Nitric oxide regulates transforming growth factor-β signaling in endothelial cells

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-beta(1) expression and endothelial dysfunction. Smad proteins are a key step in TGF-beta- initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-beta-dependent gene expression. We show that NO inhibits TGF-beta/Smad-regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1 alpha suppressed NO/ cGMP inhibition of TGF-beta-induced gene expression. Inversely, overexpression of PKG-1 alpha catalytic subunit blocked TGF-beta-induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-(1), whereas N-G- nitro-L-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-beta. Aortas from endothelial NO synthase- deficient mice showed enhanced basal TGF-beta(1) and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-beta signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-beta/Smad2 signaling by directing the proteasomal degradation of activated Smad.