Angiotensin II Activates the Smad Pathway in Vascular Smooth Muscle Cells by a Transforming Growth Factor-β–Independent Mechanism
Background— Angiotensin II (Ang II) participates in vascular fibrosis. Transforming growth factor-β (TGF-β) is considered the most important fibrotic factor, and Smad proteins are essential components of the TGF-β signaling system. Our aim was to investigate whether Ang II activates the Smad pathway in vascular cells and its potential role in fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) proteins.
Methods and Results— Systemic infusion of Ang II into Wistar rats increased aortic Smad2, phosphorylated-Smad2, and Smad4 expression, associated with CTGF upregulation. In growth-arrested vascular smooth muscle cells, Ang II treatment for 20 minutes caused Smad2 phosphorylation, nuclear translocation of phosphorylated-Smad2 and Smad4, and increased Smad DNA-binding activity. Ang II also caused Smad overexpression and Smad-dependent gene transcription. The AT1 antagonist losartan diminished Ang II-induced Smad activation. The blockade of endogenous TGF-β did not modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 diminished Ang II-induced Smad2 phosphorylation. These data show that Ang II activates the Smad pathway via AT1 receptors and MAPK activation independently of TGF-β. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II-induced CTGF promoter activation, gene and protein expression, and fibronectin and type-1 procollagen overexpression, showing that Smad activation is involved in Ang II-induced fibrosis.
Conclusions— Our results show that Ang II activates the Smad signaling system in vascular cells in vivo and in vitro. Smad proteins are involved in Ang II-induced CTGF and ECM overexpression independently of TGF-β. This novel finding suggests that Smad activation could be involved in the profibrogenic effects of Ang II in vascular diseases.
Received June 12, 2004; revision received December 15, 2004; accepted January 11, 2005.
Vascular fibrosis is one of the main features of cardiovascular diseases, including hypertension.1 The Smad proteins are essential components of the intracellular signaling pathways utilized by transforming growth factor-β (TGF-β) and participate in TGF-β–induced fibrosis.2 TGF-β presents pleiotropic effects and can both positively and negatively regulate systems involved in cell proliferation, apoptosis, migration, and synthesis of extracellular matrix (ECM) proteins, such as fibronectin, collagen, and plasminogen activator inhibitor-1 (PAI-1) in vascular smooth muscle cells (VSMCs). It therefore plays a major role in the development of cardiovascular diseases.3 On activation, TGF-β transduces its signal across the plasma membrane by binding to 2 specific serine/threonine kinase receptors, the type I and II receptors.2,4 TGF-β binds to type II receptor, which activates the type I receptor kinase, which, in turn, phosphorylates the receptor-regulated Smads (R-Smads) Smad2 and Smad3 at C-terminal serines. The R-Smads then dissociate from the receptor complex to form a heterotrimeric complex with Smad4. These complexes translocate to the nucleus and function as transcriptional regulators of target genes. The inhibitory Smad7 binds to activated type I receptor, thereby preventing phosphorylation of Smad2/3, or recruits the ubiquitin ligases Smurf1 and Smurf2 to induce proteasomal degradation of the receptor complexes.2,4
Angiotensin II (Ang II) regulates cell growth, inflammation, and fibrosis, contributing to the progression of vascular damage.1,5 The interrelation between Ang II and TGF-β is established. In different pathological settings and cell types, Ang II regulates TGF-β expression and may mediate some Ang II responses.1,5,6 ACE inhibitors and AT1 antagonists diminish tissue expression of TGF-β and fibrosis, and blockade of TGF-β diminishes Ang II-induced ECM production.1,5,6 Ang II and TGF-β share some intracellular mechanisms involved in fibrosis, including activation of protein kinases and production of growth factors.1,3,5–7 AT1 blockade diminishes Smad pathway activation in myocardial infarction in rats and in an experimental model of renal damage.8,9 However, no studies have investigated whether Ang II activates the Smad signaling pathway in VSMCs and its potential role in vascular fibrosis.
Connective tissue growth factor (CTGF) is a potent profibrotic factor implicated in fibrotic processes, including hypertension, atherosclerosis, and myocardial infarction.10 CTGF has been described as a mediator of TGF-β– and Ang II-induced fibrosis.7,10,11 In VSMCs, TGF-β and Ang II increased gene expression and production of CTGF and ECM, such as fibronectin and collagens.3,5 Recent studies have demonstrated that Smads are involved in the regulation of those genes by TGF-β.12,13 We therefore investigated whether the Smad signaling pathway mediates Ang II-induced fibrosis, evaluating CTGF and ECM regulation.
Systemic infusion of Ang II (50 ng/kg per minute; subcutaneous osmotic minipumps; n=8 rats in each group) for 3 days was performed in female Wistar rats.7 One group was treated with the AT1 antagonist losartan (10 mg/kg per day; n=8) starting 24 hours before Ang II infusion. Animals were anesthetized with 5 mg/kg xylazine (Rompun, Bayer AG) and 35 mg/kg ketamine (Ketolar, Fisher) following European guidelines. VSMCs from Wistar rat thoracic aorta were obtained14 and serum-starved for 48 hours before use.
For in vivo studies, paraffin-embedded sections of rat aorta were studied by immunohistochemistry.7 Antibodies used were as follows: Smad2, Smad4 (Santa Cruz Biotechnology, Santa Cruz, Calif); phosphorylated-Smad2 (kindly donated by Dr Moustakas, Uppsala, Sweden for immunohistochemistry and immunofluorescence, and ABCAM, UK for Western blot); anti-CTGF (Torrey Pines Biolabs, San Diego, Calif); anti-fibronectin (Chemicon International, Temecula, Calif); anti-FLAG (Sigma, Spain); and peroxidase-conjugated secondary antibodies (Amersham). Negative controls without the primary antibody or with an unrelated antibody were included to check for nonspecific staining. For in vitro studies, cells were fixed in Merckofix (Merck), treated with 0.1% Triton-X100, and incubated with primary antibodies followed by FITC-conjugated antibody.14 Nuclei were stained with 1 μg/mL propidium iodide. Samples were mounted in Mowiol 40-88 (Sigma) and examined by a laser scanning confocal microscope (Leika). In VSMCs, protein levels were also determined by Western blot.7
Analysis of Smad DNA-Binding Activity
Smad DNA-binding activity was determined in 6-μg nuclear extracts, obtained as described,14 by binding with a [γ-32P]-ATP-labeled oligonucleotide that contains consensus CAGA-box, the Smad binding sequence (5′-TCGAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAC-3′) (Sta. Cruz),15 and complexes were analyzed by electrophoretic mobility shift assay (EMSA). For competition studies, mutant CAGA-box oligo(5′-TCGAGAGCTAGATAAAAAGCTAGATATTAGCTAGATAC-3′) was used. For supershift, nuclear extracts were incubated with Smads antibodies 1 hour before incubation with labeled oligonucleotide.
Transfection, DNA Constructs, and Promoter Studies
VSMCs, in 6-well plates, were transiently transfected with FuGENE (Roche Molecular Biochemicals) and the reporter expression vectors. Smad-dependent promoter activation was evaluated by transfection of 1 μg Smad/luc (kindly donated by Dr Volgestein, Baltimore, Md15) and 0.5 μg TK-renilla as internal control (Clontech), and CTGF promoter activity was evaluated by 1 μg CTGF/secreted alkaline phosphatase (SEAP) (kindly donated by Dr Oliver, Fibrogen13) and 0.25 μg CMV-β-galactosidase (Clontech). After a 24-hour serum-starvation step, cells were stimulated for 24 hours, and conditioned media were assayed for luciferase/renilla or SEAP/β-galactosidase activity, respectively. To block Smad pathway activation, cells were transfected with PcDNA3-FLAG-Smad7 expression vector (kindly donated by Dr Massagué, Rockefeller University, New York, NY).
RNA was isolated by Trizol Invitrogen. Real-time polymerase chain reactions (PCRs) were performed on an ABI Prism 7500 sequence detection PCR system (Applied Biosystems) according to manufacturer’s protocol. CTGF and type 1 procollagen assay IDs are as follows: Rn00573960_m1 and Rn00584426_m1, respectively. These expression levels were normalized with GAPDH and 18s ribosomal RNA expression, whose assay IDs are Rn99999916_m1 and Hs99999901_s1, respectively.
The autoradiographs were scanned with the use of the GS-800 calibrated densitometer (Quantity One, Bio-Rad). Results are expressed as n-fold increase over control as mean±SEM of experiments made. Significance was established with GraphPAD Instat with the use of Student t test (GraphPAD Software), Wilcoxon test, and Student-Newman-Keuls test. Differences were considered significant when P<0.05.
Systemic Infusion of Ang II Increases Smad Expression in Aorta
In the aorta of control animals, a slight immunostaining for total Smad2 and Smad4 was observed, which was markedly increased in Ang II-infused rats (Figure 1A). In these animals enhanced accumulation of phosphorylated-Smad2 proteins in the nuclei of cells (mainly VSMCs) was found (Figure 1B). Ang II-infused rats also presented elevated CTGF expression (Figure 1A). The AT1 antagonist losartan diminished aortic Smad2, Smad4, and CTGF overexpression, suggesting that Ang II through AT1 receptor regulates Smad proteins in vivo.
Ang II Activates Smad Pathway in VSMCs
Ang II Caused Smad2 Phosphorylation
One of the initial steps of the activation of Smad pathway is the phosphorylation of R-Smads.2,4 After treatment of growth-arrested VSMCs with Ang II for 20 minutes, we observed that phosphorylated-Smad2 protein levels were increased (Western blot; Figure 2A), showing that Ang II induced Smad2 phosphorylation.
Ang II Caused Nuclear Translocation of Smad4 and Phosphorylated-Smad2
In growth-arrested VSMCs, the Smad proteins Smad4 and Smad2 are located in the cytosol. Treatment with Ang II for 20 minutes caused nuclear translocation of Smad4 (confocal microscopy; Figure 2B). This effect was similar to that found with TGF-β treatment. Stimulation with Ang II increased total phosphorylated-Smad2 staining and caused a marked translocation of phosphorylated-Smad2 from the cytosol to the nuclei (Figure 2C).
Ang II Increased Smads Production
Stimulation of VSMCs with Ang II for 48 hours caused a significant elevation of total Smad2 and Smad4 protein levels (Western blot; Figure 2D).
Ang II Increased Smad DNA-Binding Activity and Activated Smad-Dependent Gene Transcription
In the nucleus, R-Smad/Smad4 complex can activate transcription through direct binding to certain DNA sequences.15 In VSMCs, Ang II increased CAGA-box DNA-binding activity as early as 5 minutes and peaked between 15 and 20 minutes. This response was similar to that observed with TGF-β (Figure 3A). By supershift assays, the composition of Ang II-induced Smad complexes was studied (Figure 3B). Antibodies to Smad4 alone or combined Smad2/Smad4 shifted the band to a higher molecular weight. However, no effect was seen with Smad2 alone. Similar results were obtained in TGF-β–treated VSMCs. These data show that the Smad complex activated by Ang II is Smad2/Smad4 and that the protein that binds to the DNA is Smad4.
We investigated whether Ang II regulates Smad-mediated gene expression, by transient transfection with a luciferase Smad reporter plasmid (Smad/luc) that contains 4 copies of the recognition site for the Smad sequence.16 Ang II potently increased Smad promoter activity (Figure 4), as observed with TGF-β.
Ang II Activates Smad Pathway via AT1
In VSMCs, Ang II acts through 2 specific receptors, AT1 and AT2.5 The AT1 antagonist losartan significantly diminished Ang II-induced Smad activation (phosphorylation of Smad2, nuclear translocation of Smad2/Smad4, Smad overexpression, and Smad DNA-binding activation), whereas the AT2 antagonist PD123319 had no effect (Figures 2A, 2C, 2D, 3⇑C), suggesting that Ang II-induced Smad activation is mediated through AT1.
Molecular Mechanisms of Ang II-Induced Smad Activation
Ang II via AT1 receptors activates intracellular signaling systems, including mitogen-activated protein kinase (MAPK) pathway and epidermal growth factor (EGF) receptor transactivation, that participate in the regulation of fibrosis.17–20 Treatment of VSMCs with the p38 MAPK inhibitor SB203580 markedly diminished Ang II-induced Smad2 phosphorylation (Figure 2E), whereas the ERK inhibitor PD98059 had no effect. The inhibitor of EGF receptor transactivation AG1478 did not modify phosphorylated-Smad2 levels, showing that this mechanism is not involved in Ang II/Smad signaling. These data suggest that Smad2 phosphorylation, and therefore Smad pathway activation, is dependent on p38 MAPK activation.
Ang II Activates Smad Pathway by a TGF-β–Independent Mechanism
To clearly demonstrate that Ang II activates the Smad pathway independently of TGF-β, we used VSMCs from Wistar rats. These cells have been previously described to be unable to activate TGF-β in vitro.21 In control experiments, TGF-β levels were evaluated by enzyme-linked immunosorbent assay in conditioned media from Ang II-treated VSMCs. No active TGF-β levels were detected, but Ang II increased latent TGF-β production (data not shown). Blockade of endogenous TGF-β, by the extracellular matrix proteoglycan decorin or by neutralizing TGF-β antibody, did not modify Ang II-induced nuclear translocation of phosphorylated-Smad2, Smad DNA-binding activity, and promoter activation (Figures 2C, 3D, 4⇑⇑). These data show that Ang II activates the Smad pathway independently of endogenous TGF-β production or activation.
Ang II-Induced CTGF and Fibronectin Upregulation Is Mediated by Smad Activation
We evaluated whether Smad activation caused by Ang II is involved in CTGF regulation. In VSMCs, Ang II and TGF-β potently induced CTGF promoter activation (not shown) and CTGF protein synthesis.7 To block Smad actions, cells were transiently transfected with an expression vector encoding Smad7, which interferes with receptor-mediated activation of Smad2 and Smad3, known to inhibit TGF-β/Smad-mediated transcriptional effects.22 Overexpression of Smad7 (determined by anti-FLAG antibody) inhibited the inducible expression of CTGF promoter (Figure 5) and CTGF gene expression and protein production (Figure 6) caused by Ang II and TGF-β compared with treated cells transfected with empty vector. CTGF has been described as a mediator of TGF-β– and Ang II-induced fibrosis. In VSMCs we demonstrated that CTGF mediates Ang II-induced fibronectin production. Overexpression of Smad7 markedly diminished Ang II- and TGF-β–induced fibronectin production, whereas no effect was seen in cells transfected with empty vector (Figure 7A). Overexpression of Smad7 also diminished type-1 procollagen expression, measured by real-time PCR (Figure 7B). These data clearly demonstrate that Smad7 overexpression diminished Ang II-induced CTGF promoter activation and protein production of CTGF and ECM proteins, showing that the Smad pathway is involved in Ang II-induced fibrosis.
Our results show that Ang II activates the Smad signaling system in vascular cells both in vivo and in vitro. We also found that Smad proteins participate in Ang II-induced CTGF overexpression and ECM production. This novel finding suggests that Smad pathway activation may be involved in the profibrogenic effects of Ang II in vascular diseases.
To evaluate the effect of Ang II on the Smad signaling pathway in vivo, we used the model of systemic infusion of Ang II. In this model, Ang II causes vascular fibrosis that can be mediated by CTGF overexpression.7 In the aorta of control animals, we observed a weak staining of Smad proteins, which were markedly upregulated in Ang II-infused rats. Importantly, positive staining for phosphorylated-Smad2 was found to be mainly located in the nuclei of VSMCs. The increased expression of Smad proteins was associated with CTGF induction, preceding the accumulation of ECM proteins observed after 7 days,7 suggesting that the Smad signaling pathway could be a mechanism involved in vascular fibrosis caused by Ang II.
To clearly demonstrate that Ang II activates the Smad signaling pathway through direct activation of VSMCs and independent of hypertension- or hemodynamic-induced changes, we performed experiments in cultured VSMCs. In these cells, Ang II caused Smad2 phosphorylation, nuclear translocation of phosphorylated-Smad2 and Smad4, increased DNA-binding activity to CAGA-box oligonucleotide, and overexpression of Smad2 and Smad4. Ang II-induced Smad activation is very rapid (as early as 5 minutes and peaking over 15 to 20 minutes). Moreover, we demonstrated that Ang II increased Smad-dependent gene transcription by transient transfection with a reporter plasmid containing Smad promoter binding sites.
Smad proteins have been identified as TGF-β downstream intracellular signals. Smad are TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors.2,4 There are several Smad proteins. Smad2 and Smad3 are specific mediators of TGF-β/activin pathways, whereas Smad1, Smad5, and Smad8 are involved in bone morphogenetic protein (BMP) signaling.2,4 Smad4 forms hetero-oligomers with the pathway-restricted Smads and is a common mediator of TGF-β and BMP signaling. At physiological concentrations, Smad6 may selectively inhibit BMP receptor signaling, whereas Smad7 inhibits both BMP and TGF-β/activin receptor signaling. In VSMCs, we found that Ang II activates Smad2 and Smad4, showing similarities to TGF-β. We compared the response of Ang II and TGF-β, observing that both factors activate the Smad pathway with a similar kinetic response, suggesting a direct Ang II-induced Smad activation. It is well known that Ang II upregulates TGF-β mRNA expression, protein synthesis, and conversion of TGF-β to its active form.6 To demonstrate that the effect of Ang II was independent of endogenous TGF-β production or activation, we used VSMCs from Wistar rats, in which we confirmed that Ang II was unable to activate TGF-β, as reported,21 although it increased total TGF-β production. In addition, we blocked TGF-β by different methods: a neutralizing antibody against active TGF-β and decorin, a scavenger of its active form. The blockade of endogenous TGF-β did not alter nuclear translocation of phosphorylated-Smad2, Smad DNA-binding activity, and Smad-dependent promoter activation caused by Ang II. These data clearly indicate that TGF-β blockade did not modify Ang II-induced Smad pathway activation, showing a TGF-β–independent Smad signaling elicited by Ang II.
Ang II acts through its binding to specific receptors.5 AT1 is responsible for most of the pathophysiological actions of Ang II. It contributes to chronic diseases, such as hypertension, atherosclerosis, cardiac hypertrophy, and renal injury, by promoting cell growth, inflammation, and fibrosis. AT2 is involved in cell growth inhibition and renal inflammatory cell recruitment.5 The AT1 antagonist losartan diminished Smad overexpression in Ang II-infused rats and in cultured VSMCs and blocked Ang II-induced Smad signaling activation (Smad2 phosphorylation, nuclear translocation, and DNA binding). Our data clearly demonstrate that Ang II activates the Smad pathway via AT1 receptors. Ang II via AT1 regulates the expression of profibrotic growth factors (including CTGF) and ECM.5,7 Our data show that activation of Smad signaling by AT1 receptors could be a novel mechanism involved in Ang II-induced vascular fibrosis. Other data support this information. After myocardial infarction in rats, AT1 blockade attenuated the activation of TGF-β and normalized total Smad2 and Smad4 in the infarct scar. In cultured primary rat fibroblasts, Ang II via AT1 caused rapid phosphorylated-Smad2 nuclear translocation,8 suggesting that Ang II-induced Smad signaling is associated with fibrotic events in the heart. In a model of renal injury caused by ureteric obstruction, activation of Smad2 was found to be associated with interstitial fibrosis and tubule atrophy. AT1 blockade downregulated Smad2 protein and activity and attenuated the chronic tubulointerstitial injury in obstructed kidneys.9 These data indicate that Smad signaling could be a common mechanism of Ang II-mediated fibrosis in cardiovascular and renal diseases.
The activation of the Smad signaling pathway causes nuclear translocation of Smads. In the nucleus, Smads interact with transcription factors at the promoters of some genes and regulate transcription. This process is subject to regulation by other signaling pathways, such as MAPK.2,4 It is well known that many responses elicited by Ang II are mediated by MAPK.17–20 We found that p38 activation in necessary for Smad activation, showing the involvement of MAPK in this process. In VSMCs, we found that Ang II activates a Smad-dependent promoter, suggesting Smad-mediated gene transcription. Previous studies have shown that TGF-β, via Smad activation, upregulates the transcription of several genes important for cell cycle regulation, which mediate the antiproliferative response and partially explain the tumor suppressive action of TGF-β,23 as well as genes for ECM formation, such as procollagen, fibronectin, and PAI-1.12,15,24 Overexpression of some Smad proteins activates transcription of some of these genes, such as PAI-1, even in the absence of TGF-β.2,3 In CTGF promoter there is a functional Smad binding site.13 In mesangial cells, TGF-β activates CTGF via Smad,13 as we observed here in VSMCs. CTGF is an important profibrotic factor10 and is an upstream mediator of Ang II-induced vascular fibrosis.7 In VSMCs, Ang II increased CTGF mRNA expression, promoter activity, and protein production. Smad7 may function as a general negative regulator of TGF-β receptor signaling.22 Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2 and Smad3, diminished Ang II-induced CTGF promoter activation, mRNA expression, and protein production. Several works have demonstrated that overexpression of Smad7 blocks TGF-β–induced ECM production and heme oxygenase-1 expression in renal cells25–28 and in bleomycin-induced lung fibrosis.29 In VSMCs we observed that Smad7 overexpression decreased fibronectin and type 1 procollagen upregulation caused by Ang II. Our results show that the Smad pathway is involved in Ang II-induced CTGF and ECM overexpression and could contribute to the profibrogenic effects of Ang II in vascular diseases.
Other factors involved in tissue damage have been shown to activate Smad signaling. In cells transfected to express TGF-β receptor type II, insulin-like growth factor binding protein-3 (IGFBP-3) stimulated Smad2 and Smad3 phosphorylation, potentiated TGF-β1–stimulated Smad phosphorylation, and cooperated with exogenous TGF-β1 in cell growth inhibition.30 In PC12 cells, nerve growth factor led to activation of the Smad pathway independent of TGF-β.31 Advanced glycation end products caused rapid Smad2/3 activation in renal and vascular cells that was independent of TGF-β,32 suggesting the importance of Smad pathway in diabetic organ injury. Our study has demonstrated that Ang II activates Smad signaling in vascular cells, independently of TGF-β. These data provide further evidence that the Smad proteins are not exclusively activated by the classic TGF-β–triggered mechanism.
In conclusion, our data show that Ang II activates the Smad signaling system in VSMCs, independently of TGF-β. In Ang II-infused rats, aortic Smad overexpression was associated with CTGF induction and preceded ECM overexpression, suggesting that Smad proteins could be involved in Ang II-induced structural changes of the vascular wall. These data may contribute to our knowledge of the mechanisms underlying fibrosis in cardiovascular diseases.
This work was supported by grants from Fondo de Investigación Sanitaria (PI020513, PI020822), Comunidad Autónoma de Madrid (08.4/0018/2001), Red Cardiovascular (RECAVA, MP04), and Sociedad Española de Cardiología and European Project (QLG1-CT-2002-01215). J.R.-V., E.S.-L., M.R., and V.E. are fellows of FIS. We thank Mar Gonzalez Garcia-Parreño for her technical help with confocal microscopy and Alexander G. Borun for his careful reading of the manuscript.
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