Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-beta1 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-1alpha suppressed NO/cGMP inhibition of TGF-beta-induced gene expression. Inversely, overexpression of PKG-1alpha 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 NG-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-beta1 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.
"It also provides in vivo and in vitro evidence for a pro-proliferative and pro-fibrotic action of Smad3 linker region phosphorylation, which may help to clarify the contradictory findings described in different cancer cell lines , . These findings also substantially extend a previous study showing that aortas from eNOS-/- mice exhibit enhanced basal and TGF-β1 induced collagen type I expression and that endothelial cells from these animals showed increased Smad2/3 phosphorylation . "
[Show abstract][Hide abstract] ABSTRACT: Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.
PLoS ONE 12/2013; 8(12):e84063. DOI:10.1371/journal.pone.0084063 · 3.23 Impact Factor
"During high salt intake, a signaling complex (Pyk2/c-Src/PI3K) is activated, promoting endothelial production of TGF-ß and NO, the latter through Akt activation . The production of NO is facilitated by TGF-ß-mediated reduction in PTEN, and thus permits completing a negative feedback loop to regulate production of TGF-ß , , . However, the age-related increase in the “setpoint” relationship between active TGF-ß and NO and the observed resistance to change in endothelial PTEN expression despite an increase in TGF-ß raise the possibility of progressive loss of inhibition of the pathways driven by TGF-ß with aging. "
[Show abstract][Hide abstract] ABSTRACT: Aging promotes endothelial dysfunction, defined as a reduction in bioavailable nitric oxide (NO) produced by the endothelial isoform of nitric oxide synthase (NOS3). This enzyme is critically regulated by phosphorylation by protein kinase B (Akt), which in turn is regulated by the lipid phosphatase, PTEN. The present series of studies demonstrated a reduction in bioavailable NO as the age of rats increased from 1 to 12 months. At 12 months of age, rats no longer demonstrated increases in phosphorylated NOS3 in response to high dietary salt intake. Endothelial cell levels of PTEN increased with age and became refractory to change with increased salt intake. In contrast to the reduction in NO production, endothelial cell production of transforming growth factor-ß (TGF-ß) relative to NO increased progressively with age. In macrovascular endothelial cells, PTEN was regulated in a dose-dependent fashion by TGF-ß, which was further regulated by extracellular [KCl]. When combined with prior studies, the present series of experiments suggested an integral role for PTEN in endothelial cell pathobiology of aging and an important mitigating function of TGF-ß in endothelial PTEN regulation. The findings further supported a role for diet in affecting vascular function through the production of TGF-ß and NO.
PLoS ONE 11/2012; 7(11):e48715. DOI:10.1371/journal.pone.0048715 · 3.23 Impact Factor
"One relevant constitutive gene in cells involved in physiological and pathological processes is TGF-␤1, a cytokine from a large family of growth and differentiation factors involved in cell division, differentiation, migration, adhesion, extracellular matrix production and tissue homeostasis (Dallas et al., 2005; Massagué, 2008; Ruiz-Ortega et al., 2007; Shi and Massagué, 2003). Previous reports have shown that exogenous high ROS concentrations can regulate TGF-␤1 synthesis with important consequences on cellular functions (Iglesias-De La Cruz et al., 2001; Liu and Gaston Pravia, 2010; Ortega-Velázquez et al., 2004; Rhyu et al., 2005; Saura et al., 2005). Moreover, it has been demonstrated that this regulation is dependent on AP-1 transcriptional activity (Iglesias-De La Cruz et al., 2001). "
[Show abstract][Hide abstract] ABSTRACT: The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.
The international journal of biochemistry & cell biology 10/2012; 45(2). DOI:10.1016/j.biocel.2012.10.001 · 4.05 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.