Nitric Oxide Regulates Transforming Growth Factor- Signaling in Endothelial Cells
http://www.100md.com
循环研究杂志 2005年第11期
The Departmento Fisiologe猘 (M.S., B.H., M.G., L.D.-M., M.R.-P.), Universidad de Alcale? Alcale?de Henares
Nefrologe猘 (D.R.-P.), Hospital Pre猲cipe de Asturias, Alcale?de Henares
Centro Nacional de Investigaciones Cardiovasculares (C.Z.), Madrid, Spain.
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-eCinitiated signal transduction. We hypothesized that NO may regulate endothelial TGF-eCdependent gene expression. We show that NO inhibits TGF-/SmadeCregulated 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-eCinduced gene expression. Inversely, overexpression of PKG-1 catalytic subunit blocked TGF-eCinduced 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-. Aortas from endothelial NO synthaseeCdeficient 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 endothelial cells vascular remodeling transforming growth factor-
Introduction
Transforming 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.1eC3 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 chorioneCallantoic 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-eCdependent effects play a role in the pathogenesis of atherosclerosis, coronary artery disease, transplant arteriosclerosis, hypertension, diabetes, myocardial remodeling, and restenosis.10eC14 Blood vessels overexpressing TGF- develop neointimal formation,15 whereas inhibition of TGF- signaling leads to the reversion of negative remodeling associated with an increase of TGF-.16,17 However, the effects of TGF- are complex and cell-type specific. For example, the role of TGF- in atherosclerosis is not yet fully elucidated, although it seems to regulate the equilibrium between inflammatory and fibrotic processes.18,19 TGF- can also induce arteriogenesis and markedly influences angiogenic processes, possessing both pro- and antiangiogenic effects.20
The reduction of NO bioavailability associated with endothelial dysfunction is strongly correlated with cardiovascular diseases including atherosclerosis.21 Thus, we decided to test the impact of endothelial-derived NO on TGF-eCdependent gene expression in the vascular endothelium.
Materials and Methods
Materials
Recombinant human TGF-1 was from R&D Systems (Oxon, UK). Spermine-NONOate and DEA-NONOate were from Alexis (San Diego, Calif). For other chemicals, see the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.
Plasmids
Flag-tagged cGMP-dependent protein kinase 1 regulatory region (fcGK-1R) and Flag-tagged cGMP-dependent protein kinase 1 catalytic region (fcGK-1C) were kindly donated by Dr D. Browning (Medical College of Georgia, Augusta).22 3TP-Lux plasmid that encodes 3 tandem copies of TPA-response element plus the TGF-eCresponsive region of the PAI-1 gene cloned in front of a luciferase gene was a kind gift from Dr R. Harland (University of California, Berkeley).23 The plasmid containing SV40-driven Renilla luciferase gene was from Promega (Madison, Wis).
Cell Culture
Bovine aortic endothelial cells (BAEC) and human umbilical endothelial cells (HuVEC) were isolated as described.24
Murine aortic endothelial cells (MAEC) were cultured from endothelial NO synthase (eNOS) wild-type (WT) and eNOS-deficient mouse aortas as described25 (see also expanded Material and Methods section in the online data supplement).
RNA Isolation and Northern Blot Analysis
Total RNA from BAEC was isolated as described.26 For Northern analysis, a 274-bp fragment of human TGF-1, a 1600-bp fragment of collagen type I cDNA, and an 18S RNA probe were radiolabeled (Redi Prime, Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunoblotting
Immunoblotting was performed as described.26
Immunohistochemistry, Immunofluorescence, and Confocal Microscopy
Immunohistochemistry from eNOS WT and eNOS-deficient mouse aortas was performed as described25 (see also the expanded Materials and Methods section in the online data supplement).
Transient Transfection
Transfection was performed with Lipofectamine as described.26 Luciferase activity was determined using the Dual luciferase reporter kit (Promega). To determine transfection efficiency, cells were transfected with either pGL2-Control Vector containing an SV40 promoter and enhancer or a -galactosidaseeCencoding plasmid.
For cGMP-dependent protein kinase I constructs (fcGK-IC and fcGK IR) overexpressing 2 e蘥 of plasmid DNA were transiently transfected with Lipofectamine as described26 (see also the expanded Material and Methods section in the online data supplement).
Nuclear Extract Preparation and Electrophoretic Mobility-Shift Assay
Nuclear and cytosolic extracts were prepared as described27 (see also the expanded Material and Methods section in the online data supplement).
Smad Steady-State Protein Level Assays
For Smad2 stability assays, transiently transfected BAEC with Flag-tagged Smad2 were labeled for 2 hours with 50 e藽i/mL [35S]methionine (Trans [35S]-label; ICN, Barcelona, Spain) and chased in the presence or absence of 30 e蘭ol/L MG-132 for the indicated time periods. Cells were lysed and the extracts were immunoprecipitated with anti-FLAG M2-agarose affinity gel. The precipitates were electrophoresed and autoradiographed. Band intensities were quantified by densitometry.
Ubiquitination Assays
BAEC were transiently transfected with Flag-tagged Smad2 alone or Flag-Smad2 plus His-tagged ubiquitin kindly provided by Dr D. Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany). Forty-eight hours after transfection, cells were lysed and F-Smad2 was immunoprecipitated with antieCFlag-M2 antibody or anti-histidine antibody; after 4 washes with lysis buffer, the immunocomplexes were resolved by SDS-PAGE and immunoblotted with anti-Flag or anti-ubiquitin.
Statistical Analysis
Experiments were performed at least 3 times, and every condition was performed in duplicate. Comparisons were made by ANOVA and NewmaneCKeuls post hoc testing. Results are expressed as mean±SEM, with P<0.05 considered statistically significant.
Results
NO Inhibits TGF-eCDependent Gene Transactivation Through the cGMP/PKG Pathway
We determined whether NO could affect the ability of TGF- to regulate gene expression in BAEC. The NO donor, Sp-NONOate (10eC5 mol/L), significantly blocked the TGF-eCmediated upregulation of TGF-1 and collagen type I mRNAs in a dose-dependent manner (Figure 1A). Similar results were obtained with TGF-eCmediated fibronectin expression (data not shown). We also determined whether exogenous NO affects the activity of TGF-eCresponsive promoters. BAEC were cotransfected with a TGF-eCsensitive promoter (3TP-Lux) and cytomegalovirus-Renilla as a control and treated with Sp-NONOate (NO) and TGF- as above. NO inhibited 3TP-Lux promoter activity induced by TGF- in a dose-dependent matter (Figure 1B). In addition, 3TP-Lux promoter activity in HuVEC stimulated with TGF- (8x10eC4 to 8x10eC1 nmol/L) resembled the typical U-shaped profile displayed for other TGF-eCregulated effects.28 NO was able to inhibit the effects of higher concentrations of TGF-, whereas it boosted the response at lower TGF- concentrations (Figure 1C).
Next, BAEC were treated with 8-Br-cGMP (a soluble analogue of cGMP) or Rp-8-GMPS (an antagonist of PKG-1), and TGF-1 mRNA expression was analyzed by Northern blot. We found that both NO and 8-Br-cGMP inhibited TGF-eCdependent TGF-1 mRNA expression (Figure 2A), and the inhibitory effect of NO was reversed by Rp-8-GMPS (10eC6 mol/L), suggesting that the effects of NO on TGF-eCdependent gene expression are mediated by the soluble guanylate cyclase/cGMP pathway. Indeed, overexpression of the regulatory region of PKG-1 (fcGK-IR), which acts as a dominant negative for PKG-1 activity, led to the abrogation of NO effects on TGF-eCstimulated 3TP-Lux transcriptional activation (Figure 2B). Transfection with a dominant-positive construct (the catalytic region of PKG-1 that retains kinase activity in the absence of cGMP; fcGK-IC) reproduced the effects of NO on TGF-eCstimulated 3TP-Lux activity (Figure 2B). Transfection efficiency was verified by immunological detection with anti-Flag antibody (supplemental Figure I).
Taken together, these results indicate that NO is able to inhibit the transactivation of TGF-eCregulated genes through the cGMP/PKG pathway.
NO Inhibits Smad Nuclear Accumulation via PKG-1 Activation
Most of TGF- transcriptional responses depend on Smad phosphorylation and translocation to the nucleus. We found that TGF- stimulation induced Smad2/3 nuclear translocation at 15 minutes of treatment, reaching a maximum at 30 minutes, and stayed elevated for at least 2 hours. By contrast, in NO-treated cells, Smad2/3 was detectable in the nuclear fraction 90 minutes after TGF- stimulation (Figure 3A). To confirm the effect of NO on Smad2/3 phosphorylation and subsequent nuclear translocation, we detected phosphorylated Smad2 (red) and total Smad2/3 (green) by confocal microscopy. TGF- induced early Smad2 phosphorylation at 15 minutes, followed by massive migration of total Smad2/3 to the nucleus, returning to basal levels 4 hours after stimulation (Figure 3B). Pretreatment of cells with NO reduced and significantly delayed the nuclear presence of phosphorylated Smad2/3 when compared with TGF- alone (60 minutes versus 15 minutes), and the levels of activated Smad2 were never comparable to TGF-eCstimulated cells alone. These results suggest that NO influences the activation of Smad2/3 on TGF- stimulation, reducing and delaying Smad2/3 nuclear accumulation.
We also investigated the effect of NO in the DNA binding of Smad2/3 to a Smad-binding element (SBE), finding TGF-eCinducible, NO-sensitive binding activity to DNA in the nuclear fraction of treated cells (Figure 3C). The identity of the proteins binding to the SBE was revealed by supershift analysis using specific anti-Smad2 and antieCSmad2/3 antibodies. Antibody to Smad2/3 inhibited the protein binding to DNA, whereas addition of anti-Smad2 slightly supershifted the DNAeCprotein complex from TGF-eCtreated cells, indicating that Smad2 forms part of the protein complex binding to the SBE.
In addition, we found that transfection with a dominant-negative PKG-1 (fcGK-1R) reversed the NO effect on TGF-eCinduced Smad2/3 nuclear translocation, whereas transfection with a dominant-positive PKG-1 mimicked the NO effects, as detected by immunofluorescence (Figure 4A). The reduction of nuclear phospho-Smad2 levels observed in NO+TGF-eCtreated cells was reversed by preincubation with the PKG-1 inhibitor Rp-8-GMPS, as revealed by immunoblot (Figure 4B).
Endogenous Endothelial NO Controls the Transcriptional Responses to TGF-
We found in BAEC that NO deprivation (24 hours of incubation with NG-nitro-L-arginine methyl ester [L-NAME] at different concentrations) exacerbates TGF-eCinduced Smad2 phosphorylation (Figure 5A). On the other hand, bradykinin stimulation of eNOS reproduced the effect of NO on TGF-eCinduced Smad2 phosphorylation (Figure 5B). In addition, TGF-1 mRNA expression was also enhanced by NO deprivation, indicating that endogenous endothelial NO has a role in TGF-eCelicited gene expression (Figure 5C).
To investigate the physiological relevance of those results, aortic rings from WT and eNOS-deficient (KO) mice were used to test basal TGF-1 and collagen type I expression by immunohistochemistry. TGF-1 expression was found to be higher in eNOS KO aortas (Figure 6A). Collagen type I content was also increased in the eNOS KO aortas. To quantify these results, aortas were collected and TGF-1 and collagen type I expression were analyzed by immunoblot. Figure 6B shows that TGF-1 and collagen type I protein expression was significantly higher in eNOS KO aortas than in WT, indicating that NO regulates TGF-eCdependent gene expression in vivo.
To further explore the role of endogenous NO, we studied TGF-eCstimulated Smad2 phosphorylation in MAEC from eNOS KO and WT animals by confocal microscopy. TGF- enhanced Smad2 phosphorylation in MAEC from eNOS-deficient mice (Figure 6C). Preincubation with 10eC5 mol/L DEA-NONOate (NO) produced a strong inhibition of Smad2 phosphorylation in both WT and KO endothelial cells, being more pronounced in the latter. In addition, the transcriptional response to TGF- was also higher in MAEC from eNOS KO mice at the different TGF- concentrations assayed (Figure 6D).
Taken together, these results confirm the role of endothelial NO in the regulation of the transcriptional responses to TGF- in the vascular endothelium.
NO Accelerates the Degradation of Smad2/3 by the Proteasome
TGF-eCmediated Smad2/3 activation can be negatively regulated by the overexpression of I-Smads (Smads 6 and 7).4,29 To exclude this possibility, BAEC were incubated with TGF- or TGF-+Sp-NONOate (NO) for 0 to 4 hours, and Smad6 or Smad7 levels were evaluated by immunoblot; we found no differences on Smad6 or Smad7 levels (Figure 7A). A longer time-course experiment, up to 24 hours, was performed with identical results (supplemental Figure II).
TGF receptor activation leads to Smad2 degradation by the proteasome pathway.30To address whether Smad2 degradation was the underlying mechanism of NO, the level of phosphorylated Smad2 to total Smad2/3 in response to TGF- was evaluated by immunoblot. Cell preincubation with proteasome inhibitors (MG-132 and proteasome inhibitor II [PI-II]) produced a reversion of the inhibitory effect of NO on TGF-eCmediated Smad2 phosphorylation (Figure 7B), suggesting that the proteasome pathway plays a role in NO effects.
We measured the metabolic stability of a ectopically expressed Flag-tagged Smad2 (Flag-Smad2). TGF- treatment reduced Flag-Smad2 stability, and the effect was reversed by pretreatment with the proteasome inhibitor MG-132 (Figure 8A). In addition, we found that NO alone had a slight effect in the stability of Smad2. However, the effect was dramatic on Flag-Smad2 protein stability induced by TGF-, suggesting that NO effects are mediated by a decrease in Smad2 metabolic stability.
Next, Smad2 ubiquitination mediated by NO was examined by ubiquitination assays in intact cells. The effect of TGF- on Flag-Smad2 ubiquitination was examined by adding TGF- 24 hours after transfection. The results demonstrated that TGF- induced the ubiquitination of Smad2, which was enhanced by MG-132 treatment (Figure 8B, lanes 2 and 5). NO treatment further promoted the ubiquitination of Flag-Smad2, and this effect was also enhanced in the presence of MG-132 (Figure 8B, lanes 3 and 6). To better characterized this effect, we repeated the ubiquitination assays for Flag-Smad2 in the presence or the absence of His-tagged ubiquitin. Immunoprecipitation was performed using anti-His antibody followed by anti-Flag immunoblot. Total Flag-Smad2 content was detected by immunoblotting of total cell lysates. Such analysis showed that TGF- induced the conjugation of ubiquitin molecules to Flag-Smad2, and this effect was enhanced by NO treatment (Figure 8C).
Discussion
Our data suggest a new role for the NO/cGMP/PKG pathway in vascular endothelium. NO inhibits TGF- activation of endothelial gene expression by interfering with Smad signaling, leading to the degradation of Smad2 by the ubiquitin proteasome pathway.
Alterations in the local abundance of TGF-1 appear to promote vascular wall remodeling, arterial lesion growth, and vascular cell transdifferentiation. On the other hand, TGF- can also act as an antiinflammatory and antiatherogenic cytokine with a protective role in the complications of atherosclerosis. Different levels of interaction between the TGF- and NO pathways have been reported in diabetes and hypertension.31,32 TGF- can induce eNOS expression through the Smad pathway,26 and TGF- can inhibit inducible NOS by multiple mechanisms.33eC35 By contrast, stimulatory effects of NO on TGF- production have been reported in smooth muscle cells.36 We show that endothelial cells treated with an NO donor exhibit a decreased response to TGF-, resulting in a downregulation of TGF- target genes. NO targeted the Smad pathway because NO inhibited Smad2 phosphorylation and nuclear translocation. In addition, we show that NO decreases the binding of Smad to a SBE, possibly because of the decreased Smad presence in the nucleus. Because NO can decrease the affinity of several transcription factors to DNA by S-nitrosylation,37eC39 another explanation for these effects is that NO interferes with the Smad binding to SBEs.
PKG-1 inhibition reversed the inhibitory effects of NO on the TGF/Smad signaling. Moreover, overexpression of dominant-negative PKG-1 was reversed, and the expression of a dominant-positive PKG-1 mimicked the inhibitory effect of NO on Smad2 nuclear translocation. Those results corroborate our previous findings regarding PKG-1 as the main effector of the NO/cGMP pathway.40 Similarly, natriuretic peptides counteract TGF- actions through a cGMP/PKG pathway.41 cAMP-elevating agents also inhibit the TGF/Smad pathway through PKA activation.42 Taken together, a new role for cyclic mononucleotide phosphate second messengers in controlling growth factor responses can be suggested.
If NO controls basal TGF-eCdependent gene expression and signaling attributable to Smad activation, a lack of NO should increase the responses to TGF-. Our results show that endogenous NO helps to maintain endothelial TGF- signaling under a tight control, both in cultured cells and in vivo. Inhibition of endogenous NO production augmented the responses to TGF-, whereas increased eNOS activity by bradykinin mimicked the effect of NO donors in endothelial cells. In vivo, there is a marked increase in TGF-1 and collagen type I expression in the aortas from eNOS-deficient mice. In MAEC, lack of eNOS exacerbated the transcriptional responses to TGF- and increased the overall Smad2 phosphorylation. Addition of NO decreased phospho-Smad2 levels in eNOS WT MAEC, whereas it restored phosphorylation to control levels in the eNOS-null MAEC.
Long-term inhibition of NO synthesis accelerates atherosclerosis, inducing early vascular inflammation as well as cardiac fibrosis and glomerulosclerosis.43eC45 These effects had been correlated with augmented collagen deposition in arteries and hearts accompanied by an increase in TGF-1 expression. Indeed, mice lacking eNOS developed greater neointimal proliferation or showed diminished remodeling compared with control mice.46 By contrast, eNOS gene delivery protects against many of those manifestations, such as cardiac remodeling after myocardial infarction, TGF- overexpression in aortic and heart fibrosis, and luminal narrowing after coronary angioplasty.47eC49 Based on this and our results, we suggest that impaired NO signal transduction may increase TGF- responses, which could contribute to the pathogenesis of some vascular diseases.
The ubiquitineCproteasome pathway regulates the activation status of the Smad family of proteins. Smad7 acts as an adaptor protein recruiting the E3 ligase Smurfs to the TGF- receptor complex, which induces its degradation.50 Endothelial cell treatment with 2 different proteasome inhibitors reversed the inhibitory effects of NO over TGF-/Smads. The impact of NO signaling over the proteasome pathway is starting to emerge. Our previous work indicates that C-type natriuretic peptide activates the proteolytic degradation of soluble guanylate cyclase by the proteasome through a cGMP/PKGeCdependent pathway.51 Others have shown that NO increases the proteolytic activity of the 20S and 26S proteasome in endothelial cells to inhibit H2O2-induced transferrin receptor-dependent apoptosis52 and that NO can S-nitrosylate Parkin, an E3 ubiquitin ligase important in the survival of dopamine neurons in Parkinson’s disease.53 Our results clearly show that NO increased the degradation of Smad activated by TGF- in an ubiquitin-proteasomeeCdependent manner. NO reduced the metabolic life of Flag-Smad2 after TGF- stimulation, and NO increased the ubiquitination of Smad2 induced by TGF-. The exact molecular mechanism exerted by NO is still undetermined. Many of the effects described in the present report are dependent on PKG activation, and there are some reports indicating that phosphorylated proteasomes have higher activities.54 PKG might initiate a kinase cascade that could enhance the proteasomal activity, thus contributing to an accelerated Smad2 degradation.
Our results support the hypothesis that NO serves as a molecular restraint to the excessive actions of TGF-. It is possible that NO regulates TGF-eCderived gene expression in the setting of pathological TGF- activation. Alternatively, NO may help to terminate TGF- signaling. Thus, specific control of TGF- expression and signaling by endothelial NO may be required to maintain vascular wall homeostasis.
Acknowledgments
This work was supported by Comiminunided de Madrid ("CAM") (08.4/0023/2003) (to M.S. and C.Z.); Spanish Society of Nephrology ("SEN,2003") (to M.S.); Ministerio de Ciercia y Tecnologist ("MCyT") (SAF 2002-00399) (to C.Z.); and "MCyT" (BFI 2001-1036) and "CAM" (08.4/0012/2001-2) (to M.R.-P.). M.S. and C.Z. are research investigators "Rame畁 y Cajal" (MCyT).
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Nefrologe猘 (D.R.-P.), Hospital Pre猲cipe de Asturias, Alcale?de Henares
Centro Nacional de Investigaciones Cardiovasculares (C.Z.), Madrid, Spain.
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-eCinitiated signal transduction. We hypothesized that NO may regulate endothelial TGF-eCdependent gene expression. We show that NO inhibits TGF-/SmadeCregulated 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-eCinduced gene expression. Inversely, overexpression of PKG-1 catalytic subunit blocked TGF-eCinduced 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-. Aortas from endothelial NO synthaseeCdeficient 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 endothelial cells vascular remodeling transforming growth factor-
Introduction
Transforming 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.1eC3 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 chorioneCallantoic 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-eCdependent effects play a role in the pathogenesis of atherosclerosis, coronary artery disease, transplant arteriosclerosis, hypertension, diabetes, myocardial remodeling, and restenosis.10eC14 Blood vessels overexpressing TGF- develop neointimal formation,15 whereas inhibition of TGF- signaling leads to the reversion of negative remodeling associated with an increase of TGF-.16,17 However, the effects of TGF- are complex and cell-type specific. For example, the role of TGF- in atherosclerosis is not yet fully elucidated, although it seems to regulate the equilibrium between inflammatory and fibrotic processes.18,19 TGF- can also induce arteriogenesis and markedly influences angiogenic processes, possessing both pro- and antiangiogenic effects.20
The reduction of NO bioavailability associated with endothelial dysfunction is strongly correlated with cardiovascular diseases including atherosclerosis.21 Thus, we decided to test the impact of endothelial-derived NO on TGF-eCdependent gene expression in the vascular endothelium.
Materials and Methods
Materials
Recombinant human TGF-1 was from R&D Systems (Oxon, UK). Spermine-NONOate and DEA-NONOate were from Alexis (San Diego, Calif). For other chemicals, see the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.
Plasmids
Flag-tagged cGMP-dependent protein kinase 1 regulatory region (fcGK-1R) and Flag-tagged cGMP-dependent protein kinase 1 catalytic region (fcGK-1C) were kindly donated by Dr D. Browning (Medical College of Georgia, Augusta).22 3TP-Lux plasmid that encodes 3 tandem copies of TPA-response element plus the TGF-eCresponsive region of the PAI-1 gene cloned in front of a luciferase gene was a kind gift from Dr R. Harland (University of California, Berkeley).23 The plasmid containing SV40-driven Renilla luciferase gene was from Promega (Madison, Wis).
Cell Culture
Bovine aortic endothelial cells (BAEC) and human umbilical endothelial cells (HuVEC) were isolated as described.24
Murine aortic endothelial cells (MAEC) were cultured from endothelial NO synthase (eNOS) wild-type (WT) and eNOS-deficient mouse aortas as described25 (see also expanded Material and Methods section in the online data supplement).
RNA Isolation and Northern Blot Analysis
Total RNA from BAEC was isolated as described.26 For Northern analysis, a 274-bp fragment of human TGF-1, a 1600-bp fragment of collagen type I cDNA, and an 18S RNA probe were radiolabeled (Redi Prime, Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunoblotting
Immunoblotting was performed as described.26
Immunohistochemistry, Immunofluorescence, and Confocal Microscopy
Immunohistochemistry from eNOS WT and eNOS-deficient mouse aortas was performed as described25 (see also the expanded Materials and Methods section in the online data supplement).
Transient Transfection
Transfection was performed with Lipofectamine as described.26 Luciferase activity was determined using the Dual luciferase reporter kit (Promega). To determine transfection efficiency, cells were transfected with either pGL2-Control Vector containing an SV40 promoter and enhancer or a -galactosidaseeCencoding plasmid.
For cGMP-dependent protein kinase I constructs (fcGK-IC and fcGK IR) overexpressing 2 e蘥 of plasmid DNA were transiently transfected with Lipofectamine as described26 (see also the expanded Material and Methods section in the online data supplement).
Nuclear Extract Preparation and Electrophoretic Mobility-Shift Assay
Nuclear and cytosolic extracts were prepared as described27 (see also the expanded Material and Methods section in the online data supplement).
Smad Steady-State Protein Level Assays
For Smad2 stability assays, transiently transfected BAEC with Flag-tagged Smad2 were labeled for 2 hours with 50 e藽i/mL [35S]methionine (Trans [35S]-label; ICN, Barcelona, Spain) and chased in the presence or absence of 30 e蘭ol/L MG-132 for the indicated time periods. Cells were lysed and the extracts were immunoprecipitated with anti-FLAG M2-agarose affinity gel. The precipitates were electrophoresed and autoradiographed. Band intensities were quantified by densitometry.
Ubiquitination Assays
BAEC were transiently transfected with Flag-tagged Smad2 alone or Flag-Smad2 plus His-tagged ubiquitin kindly provided by Dr D. Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany). Forty-eight hours after transfection, cells were lysed and F-Smad2 was immunoprecipitated with antieCFlag-M2 antibody or anti-histidine antibody; after 4 washes with lysis buffer, the immunocomplexes were resolved by SDS-PAGE and immunoblotted with anti-Flag or anti-ubiquitin.
Statistical Analysis
Experiments were performed at least 3 times, and every condition was performed in duplicate. Comparisons were made by ANOVA and NewmaneCKeuls post hoc testing. Results are expressed as mean±SEM, with P<0.05 considered statistically significant.
Results
NO Inhibits TGF-eCDependent Gene Transactivation Through the cGMP/PKG Pathway
We determined whether NO could affect the ability of TGF- to regulate gene expression in BAEC. The NO donor, Sp-NONOate (10eC5 mol/L), significantly blocked the TGF-eCmediated upregulation of TGF-1 and collagen type I mRNAs in a dose-dependent manner (Figure 1A). Similar results were obtained with TGF-eCmediated fibronectin expression (data not shown). We also determined whether exogenous NO affects the activity of TGF-eCresponsive promoters. BAEC were cotransfected with a TGF-eCsensitive promoter (3TP-Lux) and cytomegalovirus-Renilla as a control and treated with Sp-NONOate (NO) and TGF- as above. NO inhibited 3TP-Lux promoter activity induced by TGF- in a dose-dependent matter (Figure 1B). In addition, 3TP-Lux promoter activity in HuVEC stimulated with TGF- (8x10eC4 to 8x10eC1 nmol/L) resembled the typical U-shaped profile displayed for other TGF-eCregulated effects.28 NO was able to inhibit the effects of higher concentrations of TGF-, whereas it boosted the response at lower TGF- concentrations (Figure 1C).
Next, BAEC were treated with 8-Br-cGMP (a soluble analogue of cGMP) or Rp-8-GMPS (an antagonist of PKG-1), and TGF-1 mRNA expression was analyzed by Northern blot. We found that both NO and 8-Br-cGMP inhibited TGF-eCdependent TGF-1 mRNA expression (Figure 2A), and the inhibitory effect of NO was reversed by Rp-8-GMPS (10eC6 mol/L), suggesting that the effects of NO on TGF-eCdependent gene expression are mediated by the soluble guanylate cyclase/cGMP pathway. Indeed, overexpression of the regulatory region of PKG-1 (fcGK-IR), which acts as a dominant negative for PKG-1 activity, led to the abrogation of NO effects on TGF-eCstimulated 3TP-Lux transcriptional activation (Figure 2B). Transfection with a dominant-positive construct (the catalytic region of PKG-1 that retains kinase activity in the absence of cGMP; fcGK-IC) reproduced the effects of NO on TGF-eCstimulated 3TP-Lux activity (Figure 2B). Transfection efficiency was verified by immunological detection with anti-Flag antibody (supplemental Figure I).
Taken together, these results indicate that NO is able to inhibit the transactivation of TGF-eCregulated genes through the cGMP/PKG pathway.
NO Inhibits Smad Nuclear Accumulation via PKG-1 Activation
Most of TGF- transcriptional responses depend on Smad phosphorylation and translocation to the nucleus. We found that TGF- stimulation induced Smad2/3 nuclear translocation at 15 minutes of treatment, reaching a maximum at 30 minutes, and stayed elevated for at least 2 hours. By contrast, in NO-treated cells, Smad2/3 was detectable in the nuclear fraction 90 minutes after TGF- stimulation (Figure 3A). To confirm the effect of NO on Smad2/3 phosphorylation and subsequent nuclear translocation, we detected phosphorylated Smad2 (red) and total Smad2/3 (green) by confocal microscopy. TGF- induced early Smad2 phosphorylation at 15 minutes, followed by massive migration of total Smad2/3 to the nucleus, returning to basal levels 4 hours after stimulation (Figure 3B). Pretreatment of cells with NO reduced and significantly delayed the nuclear presence of phosphorylated Smad2/3 when compared with TGF- alone (60 minutes versus 15 minutes), and the levels of activated Smad2 were never comparable to TGF-eCstimulated cells alone. These results suggest that NO influences the activation of Smad2/3 on TGF- stimulation, reducing and delaying Smad2/3 nuclear accumulation.
We also investigated the effect of NO in the DNA binding of Smad2/3 to a Smad-binding element (SBE), finding TGF-eCinducible, NO-sensitive binding activity to DNA in the nuclear fraction of treated cells (Figure 3C). The identity of the proteins binding to the SBE was revealed by supershift analysis using specific anti-Smad2 and antieCSmad2/3 antibodies. Antibody to Smad2/3 inhibited the protein binding to DNA, whereas addition of anti-Smad2 slightly supershifted the DNAeCprotein complex from TGF-eCtreated cells, indicating that Smad2 forms part of the protein complex binding to the SBE.
In addition, we found that transfection with a dominant-negative PKG-1 (fcGK-1R) reversed the NO effect on TGF-eCinduced Smad2/3 nuclear translocation, whereas transfection with a dominant-positive PKG-1 mimicked the NO effects, as detected by immunofluorescence (Figure 4A). The reduction of nuclear phospho-Smad2 levels observed in NO+TGF-eCtreated cells was reversed by preincubation with the PKG-1 inhibitor Rp-8-GMPS, as revealed by immunoblot (Figure 4B).
Endogenous Endothelial NO Controls the Transcriptional Responses to TGF-
We found in BAEC that NO deprivation (24 hours of incubation with NG-nitro-L-arginine methyl ester [L-NAME] at different concentrations) exacerbates TGF-eCinduced Smad2 phosphorylation (Figure 5A). On the other hand, bradykinin stimulation of eNOS reproduced the effect of NO on TGF-eCinduced Smad2 phosphorylation (Figure 5B). In addition, TGF-1 mRNA expression was also enhanced by NO deprivation, indicating that endogenous endothelial NO has a role in TGF-eCelicited gene expression (Figure 5C).
To investigate the physiological relevance of those results, aortic rings from WT and eNOS-deficient (KO) mice were used to test basal TGF-1 and collagen type I expression by immunohistochemistry. TGF-1 expression was found to be higher in eNOS KO aortas (Figure 6A). Collagen type I content was also increased in the eNOS KO aortas. To quantify these results, aortas were collected and TGF-1 and collagen type I expression were analyzed by immunoblot. Figure 6B shows that TGF-1 and collagen type I protein expression was significantly higher in eNOS KO aortas than in WT, indicating that NO regulates TGF-eCdependent gene expression in vivo.
To further explore the role of endogenous NO, we studied TGF-eCstimulated Smad2 phosphorylation in MAEC from eNOS KO and WT animals by confocal microscopy. TGF- enhanced Smad2 phosphorylation in MAEC from eNOS-deficient mice (Figure 6C). Preincubation with 10eC5 mol/L DEA-NONOate (NO) produced a strong inhibition of Smad2 phosphorylation in both WT and KO endothelial cells, being more pronounced in the latter. In addition, the transcriptional response to TGF- was also higher in MAEC from eNOS KO mice at the different TGF- concentrations assayed (Figure 6D).
Taken together, these results confirm the role of endothelial NO in the regulation of the transcriptional responses to TGF- in the vascular endothelium.
NO Accelerates the Degradation of Smad2/3 by the Proteasome
TGF-eCmediated Smad2/3 activation can be negatively regulated by the overexpression of I-Smads (Smads 6 and 7).4,29 To exclude this possibility, BAEC were incubated with TGF- or TGF-+Sp-NONOate (NO) for 0 to 4 hours, and Smad6 or Smad7 levels were evaluated by immunoblot; we found no differences on Smad6 or Smad7 levels (Figure 7A). A longer time-course experiment, up to 24 hours, was performed with identical results (supplemental Figure II).
TGF receptor activation leads to Smad2 degradation by the proteasome pathway.30To address whether Smad2 degradation was the underlying mechanism of NO, the level of phosphorylated Smad2 to total Smad2/3 in response to TGF- was evaluated by immunoblot. Cell preincubation with proteasome inhibitors (MG-132 and proteasome inhibitor II [PI-II]) produced a reversion of the inhibitory effect of NO on TGF-eCmediated Smad2 phosphorylation (Figure 7B), suggesting that the proteasome pathway plays a role in NO effects.
We measured the metabolic stability of a ectopically expressed Flag-tagged Smad2 (Flag-Smad2). TGF- treatment reduced Flag-Smad2 stability, and the effect was reversed by pretreatment with the proteasome inhibitor MG-132 (Figure 8A). In addition, we found that NO alone had a slight effect in the stability of Smad2. However, the effect was dramatic on Flag-Smad2 protein stability induced by TGF-, suggesting that NO effects are mediated by a decrease in Smad2 metabolic stability.
Next, Smad2 ubiquitination mediated by NO was examined by ubiquitination assays in intact cells. The effect of TGF- on Flag-Smad2 ubiquitination was examined by adding TGF- 24 hours after transfection. The results demonstrated that TGF- induced the ubiquitination of Smad2, which was enhanced by MG-132 treatment (Figure 8B, lanes 2 and 5). NO treatment further promoted the ubiquitination of Flag-Smad2, and this effect was also enhanced in the presence of MG-132 (Figure 8B, lanes 3 and 6). To better characterized this effect, we repeated the ubiquitination assays for Flag-Smad2 in the presence or the absence of His-tagged ubiquitin. Immunoprecipitation was performed using anti-His antibody followed by anti-Flag immunoblot. Total Flag-Smad2 content was detected by immunoblotting of total cell lysates. Such analysis showed that TGF- induced the conjugation of ubiquitin molecules to Flag-Smad2, and this effect was enhanced by NO treatment (Figure 8C).
Discussion
Our data suggest a new role for the NO/cGMP/PKG pathway in vascular endothelium. NO inhibits TGF- activation of endothelial gene expression by interfering with Smad signaling, leading to the degradation of Smad2 by the ubiquitin proteasome pathway.
Alterations in the local abundance of TGF-1 appear to promote vascular wall remodeling, arterial lesion growth, and vascular cell transdifferentiation. On the other hand, TGF- can also act as an antiinflammatory and antiatherogenic cytokine with a protective role in the complications of atherosclerosis. Different levels of interaction between the TGF- and NO pathways have been reported in diabetes and hypertension.31,32 TGF- can induce eNOS expression through the Smad pathway,26 and TGF- can inhibit inducible NOS by multiple mechanisms.33eC35 By contrast, stimulatory effects of NO on TGF- production have been reported in smooth muscle cells.36 We show that endothelial cells treated with an NO donor exhibit a decreased response to TGF-, resulting in a downregulation of TGF- target genes. NO targeted the Smad pathway because NO inhibited Smad2 phosphorylation and nuclear translocation. In addition, we show that NO decreases the binding of Smad to a SBE, possibly because of the decreased Smad presence in the nucleus. Because NO can decrease the affinity of several transcription factors to DNA by S-nitrosylation,37eC39 another explanation for these effects is that NO interferes with the Smad binding to SBEs.
PKG-1 inhibition reversed the inhibitory effects of NO on the TGF/Smad signaling. Moreover, overexpression of dominant-negative PKG-1 was reversed, and the expression of a dominant-positive PKG-1 mimicked the inhibitory effect of NO on Smad2 nuclear translocation. Those results corroborate our previous findings regarding PKG-1 as the main effector of the NO/cGMP pathway.40 Similarly, natriuretic peptides counteract TGF- actions through a cGMP/PKG pathway.41 cAMP-elevating agents also inhibit the TGF/Smad pathway through PKA activation.42 Taken together, a new role for cyclic mononucleotide phosphate second messengers in controlling growth factor responses can be suggested.
If NO controls basal TGF-eCdependent gene expression and signaling attributable to Smad activation, a lack of NO should increase the responses to TGF-. Our results show that endogenous NO helps to maintain endothelial TGF- signaling under a tight control, both in cultured cells and in vivo. Inhibition of endogenous NO production augmented the responses to TGF-, whereas increased eNOS activity by bradykinin mimicked the effect of NO donors in endothelial cells. In vivo, there is a marked increase in TGF-1 and collagen type I expression in the aortas from eNOS-deficient mice. In MAEC, lack of eNOS exacerbated the transcriptional responses to TGF- and increased the overall Smad2 phosphorylation. Addition of NO decreased phospho-Smad2 levels in eNOS WT MAEC, whereas it restored phosphorylation to control levels in the eNOS-null MAEC.
Long-term inhibition of NO synthesis accelerates atherosclerosis, inducing early vascular inflammation as well as cardiac fibrosis and glomerulosclerosis.43eC45 These effects had been correlated with augmented collagen deposition in arteries and hearts accompanied by an increase in TGF-1 expression. Indeed, mice lacking eNOS developed greater neointimal proliferation or showed diminished remodeling compared with control mice.46 By contrast, eNOS gene delivery protects against many of those manifestations, such as cardiac remodeling after myocardial infarction, TGF- overexpression in aortic and heart fibrosis, and luminal narrowing after coronary angioplasty.47eC49 Based on this and our results, we suggest that impaired NO signal transduction may increase TGF- responses, which could contribute to the pathogenesis of some vascular diseases.
The ubiquitineCproteasome pathway regulates the activation status of the Smad family of proteins. Smad7 acts as an adaptor protein recruiting the E3 ligase Smurfs to the TGF- receptor complex, which induces its degradation.50 Endothelial cell treatment with 2 different proteasome inhibitors reversed the inhibitory effects of NO over TGF-/Smads. The impact of NO signaling over the proteasome pathway is starting to emerge. Our previous work indicates that C-type natriuretic peptide activates the proteolytic degradation of soluble guanylate cyclase by the proteasome through a cGMP/PKGeCdependent pathway.51 Others have shown that NO increases the proteolytic activity of the 20S and 26S proteasome in endothelial cells to inhibit H2O2-induced transferrin receptor-dependent apoptosis52 and that NO can S-nitrosylate Parkin, an E3 ubiquitin ligase important in the survival of dopamine neurons in Parkinson’s disease.53 Our results clearly show that NO increased the degradation of Smad activated by TGF- in an ubiquitin-proteasomeeCdependent manner. NO reduced the metabolic life of Flag-Smad2 after TGF- stimulation, and NO increased the ubiquitination of Smad2 induced by TGF-. The exact molecular mechanism exerted by NO is still undetermined. Many of the effects described in the present report are dependent on PKG activation, and there are some reports indicating that phosphorylated proteasomes have higher activities.54 PKG might initiate a kinase cascade that could enhance the proteasomal activity, thus contributing to an accelerated Smad2 degradation.
Our results support the hypothesis that NO serves as a molecular restraint to the excessive actions of TGF-. It is possible that NO regulates TGF-eCderived gene expression in the setting of pathological TGF- activation. Alternatively, NO may help to terminate TGF- signaling. Thus, specific control of TGF- expression and signaling by endothelial NO may be required to maintain vascular wall homeostasis.
Acknowledgments
This work was supported by Comiminunided de Madrid ("CAM") (08.4/0023/2003) (to M.S. and C.Z.); Spanish Society of Nephrology ("SEN,2003") (to M.S.); Ministerio de Ciercia y Tecnologist ("MCyT") (SAF 2002-00399) (to C.Z.); and "MCyT" (BFI 2001-1036) and "CAM" (08.4/0012/2001-2) (to M.R.-P.). M.S. and C.Z. are research investigators "Rame畁 y Cajal" (MCyT).
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