High molecular weight hyaluronic acid regulates MMP13 expression in chondrocytes via DUSP10/MKP5

To determine the effect of high molecular weight hyaluronic acid (HA) on matrix metalloproteinase 13 (MMP13) expression induced by tumor necrosis factor α (TNF‐α) in chondrocytes. Human chondrocytic C28/I2 cells were incubated with TNF‐α and HA. In some experiments, the cells were pre‐incubated with a CD44 function‐blocking monoclonal antibody (CD44 mAb) prior to addition of TNF‐α and HA. The expression of MMP13 was determined by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and an enzyme linked immunosorbent assay, while the phosphorylation of signaling molecules was measured by western blot analysis. The transcriptional activity of activator protein 1 (AP‐1) was analyzed by a reporter assay. To further clarify the molecular mechanisms of HA in MMP13 regulation, the expression level of dual‐specificity protein phosphatase 10 (DUSP10)/mitogen‐activated protein kinases phosphatase 5 (MKP5) in HA‐treated chondrocytes was assessed by real‐time RT‐PCR, western blotting, and immunofluorescence microscopy. HA decreased MMP13 mRNA and protein expression induced by TNF‐α. Blockage of HA‐CD44 binding by CD44 mAb suppressed HA‐mediated inhibition of MMP13. HA inhibited transient phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun NH₂‐terminal kinase (JNK) induced by TNF‐α. Reporter assay findings also revealed that pre‐treatment with HA inhibited the transcriptional activity of AP‐1 mediated by TNF‐α. Moreover, HA induced the expression of DUSP10/MKP5, a negative regulator of p38 MAPK and JNK pathways. These results indicate that HA‐CD44 interactions downregulate TNF‐α‐induced MMP13 expression via regulation of DUSP10/MKP5, suggesting that HA plays an important role as a regulatory factor in cartilage degradation.