Transforming growth factor beta (TGF-beta) belongs to a family of inflammatory cytokines involved in the regulation of cellular differentiation, proliferation, migration, and protein expression. The ability of TGF-beta to alter endothelial cell (EC) phenotype suggests its role in the regulation of vascular endothelial integrity. We hypothesized that TGF-beta-induced activation of ALK-receptor/SMAD complexes may initiate specific signaling pathways leading to increased EC contractility and EC barrier dysfunction. Using siRNA approach, we showed that depletion of ALK5 and SMAD4, but not ALK1 receptors attenuated TGF-beta-induced decreases in transendothelial electrical resistance (TER) (49.5 ± 11.8% and 39.7 ± 8.7% inhibition, respectively, p<0.05, n = 5) suggesting direct involvement of ALK5- and Smad-mediated signaling in EC barrier regulation. Consistently, EC treatment with SB 431542 (inhibitor of ALK5 but not ALK1 receptors) attenuated TGF-beta-induced TER decreases (78.1 ± 14.9% inhibition, p<0.05, n = 6). TGF-beta-induced TER decrease was accompanied by paracellular gap formation and EC contraction judged by actin stress fiber formation and increased phoshorylation of myosin light chains (MLC) and MLC phosphatase. These changes were attenuated by inhibition of Rho kinase (51.6 ± 15.3% inhibition of permeability, 75.3 ± 16.2% inhibition of stress fiber formation and 79.5 ± 15.4% inhibition of gap formation), but not by MLC kinase inhibitors, suggesting a leading role for the Rho/Rho kinase-mediated pathway. Furthermore, TGF-beta induced p38 MAP kinase activation and phosphorylation of regulatory cytoskeletal protein HSP-27, whereas molecular and pharmacological inhibition of p38 MAPK pathway attenuated HSP-27 phosphorylation and TGF-beta-induced EC permeability (64.8 ± 11.6% inhibition). Finally, elevation of intracellular cAMP levels attenuated TGF-beta-induced Rho activation and EC barrier dysfunction (88.7 ± 7.3% inhibition of TGF-beta-induced permeability), which was reversed by protein kinase A inhibitors. Thus, our data suggest ALK5/SMAD4 - Rho/p38 MAPK - MLC/HSP27-mediated mechanism of TGF-beta-mediated EC remodeling and barrier dysfunction.
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