MMP-13 Regulates Matrix Binding and Internalization of NG2/CSGP4 in Mandibular Fibrochondrocytes

Abstract

Objective: Post-traumatic osteoarthritis (OA) is associated with cartilage degradation resulting in changes in cell-matrix mediated signaling after injury. Matrix metalloproteinase-13 (MMP13) is a critical enzyme in this process, degrading the extracellular matrix and modifying receptors. One of the receptors altered by MMP13 is a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). In the temporomandibular joint (TMJ), NG2/CSPG4 is membrane bound and binds with type VI collagen. During OA, the NG2/CSPG4 intracellular domain is internalized into the cytosol. While NG2/CSPG4 is a known substrate for MMP13, there are important gaps in knowledge related to how MMP-13 modifies NG2/CSPG4 after injury. The hypothesis of this study is that MMP13 regulates NG2/CSPG4 interactions with the extracellular matrix, the internalization of the intracellular domain, and key cartilage signaling pathways during mechanical loading and OA progression. Methods: To characterize MMP13 and NG2/CSPG4 levels during the progression of TMJ OA, we used a surgical destabilization preclinical murine model (approved under UIC ACC #20-068) and compared them with clinical samples from human patients receiving total joint replacement for advanced stage TMJ OA (approved under UIC IRB #2017-0033). To mechanistically interrogate MMP-13-NG2/CSPG4, we cultured primary mandibular fibrochondrocytes and treated them with an MMP13 inhibitor (CAS 544678-85-5) at 15 nM for 24 hours or vehicle control. To evaluate matrix binding, we performed immunocytochemistry using an antibody against the NG2/CSPG4 ectodomain on decellularized coverslips. To mechanically load samples, cell-agarose-collagen scaffolds received a constrained static compression treatment for 2 hours at 2.5 N with and without the MMP13. Gene expression changes were quantified using RT-qPCR and protein changes were quantified with western blot. Data were evaluated using a one-way ANOVA. Results: Our results illustrate that MMP13 is present in preclinical and clinical samples a spatiotemporally consistent pattern with NG2/CSPG4 internalization. In vitro, the inhibition of MMP13 impairs the ability of the NG2/CSPG4 ectodomain to be retained in the extracellular matrix. Mechanical loading of the cells activates MMP13, leading to clearance of membrane associated NG2/CSPG4. Inhibition of MMP13 during mechanical loading promotes the accumulation of membrane associated NG2/CSPG4 and a decrease in internalized NG2/CSPG4 (n = 4; p < 0.05). MMP13 did not cause the complete proteolytic degradation of the ectodomain. This mechanically sensitive MMP-13-NG2/CSPG4 axis regulates the expression of key mineralization and OA genes including BMP2 and PTHrP (n = 4; p < 0.05). Conclusions: Together, these findings indicate that MMP13 mediated cleavage of NG2/CSPG4 potentiates the internalization of the intracellular domain and regulates genes implicated in the mineralization signaling axis during growth and degenerative diseases such as OA. Funding support from NIH/NIDCR 1R01DE029835-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.