Levels of activated protein C (APC) are elevated in the synovial fluid of patients with osteoarthritis (OA), and increased APC levels are correlated with the levels of active matrix metalloproteinase 2 (MMP-2). This study sought to investigate whether APC is a relevant protein for activation of MMPs in the degradation of human OA cartilage, and to elucidate its mechanisms of action.
Human articular cartilage was cultured with or without interleukin-1α (IL-1α), in the presence or absence of APC or protein C, and an MMP or serine proteinase inhibitor. Aggrecan and collagen release and chondrocyte gene expression levels were quantified. Aggrecanase and MMP cleavage of aggrecan was examined with neoepitope-specific antibodies, and MMP activity was measured using gelatin zymography and fluorogenic peptide assay.
In human OA cartilage, APC induced aggrecan and collagen release, whereas in non-OA cartilage, costimulation with IL-1α was required. Inhibition of MMP activity reduced APC-induced cartilage proteolysis, and MMP-induced aggrecanolysis was confirmed by Western blotting. In cultures with APC alone, the activity of MMPs 2, 9, and 13 was significantly increased in OA cartilage, although APC could not directly activate MMPs 2 or 9. Expression of MMP1, MMP2, MMP9, MMP13, TIMP1, and TIMP3 was not altered by APC in OA cartilage. Human OA chondrocytes expressed messenger RNA for protein C, endothelial protein C receptor, thrombomodulin, and protease-activated receptor 1, but these were unaltered or down-regulated by APC. The induction of MMP activation and cartilage degradation by APC was dependent on its serine protease activity.
APC is a physiologically relevant activator of MMPs and cartilage breakdown in human OA. The effects of APC are dependent on its proteolytic activity and as-yet-undefined cell and/or cartilage matrix factors, and inhibition of this pathway may provide a novel therapeutic target to halt the progression of cartilage damage in OA.