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 (TNF-) in chondrocytes. Human chondrocytic C28/I2 cells were incubated with TNF- and HA. In some experiments, the cells were pre-incubated with a CD44 function-blocking monoclonal antibody (CD44 mAb) prior to addition of TNF- 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-. 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-. Reporter assay findings also revealed that pre-treatment with HA inhibited the transcriptional activity of AP-1 mediated by TNF-. 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--induced MMP13 expression via regulation of DUSP10/MKP5, suggesting that HA plays an important role as a regulatory factor in cartilage degradation. (c) 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:331-339, 2017.