Tumor necrosis factor-α alters integrins and metalloprotease ADAM12 levels and signaling in differentiating myoblasts

The extracellular matrix (ECM) is important in the regulation of myogenesis. We hypothesized that tumor necrosis factor-alpha (TNF-alpha) modifies ECM during differentiation of mouse C2C12 myoblasts. Exogenous TNF-alpha (1 ng/ml) stimulated myoblast fusion on the 3rd day (by 160% vs control) but not on the 5th day of myogenesis. The level of integrin alpha 5 was significantly augmented by TNF-alpha during 5 day-differentiation; however, integrin beta 1 was higher than control only on the 3rd day of cytokine treatment. Both the abundance of integrin alpha 5 bound to actin and the level of integrin (31 complexed with integrin alpha 5 increased in the presence of TNF-alpha, especially on the 3rd day of differentiation. Similarly, the stimulatory effects of TNF-alpha on integrin alpha 3, metalloprotease ADAM12 and kinases related to integrins, FAK and ILK, were limited to the 3rd day of differentiation. We concluded that TNF-alpha-induced changes in ECM components in differentiating myogenic cells, i.e. i) increased expression of integrin alpha 5, beta 1, alpha 3, and metalloprotease ADAM12, ii) enhanced formation of alpha 5 beta 1 integrin receptors and interaction of integrin alpha 5-cytoskeleton, and iii) increased expression of kinases associated with integrin signaling, FAK and ILK, were temporarily associated with the onset of myocyte fusion.