Nitric Oxide Regulates Transforming Growth Factor-&bgr; Signaling in Endothelial Cells

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-&bgr;1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-&bgr;–initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-&bgr;–dependent gene expression. We show that NO inhibits TGF-&bgr;/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&agr; suppressed NO/cGMP inhibition of TGF-&bgr;–induced gene expression. Inversely, overexpression of PKG-1&agr; catalytic subunit blocked TGF-&bgr;–induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas NG-nitro-l-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-&bgr;. Aortas from endothelial NO synthase–deficient mice showed enhanced basal TGF-&bgr;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-&bgr; 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-&bgr;/Smad2 signaling by directing the proteasomal degradation of activated Smad.

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