Reactive oxygen species regulate fibronectin fragments-induced map kinase signaling and metalloproteinase 13 release in human chondrocytes through nadph oxidase 2 and endocytosis of chondrocyte integrins

Purpose: OA is characterized by the degradation of cartilage extracellular matrix (ECM), resulting in the production of ECM protein fragments. One of these, fibronectin fragments (FN-f), found in OA cartilage and synovial fluid, promotes further cartilage matrix destruction through activating the α5β1 integrin and increasing MMP generation. While reactive oxygen species (ROS) are necessary second messengers in this process, the specific cellular source for ROS is unclear. The purpose of this study was to investigate the role of NADPH oxidase (Nox) as an ROS generator in chondrocytes and to study the localization of Nox-induced ROS following α5β1 integrin endocytosis. Methods: Primary human chondrocytes were isolated from ankle joints from tissue donors by sequential digestion. Culture media was changed from DMEM/F12 with 10% fetal bovine serum to serum-free before FN-f treatment. Purified endotoxin-free recombinant FN-f (FN7-10) of 42 kDa and EGFP fusion FN-f that both contain an α5β1 integrin RGD binding site were generated. Chondrocytes were treated with 1 μM FN-f for30 minutes to study the activation of cell signaling or overnight to study the production of MMPs, or with 1 μM FN-f-EGFP for indicated time periods to study integrin endocytosis. Transduction of chondrocytes with adenoviral constructs encoding peroxiredoxin 2 (PRX2) and cytosolic catalase (cCAT) were used to reduce cytosolic H2O2. Several pretreatment approaches were implemented in this study prior to FN-f stimulation, including free radical scavenger Tempol (100, 200, and 500 μM), pan Nox inhibitors VAS2870 (10 μM for cell signaling and 2 μM for MMP studies) and APX-115 (5 μM), scrambled ds-tat (2 μM), Nox2-specific inhibitor gp91ds-tat (2 μM), dual Nox1 and Nox4 inhibitor GKT137831 (20 μM), and dynamin inhibitor Dynasore (40, 60, 80 μM), which prevents endocytosis. Cell lysates or conditioned media were analyzed by immunoblotting to assess MMP-13 generation and MAP-kinase activation (JNK, ERK, and p38). To assess Nox isoform expression, seven Nox isoforms and subunits of Nox2 and Nox4, namely p22phox, p40phox, p47phox, and p67phox were analyzed by qPCR. Protein levels of Nox2 and Nox4 were analyzed by immunoblotting. Chondrocytes underwent nucleofection for the reporter constructs, including integrin α5-GFP, integrin α5-mCherry, and Rab5CA(Q79L)-mCherry. Alexa Fluor 568 conjugated transferrin followed by DiO plasma membrane labeling was used to study chondrocyte endocytosis using confocal microscopy. Results: Pretreatment with Tempol and adenoviral overexpression of antioxidants PRX2 and cCAT reduced the FN-f induced MAP kinase pathway and MMP-13 generation. Nox2 and Nox4 were identified as the two major Nox isoforms expressed in human articular chondrocytes (Fig 1a). Pan-Nox inhibitors, including VAS2870 (Fig 1c) and APX-115 (Fig 1d), and the Nox2-specific inhibitor gp91ds-tat (Fig 1e) abrogated FN-f-induced signaling and MMP-13 generation, whereas the dual Nox1 and Nox4 inhibitor GKT 137831 (Fig 1f) exhibited no effect, which indicated that Nox2 was necessary. The α5β1 integrin was endocytosed in response to FN-f stimulation in a time-dependent pattern (Fig 2a). Similar to FN-f, EGFP fusion FN-f activated MAP kinases and led to excessive MMP-13 generation in chondrocytes. FN-f-EGFP was endocytosed by chondrocytes together with α5β1 integrin, which was verified by confocal microscopy (Fig 2b) and immunoblotting. Following Dynasore pretreatment, a significant reduction in cellular endocytosis (Fig 2c) was observed as well as a decrease in FN-f induced MAP kinase activation and MMP-13 generation. Conclusions: We propose that FN-f induced ROS in chondrocytes are generated through Nox2 via integrin endocytosis, which regulates MAP kinase activation and MMP-13 generation. To further test this hypothesis, we are determining if Nox2 and the α5β1 integrin are co-localized in endosomes in response to FN-f and if this is a site for the activation of MAP kinases. Elucidating the mechanism of FN-f signaling to induce matrix degradation will lead to the discovery of new therapeutic targets to inhibit this process in arthritis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)