Response gene to complement 32 interacts with SMAD3 to promote TGF-β-induced extracellular matrix production in multiple sclerosis

Abstract

Abstract We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growth factor-β (TGF-β)-induced astrocyte reactivity and extracellular matrix production. However, the molecular mechanisms underlying RGC-32 expression and function remain to be elucidated. In the present study, we found that TGF-β induced expression and nuclear translocation of both RGC-32 and SMAD3 in astrocytes. Using specific pathways inhibitors, we have found that RGC-32 induction is mediated by SMAD-dependent and independent mechanisms. We investigated whether RGC-32 interacts with SMAD2 or SMAD3 in astrocytes and if this association plays a role in extracellular matrix synthesis. Co-immunoprecipitation showed that SMAD3, but not SMAD2 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of SMAD3 phosphorylation, significantly reduced RGC-32 translocation to the nucleus, indicating that SMAD3 phosphorylation is required for RGC-32 nuclear translocation. On the other hand, silencing RGC-32 with siRNA had no effect on SMAD3 nuclear translocation. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the SMAD3 nuclear translocation was unaffected. The inhibition of SMAD3 phosphorylation also blocked the RGC-32-induced collagen type I expression, indicating a synergistic action between RGC-32 and SMAD3 to induce the expression of collagen type I. Taken together, our data demonstrate for the first time that RGC-32 interacts with SMAD3 to mediate the extracellular matrix production in astrocytes. These data suggest that a similar mechanism might be involved in mediation of gliosis seen in chronic multiple sclerosis lesions.