriate concentration of p38 MAPK inhibitor,ERK inhibitor and JNK inhibitor were tested whether they could decrease the downstream genes through the translocation of NFAT5 or not.Results: Although cell growth rate became slower and slower with the augment of osmotic stress,there were no significant differences in their cellular viability.Western blot and immunofluorescence results showed that hypertonicity raised NFAT5 expression,and transferred it from cytoplasm to the nucleus.In the level of mRNA,RT-qPCR results reflected that hyperosmotic stress could regulate downstream genes,containing MTF1 and MMP13,which were associated with NFAT5.The expressions of NFAT5,MTF1 and its downstream gene,MMP13 were promoted after artilage chondrocytes were applied 12% cyclic tensile strain for 3 h,6 h and 12 h.ERK inhibitor and p38 MAPK inhibitor could regulate the transcription activity of NFAT5,and significantly reduced the effect of hypertonicity to MMP13.Conclusion: Hypertonicity and mechanical loading could increase the expressions of MTF1 and MMP13 in cartilage chondrocytes of SD rats may actualized by NFAT5 translocation,resulting in degradation of extracellular matrix,ultimately lead to formation of cartilage degeneration and osteoarthritis.