MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation – divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation

Abstract

Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated cartilage degradation. Cartilage degradation was induced in bovine articular cartilage explants by oncostatin M (OSM) and tumor necrosis factor (TNF), in the presence or absence of specific inhibitors of the mitogen-activated protein kinases (MAPKs) P38, P44/42 and Src family. Toxicity was followed by the AlamarBlue colorimetric assay. MMP-activity was assessed using a fluorescent substrate assay and MMP-9 and -2 activities by gelatinase zymography. MMP-mediated collagen type II degradation and MMP as well as aggrecanase-mediated aggrecan degradation was investigated with specific ELISA and hydroxyproline release by standard methods. The findings were verified by immunohistochemistry and histology. Stimulation of cartilage degradation by OSM+TNF resulted in 100-fold induction of CTX-II release (P<0.01). This was dose-dependently inhibited by MAPK P38 inhibitors and by the MAPK P44/42 inhibitors. MMP-activity and expression was significantly decreased, as evaluated by cleavage of fluorescence MMP-substrate and zymography. Immunohistochemistry confirmed these findings. Interestingly, only the P44/42 inhibitors abrogated aggrecanase-mediated aggrecan degradation. We found that inhibition of MAPK P38, P44/42 and Src family abrogated proteolytic cartilage degradation by blocking MMP synthesis and activity. However, only MAPK P44/42 was essential for aggrecanase-mediated aggrecan degradation. These data suggest that various aspects of cartilage degradation can be targeted independently by inhibiting specific upstream signaling pathway.