Angiotensin II Induces Connective Tissue Growth Factor and Collagen I Expression via Transforming Growth Factor–β–Dependent and –Independent Smad Pathways
The Role of Smad3
Connective tissue growth factor (CTGF) plays a critical role in angiotensin II (Ang II)–mediated hypertensive nephropathy. The present study investigated the mechanisms and specific roles of individual Smads in Ang II–induced CTGF and collagen I expression in tubular epithelial cells with deletion of transforming growth factor (TGF)-β1, overexpression of Smad7, or knockdown of Smad2 or Smad3. We found that Ang II–induced tubular CTGF and collagen I mRNA and protein expressions were regulated positively by phosphorylated Smad2/3 but negatively by Smad7 because overexpression of Smad7-abolished Ang II–induced Smad2/3 phosphorylation and upregulation of CTGF and collagen I in vitro and in a rat model of remnant kidney disease. Additional studies revealed that, in addition to a late (24-hour) TGF-β–dependent Smad2/3 activation, Ang II also induced a rapid activation of Smad2/3 at 15 minutes and expression of CTGF and collagen I in tubular epithelial cells lacking the TGF-β gene, which was blocked by the addition of an Ang II type 1 receptor antagonist (losartan) and inhibitors to extracellular signal–regulated kinase 1/2 (PD98059) and p38 (SB203580) but not by inhibitors to Ang II type 2 receptor (PD123319) or c-Jun N-terminal kinase (SP600125), demonstrating a TGF-β–independent, Ang II type 1 receptor–mediated extracellular signal–regulated kinase/p38 mitogen-activated protein kinase cross-talk pathway in Ang II–mediated CTGF and collagen I expression. Importantly, the ability of knockdown of Smad3, but not Smad2, to inhibit Ang II–induced CTGF and collagen I expression further revealed an essential role for Smad3 in Ang II–mediated renal fibrosis. In conclusion, Ang II induces tubular CTGF expression and renal fibrosis via the TGF-β–dependent and –independent Smad3 signaling pathways, suggesting that targeting Smad3 may have therapeutic potential for hypertensive nephropathy.
Increasing evidence shows that angiotensin II (Ang II) is a key mediator of chronic renal diseases, in particular, of hypertensive and diabetic nephropathy.1 This is demonstrated by the findings that blockade of Ang II with Ang II-converting enzyme inhibitor and/or Ang II type 1 (AT1) receptor antagonist slows the progression of renal diseases in both experimental and human kidney diseases.2 It is now understood that Ang II mediates renal fibrosis by stimulating endogenous synthesis of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF).3,4⇓ However, the signaling mechanisms by which Ang II induces these fibrogenetic factors in the kidney remain largely unclear.
CTGF, a member of the CCN family, is a downstream mediator of TGF-β1 and plays a critical role in renal fibrosis. This is supported by the findings that blockade of CTGF by an antisense strategy is able to attenuate renal fibrosis in obstructive nephropathy,5 remnant kidney disease induced in TGF-β1 transgenic mice,6 and tubular epithelial-myofibroblast transition in response to TGF-β and advanced glycation end products.7,8⇓ Direct evidence for a role of CTGF in kidney disease comes from a recent study in the type 1 diabetic mouse model that cell-specific overexpression of CTGF in podocytes of CTGF transgenic mice is able to intensify proteinuria and mesangial expansion.9 CTGF is also upregulated in the kidney and cardiovascular tissues after Ang II stimulation, which is blocked by the AT1 antagonist in vivo and in vitro,10,11⇓ demonstrating a causal role for Ang II in CTGF expression.
It is generally accepted that Ang II–induced CTGF expression is mediated via the TGF-β1–dependent pathway.3,4⇓ The Smad proteins, including Smad2, Smad3, and Smad7, are essential components of downstream TGF-β signaling, which either positively (via activation of Smad2/3) or negatively (through the negative feedback mechanism of Smad7) regulates biological activities of TGF-β1.12 We and other investigators have shown recently that Ang II activates the Smad signaling pathway in vascular smooth muscle cells via both TGF-β–dependent and –independent mechanisms.13,14⇓ These observations suggest that additional signaling mechanisms independent of TGF-β may be required for Ang II–induced CTGF expression.
It is now clear that CTGF is a target gene of TGF-β/Smad signaling15,16⇓ and that Ang II is able to activate TGF-β/Smad signaling to mediate vascular fibrosis independent of TGF-β through the AT1-extracellular signal–regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) signaling mechanism13,14⇓ and also to mediate renal fibrosis in a TGF-β–independent process.17 We, thus, hypothesized that, in addition to the TGF-β–dependent mechanism, Ang II may also stimulate CTGF expression to induce renal fibrosis in tubular epithelial cells (TECs) via the TGF-β–independent ERK/p38 MAPK-Smad signaling pathway.
Materials and Methods
Cell Culture Models
A normal rat kidney tubular epithelial cell line (NRK52E) was stimulated with Ang II to detect the activation of the Smad2/3 and MAPK pathways, as well as expression of CTGF, TGF-β1, and collagen I. The effects of endogenous TGF-β1 on Ang II–induced Smad2/3 phosphorylation and CTGF and collagen I expressions were examined in NRK52E cells pretreated with or without an anti–TGF-β1 neutralizing antibody or in TECs obtained from the kidneys of TGF-β1 wild-type (WT) and knockout (KO) mice (generously provided by Prof. J. B. Kopp, National Institutes of Health, Bethesda, MD). To evaluate the importance of the Smad signaling pathway in Ang II–induced CTGF and collagen I expressions, a doxycycline-regulated Flagged M2-Smad7–expressing NRK52E cell line was used.18 To delineate the differential roles of Smad2 and Smad3 in regulating Ang II–induced CTGF and collagen I expressions, cell lines with stable knockdown of Smad2 or Smad3 were established by expressing either Smad2 or Smad3 small-interfering RNA (siRNA). The details of cell culture conditions and the siRNA constructs were presented in the online Supplemental Data (please see http://hyper.ahajournals.org).
Animal Model of Remnant Kidney Disease Treated With Smad7 Gene Transfer
Male Sprague-Dawley rats at 6 weeks of age, weighing 220 to 250 g, were obtained from Harlan (Indianapolis, IN). A progressive renal disease model with hypertension was induced by 5/6 subtotal nephrectomy, and groups of 6 animals received gene therapy with doxycycline-inducible Smad7 or empty vectors mediated by an ultrasound-microbubble technique, as described previously.19 All of the procedures were performed in accordance with institutional guidelines for animal care.
Real-Time RT-PCR and Western Blot Analyses
Real-time PCR and Western blotting were performed following the protocol as described previously.14 The materials and methods were detailed in the online Data Supplement.
Data are expressed as mean±SEM. Statistical analysis was performed by 1-way ANOVA followed by a Tukey comparison program (a t test between groups) from GraphPad Prism 3.0 (GraphPad Software). P<0.05 indicates a statistically significant difference.
Ang II Induces Tubular CTGF Expression via the TGF-β–Dependent and –Independent Smad Signaling Pathways
Consistent with the previous findings,10 Ang II induced CTGF and collagen I mRNA and protein expression by TECs (NRK52E) in a time- and dose-dependent manner (please see the online Data Supplement). We next investigated whether Ang II stimulates tubular CTGF expression via a TGF-β1–dependent or –independent mechanism in TGF-β1 WT and KO mouse TECs. As shown in Figure 1, real-time PCR and Western blot analyses showed that Ang II induced CTGF mRNA expression at 6 hours (Figure 1A) and protein expression at 24 hours (Figure 1B) in TGF-β1 WT TECs. Surprisingly, Ang II also caused equal levels of CTGF mRNA and protein expression in TGF-β1 KO TECs (Figure 1A and 1B). This was associated with a rapid phosphorylation of Smad2/3 at 15 to 30 minutes in both TGF-β1 WT and KO TECs (Figure 1C). Interestingly, Ang II induced a second peak of Smad2/3 phosphorylation at 24 hours in TGF-β1 WT but not in KO TECs (Figure 1C), suggesting that Ang II caused a rapid Smad2/3 activation (15 to 30 minutes) via the TGF-β–independent pathway, whereas Ang II activated Smad2/3 at 24 hours through the TGF-β–dependent mechanism. This was confirmed by the finding that Ang II was able to induce endogenous TGF-β1 expression at 24 hours as measured by ELISA (Figure 2A), and the use of a neutralizing TGF-β antibody was able to block Ang II–induced Smad2/3 phosphorylation at 24 hours but not at 15 minutes (Figure 2B).
Ang II Induces CTGF Expression via the AT1-ERK/p38-Smad Cross-Talk Pathway
We next explored the signaling mechanism by which Ang II induces the early, TGF-independent Smad signaling pathway by testing the hypothesis that Ang II may act by stimulating the MAPK to cause Smad2/3 phosphorylation and CTGF expression. As shown in Figure 3A, Ang II was able to activate 3 members of MAPKs, including ERK1/2, p38, and c-Jun N-terminal kinase (JNK), being significant at 5 minutes and peaking at 15 minutes. This preceded or paralleled the early phosphorylation of Smad2/3 at 15 minutes, as shown in Figure 1C. To investigate the relationship between the phosphorylation of ERK/p38/JNK MAPKs and activation of Smad2/3, we examined whether Ang II binds AT1 or Ang II type 2 (AT2) receptors to activate the early, TGF-β1–independent Smad signaling directly via the MAPK-Smad cross-talk pathway. As shown in Figure 3B, Western blot analysis showed that Ang II signaled through AT1, not AT2, to induce a rapid phosphorylation of Smad 2/3 at 15 minutes because this was blocked by the AT1 receptor antagonist (losartan; 1 μmol/L), not by the AT2 inhibitor PD123319 (1 μmol/L). We also found that blockade of ERK1/2 and p38 but not JNK was able to inhibit Ang II–induced Smad2/3 phosphorylation.
The functional role of the AT1-ERK/p38-Smad cross-talk pathway in Ang II–induced CTGF and collagen I expression in TECs is shown in Figures 4 and 5⇓. Real-time PCR and Western blot analyses revealed that Ang II–induced tubular CTGF and collagen I mRNA (at 6 hours) and protein (24 hours) expressions were blocked by an AT1 receptor antagonist, pharmacological inhibitors to MAPK/ERK kinase (MEK) 1, the upstream ERK1/2 kinase, and p38 but not by the AT2 receptor blocker and JNK inhibitor, demonstrating a critical role for the AT1-ERK/p38 MAPK-Smad cross-talk pathway in Ang II–induced tubular CTGF and collagen matrix expression.
Smad7 Negatively Regulates Ang II–Induced Smad2/3 Activation and CTGF and Collagen Matrix Expressions In Vitro and In Vivo
We next tested a critical role of the Smad signaling pathway and the specific role for Smad7 in Ang II–induced tubular CTGF and collagen I expression in vitro in a stable, doxycycline-regulated Flagged M2-Smad7–expressing NRK52E TEC line. As shown in Figure 6A, an addition of doxycycline (2 μg/mL) induced Flagged M2-Smad7 transgene expression as identified with the anti–Flag-M2 antibody. Overexpression of Smad7 completely inhibited both the early (5- to 30-minute) and late (24-hour) phosphorylation of Smad2/3 and the abrogation of CTGF and collagen I mRNA and protein expressions induced by Ang II (Figure 6B and 6C).
The critical role of Smad signaling and the negative regulating role of Smad7 in CTGF and collagen matrix expressions were further examined in a rat model of remnant kidney disease in which Smad7 was overexpressed by ultrasound-microbubble–mediated gene transfer of the inducible Smad7, as described previously.19 We have shown previously that overexpression of Smad7 is capable of inhibiting the activation of Smad2/3 and collagen matrix expression in the disease model.19 In the present study, we also found that inhibition of renal fibrosis in this disease model was associated with blockade of CTGF expression in both mRNA and protein levels (Figure 6D).
Smad3, Not Smad2, Is the Key Mediator of TGF-β/Smad Signaling for Ang II–Induced Tubular CTGF and Collagen I Expression
To further dissect the differential roles of Smad2 and Smad3 in Ang II–induced CTGF and collagen I expressions, we established Smad2 or Smad3 knockdown cell lines in NRK52E cells by stably expressing Smad2 or Smad3 siRNA. As shown in Figure 7A and 7B, real-time PCR and Western blot showed that TECs stably expressing Smad2 siRNA or Smad3 siRNA resulted in a selective inhibition of Smad2 or Smad3 expression at mRNA and protein levels. Interestingly, Ang II-induced CTGF and collagen I mRNA upregulations were prevented in Smad3, not Smad2, knockdown TECs (Figure 7C). Similarly, Western blot analysis also showed that knockdown of Smad3, not Smad2, abolished Ang II–induced tubular CTGF and collagen I protein expressions at 24 hours (Figure 7D).
It is now well accepted that Ang II is a key mediator in hypertensive and diabetic nephropathy.1–4⇓⇓⇓ Generally, CTGF is considered a downstream mediator of TGF-β1.3,4⇓ It is well accepted that TGF-β1 signals through a heteromeric receptor complex of the type I and type II receptors to activate the downstream intracellular mediators Smad2 and Smad3 to exert its biological effects.12 Because CTGF is one of the responsive genes of TGF-β/Smad signaling,15,16⇓ activation of TGF-β/Smad signaling results in upregulation of CTGF. Because Ang II is able to induce TGF-β1 expression,3,4⇓ therefore, it is generally believed that Ang II induces CTGF expression via TGF-β–dependent Smad signaling.3,4⇓ A significant finding in the present study was that Ang II could directly induce CTGF, as well as collagen I, expression through a TGF-β–independent Smad signaling pathway via the AT1-ERK/p38 MAPK cross-talk pathway. This was supported by the findings that Ang II was able to activate the early (15-minute), but not the late (24-hour), Smad2/3 phosphorylation in TECs lacking the TGF-β1 gene and that blockade of the AT1 receptor, ERK1/2, and p38, but not the AT2 receptor and JNK MAPK was capable of inhibiting Ang II–induced activation of Smad2/3 and CTGF, as well as collagen I expression. This notion is further supported by evidence of direct interactions between ERK/p38 MAPKs and Smads.20–22⇓⇓ Thus, additional phosphorylation of Smads by ERK/p38 signals is necessary for full activity of Smad signaling in response to Ang II.
The ability of overexpression of Smad7 to inhibit Smad2/3 phosphorylation and tubular CTGF and collagen I expressions in response to Ang II in vitro and in a rat remnant kidney disease further demonstrated a critical role of Smad signaling in Ang II–mediated renal fibrosis. This was consistent with the previous report in vascular smooth muscle cells and TECs in which expression of Smad7 inhibits Ang II or TGF-β–induced CTGF expression in vitro and in vivo.13,18,19⇓⇓ Thus, it appears that a Smad-dependent Ang II signaling pathway is required for CTGF and collagen matrix expressions, whereas Smad7 may play a negative regulating role in Ang II–induced tubular CTGF expression and renal fibrosis.
Another important finding in this study was that Smad3, but not Smad2, was essential for Ang II-induced CTGF and collagen I expressions in tubular epithelial cells. Although Smad2 and Smad3 share >90% homology in their amino acid sequences and both are downstream mediators for TGF-β1 signaling, Smad2 and Smad3 have differential roles because of 2 major differences in their structures. It is shown that the additional 30 amino acids in the MH1 domain of Smad2 prevent Smad2 from direct binding to DNA, whereas the transactivation domain in the linker region of Smad3 allows Smad3 to directly bind to DNA and regulate the target genes.23,24⇓ Because the TGF-β regulatory element and Smad binding element are found in the CTGF promoters,15,16,25⇓⇓ many fibrogenic genes, including most collagen genes, have been shown to be Smad3 dependent.26,27⇓ Thus, Smad3 may directly mediate the transcription of CTGF and collagen genes. A critical role for Smad3 in TGF-β1–induced CTGF expression has been reported by the findings that TGF-β1–induced CTGF mRNA expression is significantly reduced in the fibroblast derived from Smad3 KO mice.16,28⇓ The present study added new evidence that Smad3, not Smad2, played an essential role in Ang II–induced tubular CTGF and collagen I expressions.
Results obtained from this study demonstrate that, after binding to the AT1 receptor, Ang II mediates CTGF and collagen matrix expression via a rapid activation of Smad2/3 through the ERK1/2 and p38 MAPK cross-talk pathway within 30 minutes, in addition to the TGF-β–dependent Smad signaling pathway at 24 hours. Overexpression of Smad7 is capable of blocking Ang II–induced activation of Smad2/3, thereby inhibiting CTGF and collagen matrix expressions. The ability of blocking Smad3, but not Smad2, to inhibit Ang II–mediated CTGF and fibrosis responses indicates that targeting Smad3 by overexpressing Smad7 or by specific siRNA to Smad3 may be a novel therapeutic strategy for hypertensive complications.
Sources of Funding
This work has been supported by grants from the Research Grant Council of Hong Kong (RGC GRF 768207 and 767508) and Baxter Renal Discovery Programs.
- Received May 21, 2009.
- Revision received June 6, 2009.
- Accepted July 10, 2009.
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- ↵Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ, Lan HY. Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol. 2002; 13: 1464–1472.
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