Limitation by Rho-kinase and Rac of transforming growth factor-β-induced interleukin-6 release from astrocytes

Transforming growth factor (TGF)-β stimulates release of interleukin (IL)-6, which is recognized to function as both a pro- and anti- inflammatory cytokine in the central nervous system, from astrocytes. It is generally recognized that effects of TGF-β are mediated through Smad-independent as well as Smad-dependent pathways. Small GTPases regulate a variety of cell functions. In the present study, we investigated whether or not Rho-kinase, a downstream effector of Rho, and Rac are implicated in TGF-β-stimulated IL-6 release from astrocytes (C8D1A cells). Y-27632 or fasudil (Rho-kinase inhibitors) or NSC23766 (an inhibitor of Rac-guanine nucleotide exchange factor interaction) significantly enhanced TGF-β-stimulated IL-6 release from these cells. TGF-β-stimulated IL-6 release was markedly upregulated in RhoA- or Rac-knockdown C8D1A cells. We found that SIS3 (a specific inhibitor of TGF-β-dependent Smad3 phosphorylation) or LY364947 (a TGF-β type I receptor kinase inhibitor) significantly reduced the IL-6 release. However, TGF-β-induced-Smad2 and Smad3 phosphorylation was not affected by Y-27632, fasudil or NSC23766. In conclusion, our results strongly suggest that Rho-kinase and Rac limit TGF-β-induced IL-6 release from astrocytes, and the suppressive effects are exerted independently of the Smad pathway or at a point downstream of Smad2/3 complex.