Cell-Matrix Interactions Regulating the Migration of Mandibular Fibrochondrocytes

BACKGROUND: Cell migration is critical for the injury response of the temporomandibular joint. Neuron/Glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that interacts with β1-integrins and influences cell migration. NG2/CSPG4 regulates the mTOR signaling pathway, an established signaling axis that coordinates cell survival, proliferation, and migration. OBJECTIVE: The objective of this study is to determine if the NG2/CSPG4 impacts cell migration in an mTOR dependent manner in mandibular fibrochondrocytes. METHODS: Primary mandibular fibrochondrocytes were collected from the condylar cartilages of wild-type (WT) and NG2/CSPG4 knockout (KO) mice. Cells were digested in a type II collagenase suspended at 3 mg/ml for 45 minutes and 1.5mg/ml overnight. Isolated cells were plated in a 3-well culture insert for cell migration (Ibidi, Gräfelfing) using 55x103 cells and grown to 70-80% confluence. To evaluate the role of mTOR signaling, WT and NG2/CSPG4 KO cells were treated with and without the mTOR modulators MHY1485 (mTOR agonist, Calbiochem) and Rapamycin (mTOR antagonist, Sigma) in low-serum media for 24 hours. To evaluate the role of NG2/CSPG4 ectodomain binding, WT cells were treated with anti-NG2/CSPG4 monoclonal antibodies. To quantify cell migrations, the inserts were removed and imaged using a live cell with phase contrast microscopy (Leica, DMI6000B). Data were analyzed in ImageJ by manually tracing the leading edges and calculating cell-free areas at 0, 1, 4, 12, and 24 hours. All values were statistically compared using a one-way ANOVA with bonferroni corrections. RESULTS: Serum starvation significantly slowed the migration rate of WT, but not NG2/CSPG4 KO cells (p<0.05; n=4). NG2/CSPG4 knockout increased the migration rate of mandibular fibrochondrocytes in low serum (p<0.05; n=4). MHY 1485 attenuated the migration of NG2/CSPG4 KO, but not WT cells. Rapamycin significantly suppressed the migration of both NG2/CSPG4 KO and WT cells at all time points (p<0.05; n=4). NG2/CSPG4 antibody treatment slightly attenuated the migration of mandibular fibrochondrocytes, but not more than the IgG control. CONCLUSION: NG2/CSPG4 negatively regulates the migration of mandibular fibrochondrocytes in an mTOR dependent manner in serum starvation conditions. This novel signaling axis could be a potentially valuable therapeutic target for the mobilization of reparative migratory cells following traumatic injury to the joint.