Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-alpha promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase dependent manner. Consistent with this observation, TNF-alpha mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-kappa B RelA/p65 and subsequent DNA binding activity, with no significant effects on I kappa B alpha degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.