Nitric Oxide Regulates Transforming Growth Factor-β Signaling in Endothelial Cells

Abstract

Abstract-Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-␤ 1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-␤-initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-␤-dependent gene expression. We show that NO inhibits TGF-␤/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␣ suppressed NO/cGMP inhibition of TGF-␤-induced gene expression. Inversely, overexpression of PKG-1␣ catalytic subunit blocked TGF-␤-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-␤. Aortas from endothelial NO synthase-deficient mice showed enhanced basal TGF-␤ 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-␤ 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-␤/Smad2 signaling by directing the proteasomal degradation of activated Smad. Key Words: nitric oxide Ⅲ endothelial cells Ⅲ vascular remodeling Ⅲ transforming growth factor-␤ T ransforming growth factor (TGF)-␤ plays a major role in the vascular response to injury by controlling both cellular proliferation and extracellular matrix turnover through the Smad-signaling pathway. [1][2][3] Ligand binding leads to phosphorylation and nuclear translocation of receptor-activated Smads (R-Smads), Smad2/3, which modulate the transcription of a large number of genes. Smad7 and Smad6, inhibitory Smads (I-Smad), antagonize TGF-␤ signaling. Smad7/6 expression is induced by TGF-␤ in the endothelium, providing an autoregulatory negative feedback loop on TGF-␤ signaling. 4 The response to TGF-␤ in the cardiovascular system is tightly controlled. Mice deficient in TGF-␤ 1 die in utero because of vascular defects. 5 Smad1-deficient mice fail to establish chorion-allantoic circulation, whereas Smad5-deficient embryos have defects in yolk sac vasculature with enlarged blood vessels. 6,7 Mice deficient in the accessory receptor endoglin exhibit embryonic lethality, with cardiovascular and angiogenesis defects associated with abnormal vascular smooth muscle cell development. 8 Smad6-deficient mice develop aortic ossification and elevated blood pressure. 9 TGF-␤ is highly expressed in injured arteries, and TGF-␤-dependent effects play a role in the pathogenesis of atherosclerosis, coronary artery disease, transplant arteriosclerosis, hypertension, diabetes, myocardial remodeling, and restenosis. 10 -14 Blood vessels overexpressing TG...

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats

“…12,33,37,38 This assumption is based on the known inhibitory effects of cGMP on collagen synthesis and the TGFb1 pathway, and its vasculoprotective effects on arterial SMC. [14][15][16][17]20,[45][46][47] However, the anti-apoptotic and pro-proliferative effects of sildenafil that we found in the corporal smooth muscle do not agree with the fact that cGMP inhibits vascular SMC proliferation. 48,49 Since the effects of BCNR on the corporal histology and pharmacokinetics are the same as those seen in the aged rat, we assume that cGMP effects on corporal SMC may be modulated by some specific features in the corporal SMC themselves or the tissue milieu, that are not operative in the vascular SMC.…”

“…[11][12][13] It is likely that this occurs through the maintenance of high levels of cGMP, since this compound reduces collagen synthesis and the activation of the pro-fibrotic TGFb1 pathway and protects SMC from apoptosis, while stimulating the spontaneous induction of inducible nitric oxide synthase (iNOS, also known as NOS2). [14][15][16][17][18][19][20][21][22][23] The expression of iNOS in certain non-immunological tissues is assumed to be a defense mechanism against fibrosis. [24][25][26][27][28][29] The nitric oxide produced by iNOS, besides inhibiting collagen synthesis and the TGFb1 pathway, also quenches reactive oxygen species, and in some cases, the differentiation of fibroblasts to myofibroblasts, the cells that produce collagen in many fibrotic conditions.…”

Mechanism of the protective effects of the combined treatment with rhynchophylla total alkaloids and sinapine thiocyanate against a prothrombotic state caused by vascular endothelial cell inflammatory damage

“…Furthermore, TGF-β1 upregulates PAI-1 expression at the transcriptional level via the TGF-β1/SMADs and TGF-β1/MAPK signaling pathways (27,28). PAI is a serine protease inhibitor.…”

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: Studies on TGF-β1 regulation

“…Previous reports have showed that ROS can regulate TGF-β1 synthesis GONZÁLEZ-RAMOS ET AL. 4 (5,12,17,23,25,32) also through the activation of the transcription factor AP-1 (12). Taking into account this fact, we hypothesized that basal concentrations of ROS could be involved in the constitutive expression of TGF-β1.…”