Inhibition of NADPH Oxidases Abrogates Fibronectin Fragment Induced Integrin Signaling and Matrix Metalloproteinase 13 Release in Human Chondrocytes

Purpose: OA is characterized by the degradation of cartilage extracellular matrix (ECM), including the production of ECM protein fragments. One of these, fibronectin fragments (FN-f), found in OA cartilage and synovial fluid, promotes cartilage matrix destruction through activating the α5β1 integrin and increasing MMP production. Reactive oxygen species (ROS) are necessary second messengers in the chondrocyte integrin signaling pathways that mediate MMP-13 production in response to FN-f stimulation. NADPH oxidases (Noxes) generate ROS in response to a variety of extracellular stimuli. The purpose of this study was to investigate the role of Noxes as mediators of ROS production and α5β1 signaling in human chondrocytes in response to FN-f. Methods: Primary human chondrocytes were isolated from normal donor tissue or OA tissue removed at the time of knee arthroplasty. Prior to FN-f stimulation, media was changed to serum-free media. Chondrocytes were treated with 10 μM VAS2870 (pan-Nox inhibitor) or 20 μM GKT 137831(dual Nox1/4 inhibitor) for 1 h before the treatment of 1 μM FN-f overnight (to study MMP release) or 30 min (to study the activation of cell signaling). Oxygen is the substrate of Nox reactions. To assess the effect of oxygen tension on FN-f induced MMP release, chondrocyte monolayers from same donors were cultured in normoxic (20%) or low oxygen (3%) conditions. Cell lysates or conditioned media were collected and analyzed by immunoblotting to assess MMP-13 production and the activation of MAP-kinases (JNK, ERK, and P38) following FN-f stimulus. To assess Nox isoform expression, seven described Nox isoforms and subunits of Nox2 and Nox4 p22phox, p67phox, p47phox and p40phox were analyzed by Real-Time PCR. Protein levels of Nox2 and Nox4 in response to FN-f were analyzed by immunoblotting. Results: FN-f induced MAP-kinases (JNK, ERK, and p38) signaling and MMP-13 production by normal chondrocytes were inhibited by the pan Nox inhibitor VAS2870 but not by the dual Nox1/Nox4 inhibitor GKT 137831 (Fig 1) suggesting Noxes other than Nox1/4 were required for FN-f activity. Similar findings were noted with OA chondrocytes. Low oxygen conditions partially decreased FN-f induced MMP-13 release as compared to normoxic conditions but Nox inhibition with VAS2870 was still effective (Fig.2). Moreover, we found Nox2 and Nox4 were highly expressed within human chondrocytes. FN-f stimulated the mRNA expression of Nox subunits, including p22phox and p67phox. FN-f stimulation did not affect Nox2 protein levels but increased Nox4 at early time points (30 min) and gradually led to Nox4 downregulation. Conclusions: Collectively, these results indicate that NADPH oxidase activity is required for ROS production in response to FN-f that leads to MAP kinase activation and MMP-13 production. Inhibitor studies suggested Nox2 but not Nox1 or Nox4 was required. Future studies will investigate the role of Noxes in the development of OA in vivo.View Large Image Figure ViewerDownload Hi-res image Download (PPT)