Published online before print February 9, 2006, doi: 10.1161/01.ATV.0000209517.00220.cd
© 2006 American Heart Association, Inc.
Vascular Biology |
PKC
Is Necessary for Smad3 Expression and Transforming Growth Factor ß–Induced Fibronectin Synthesis in Vascular Smooth Muscle Cells
From the Division of Vascular Surgery (E.J.R., R.P.H., K.S., P.L.F., B.L., K.C.K.), New York Presbyterian Hospital, Cornell University, Weill Medical School and Columbia University, College of Physicians and Surgeons, New York, NY; the Department of Molecular and Cellular Biology (K.I.N.), Medical Institute of Bioregulation, Kyushu University, Japan; and the Department of Developmental Biology (K.N.), Graduate School of Medicine, Tohoku University School of Medicine, Japan.
Correspondence to K. Craig Kent, MD, Department of Surgery, New York Presbyterian Hospital, Weill Medical College, Cornell University, 525 E 68th St, Room 707, New York, NY 10021. E-mail kckent{at}med.cornell.edu
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Abstract |
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Objective— The purpose of these studies is to investigate the mechanism by which transforming growth factor (TGF)ß1 regulates the synthesis of the extracellular matrix protein fibronectin (FN).
Methods and Results— TGFß1 elicited a time-dependent induction of FN protein and mRNA in A10 rat aortic smooth muscle cells (SMCs). Ectopic expression of Smad3 in A10 cells stimulated both basal and TGFß1-induced FN expression, whereas expression of Smad7 eliminated the TGFß response. Because TGFß activated PKC
in SMCs, we tested the role of PKC in regulation of FN expression. Inhibition of PKC activity by rottlerin or dominant-negative adenovirus (AdPKC DN) blocked TGFß1’s induction of FN, whereas overexpression of PKC enhanced TGFß’s effect. Moreover, aortic SMCs isolated from PKC–/– mice exhibited diminished FN induction in response to TGFß. Furthermore, we found that Smad3 protein and mRNA were markedly reduced in AdPKC DN-treated A10 cells and in PKC null cells. Finally, restoring Smad3 in rottlerin-treated A10 and PKC null cells rescues the ability of TGFß to upregulate FN protein and mRNA expression.
Conclusion— Our data suggest that TGFß-activated PKC is critical to maintain normal expression of Smad3, which in turn is required for the induction of fibronectin. PKC represents a promising target for treating the fibroproliferative response after arterial injury.
The mechanism by which TGFß regulates the synthesis of fibronectin remains inconclusive. Our data suggest that TGFß-activated PKC is critical to maintain Smad3 levels, which in turn are required for fibronectin expression. Thus, PKC may serve as a novel therapeutic target to prevent the fibroproliferative response after arterial injury.
Key Words: extracellular matrix • fibronectin • intimal hyperplasia • protein kinase C delta • TGF beta
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Introduction |
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Extracellular matrix (ECM) proteins play an important role in intimal hyperplasia by contributing up to 80% of the mass in a restenotic plaque.1–3 Additionally, alteration of matrix composition during the formation of a restenotic lesion regulates vascular smooth muscle cell (VSMC) responses including survival, migration, proliferation, and additional production of ECM, all which significantly contribute to restenosis.4
TGF beta (TGFß), a well recognized stimulus of matrix synthesis,5 has been implicated in the fibroproliferative response to vascular injury. Furthermore, higher levels of TGFß are observed in restenotic arteries after percutaneous angioplasty,6 whereas inhibition of TGFß prevents accumulation of ECM and intimal hyperplasia.7
TGFß transmits its signal via transmembrane receptors. The Smads are a series of proteins downstream from the TGFß receptor that transduces its signal to the nucleus. On ligand binding, Smad3 is phosphorylated and translocates to the nucleus where it regulates expression of TGFß target genes.8 Inhibitory Smads, such as Smad7, prevent Smad3 phosphorylation, its subsequent nuclear translocation, and therefore limit TGFß signaling.9
In addition to Smad3, accumulating data suggest that protein kinase C (PKC) pathways might converge to modulate TGFß target genes. In particular, the novel PKC isoform, protein kinase C delta (PKC), is specifically activated by TGFß in mesangial cells and modulates their expression of collagen via interaction with the Smad pathway.10,11 Moreover, PKC has been shown to be essential in TGFß induced elastin synthesis.12
As a profibrotic factor, TGFß appears to be responsible for the accumulation of most ECM proteins by inducing their synthesis. However, the mechanism by which TGFß regulates gene expression of the matrix protein fibronectin remains in question. Conflicting data have been reported regarding whether a Smad-dependent signaling pathway is required for TGFß-stimulated fibronectin production.
Fibronectin is a multifunctional extracellular matrix glycoprotein present in all layers of the arterial wall.13 It is synthesized by endothelial cells,14 SMCs,15 and fibroblasts16 as a disulphide-linked dimer that binds collagen, fibrin, and proteoglycans via specific domains as well as vascular cells through specific integrins.17 Fibronectin production by SMCs has been demonstrated in restenotic lesions after balloon angioplasty,18 whereas electron microscopy has demonstrated newly deposited fibronectin associated with synthetic SMCs during early atherosclerotic and restenotic lesions19,20 Although the precise role on fibronectin in restenosis remains to be elucidated, it potentially is critical to numerous cellular phenomena, eg, adhesion, migration, and differentiation.21
In the current study, we investigated the mechanisms by which TGFß1 increases VSMC fibronectin production. We found that TGFß1-induced fibronectin production in SMCs requires Smad3. Furthermore, we demonstrated that TGFß1 activates PKC in SMCs and that this activation plays a role in TGFß1-stimulated fibronectin production by regulating Smad3.
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Materials and Methods |
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General Materials
TGFß1 was obtained from R & D Systems. Dulbecco’s Modified Eagles Medium (DMEM) and cell culture reagents were from Gibco BRL Life Technologies. Chemicals, if not specified, were purchased from Sigma Chemical Co. Fibronectin antibody was obtained from Calbiochem, whereas Smad3, Smad7, and PKC
antibodies were from Santa Cruz Biotechnology.
Cell Culture
Rat aortic A10 SMCs, obtained from American Tissue Culture Collection, were grown as recommended, in DMEM modified to contain 4 mmol/L L-glutamine, 4.5 g/L glucose, 1 mmol/L sodium pyruvate, and 1.5 g/L sodium bicarbonate supplemented with 10% fetal bovine serum (FBS) and antibiotics. The generation of PKC target deletion was described elsewhere.22 Mouse aortic SMCs were isolated from the thoracic aorta of both PKC–/– and PKC+/+ male mice22 based on a protocol described by Clowes et al23 and maintained in DMEM containing 10% FBS at 37°C with 5% CO2.
Construction of Adenoviral Vectors and Infection
A recombinant adenoviral vector was constructed to express PKC, Smad3, or Smad7. Briefly, a DNA fragment containing the desired sequence was generated by polymerase chain reaction (PCR) using the human cDNA as a template. After DNA sequencing, the PCR product was then cloned into an E1- and E3-deficient adenoviral vector (pEasy). Adenoviruses were propagated in HEK 293 cells and purified by CsCl density gradient centrifugation. A recombinant adenoviral vector containing the dominant-negative PKC24 was obtained from Dr T. Biden (Garvan Institute). SMCs were infected with adenoviruses as described previously.25
Immunoblotting
Protein extracts were resolved by electrophoresis as described previously.25 Equal amount of protein extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane and blotted with antibodies. Labeled proteins are visualized with an ECL system (Amersham Biosciences).
Fibronectin Assay by Immunoblotting
SMCs were grown to confluence in media containing 10% serum. Cells were then starved for 24 hours in 0.5% serum media and then stimulated with TGFß. For measurement of fibronectin in the cell media, 500 mL samples were diluted 1:5 in DMEM. Equal amounts were diluted 1:2 in Laemmli sample buffer, boiled at 100°C for 5 minutes, and then subjected to immunoblotting protocol as described previously.5,26
PKC Translocation Assay
As described previously,27 cells were scraped into a detergent-free buffer and then disrupted with a Dounce homogenizer. After centrifugation at 100 000g, the supernatant, representing the cytosolic fraction, was saved; the pellet, representing the particulate fraction, was resuspended in buffer. After incubation on ice, the pellet was centrifuged at 18 000g for 10 minutes to remove insoluble material. The supernatant was saved as the soluble membrane fraction. After determination of the protein content, each fraction was analyzed by immunoblotting as described.
Quantitative Reverse-Transcribed PCR
Total RNA was isolated using RNA Aqueous (Ambion) and reversed-transcribed using a reverse-transcriptase kit and probes from Applied Biosystems as previously described.25 Quantification of mRNA was performed using the ABI Prism7700 (Applied Biosystems).
Statistical Analysis
Values were expressed as a fold increase (mean±standard error). Unpaired Student t test was used to evaluate the statistical differences between control and treated groups. Values of P<0.05 were considered significant. All experiments were repeated at least three times.
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Results |
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Smad3 Increases Fibronectin Gene Expression
Smad3 is a well-described mediator of TGFß1-dependent gene targets, however, whether Smad3 is involved in regulation of fibronectin expression is an area of controversy. We began our studies by confirming TGFß’s induction of fibronectin in A10 SMCs, a rat aortic cell line. A10 cells were treated with TGFß1 (10 ng/mL) for up to 48 hours. Equal amounts of cell medium were then harvested and examined by immunoblotting with fibronectin antibody. As we previously reported in human arterial SMCs, TGFß1 significantly stimulated fibronectin production in A10 SMCs in a time-dependent manner (please see the supplemental materials, available online at http://atvb.ahajournals.org). We next examined the effect of TGFß1 on fibronectin gene expression by quantifying fibronectin type 1 mRNA (FN mRNA) levels via reverse-transcribed PCR. TGFß1 (10 ng/mL) elicited a rapid increase in the steady-state level of FN mRNA, which peaked after 3 hours (please see the supplemental materials). After establishing the TGFß response in A10 cells, we ectopically expressed Smad3 using a replication deficient adenovirus (AdSmad3). A10 cells were infected with AdSmad3 or empty viral vector (AdNull) as described in Materials and Methods. After treatment with TGFß1 (10 ng/mL for 48 hours [protein] or 12 hours [mRNA]), we analyzed fibronectin expression using both Western blotting and reverse-transcribed PCR analyses. AdSmad3 significantly increased basal fibronectin production evident by elevated levels of fibronectin protein and mRNA (Figure 1A and 1B). Additionally, overexpression of Smad3 further enhanced the ability of TGFß to stimulate fibronectin expression (Figure 1A and 1B). To further test the hypothesis that TGFß regulates fibronectin in vascular SMCs via a Smad-dependent pathway, we constructed an adenoviral vector to express the inhibitory Smad, Smad7, which is known to block the TGFß-dependent activation of Smad3 as well as Smad2. A10 cells were infected with AdSmad7 or AdNull and then treated with TGFß1(10 ng/mL for 48 hours [protein] or 12 hours [mRNA]). Expression of Smad7 completely blocked TGFß1’s induction of fibronectin protein and mRNA (Figure 1C and 1D). These data support that the Smad signaling pathway is required for TGFß1-induced fibronectin production
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Activation of PKC by TGFß1
Because the novel PKC isoform PKC has been implicated in the regulation of TGFß-dependent elastin and collagen expression, we next tested whether PKC plays a role in fibronectin by examining PKC activation in SMCs after TGFß treatment. After increasing periods of TGFß1 treatment (10 ng/mL), we evaluated PKC activation by examining PKC membrane translocation, which corresponds to increased PKC activity.10,28 TGFß1 induced an increase in membrane-associated PKC that was detectable 30 minutes after administration (please see htt://atvb.ahajournals.org). Accordingly, this increase in membrane associated PKC was accompanied by a decline in the cytosolic fraction. These data suggest that PKC is activated in SMCs in response to TGFß1.
PKC Activity Is Required for TGFß-Stimulated Fibronectin Gene Expression
To determine whether PKC plays a role in TGFß1-stimulated fibronectin expression, A10 SMCs were pretreated for 1 hour with increasing doses of rottlerin, a selective PKC inhibitor. After TGFß1 treatment (10 ng/mL for 48 hours), fibronectin protein levels were evaluated. Rottlerin, in concentrations as low as 0.5 µmol/L, significantly diminished TGFß’s induction of fibronectin, with maximal effect at 2 µmol/L (Figure 2A). Similarly, 1 µmol/L rottlerin completely eliminated TGFß’s (10 ng/mL for 12 hours) induction of FN mRNA (Figure 2B). To confirm our results obtained with the chemical inhibitor, we also inhibited PKC through the use of a dominant negative adenovirus (AdPKCDN).24 A10 cells were infected with AdPKC DN or AdNull followed by treatment with TGFß1. Inhibition of PKC, via the dominant-negative adenovirus, blocked TGFß1-induced fibronectin protein and mRNA (Figure 2C and 2D). To exclude any possibility that downregulation of Smad3 expression might be related to the use of an adenovirus, we used PKC "knockout" SMCs as an alternative approach to inhibit PKC activity. Vascular SMCs isolated from PKC null mice exhibited a reduced TGFß induced FN expression compared with SMCs isolated from their wild-type littermates (Figure 2E and 2F). Lastly, we ectopically expressed PKC using adenoviral transfection (AdPKC) in A10 cells. After treatment with TGFß1, overexpression of PKC increased fibronectin production evident by elevated levels of fibronectin protein and mRNA (Figure 2G and 2H). Taken together, these data support a role for PKC in TGFß1-stimulated fibronectin production.
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Regulation of Smad3 by PKC
Next, we sought to understand how PKC participates in TGFß signaling, in particular, how this kinase interacts with the Smad pathway. We began by evaluating Smad3 protein levels in A10 cells in which PKC was inhibited with the dominant-negative mutant (AdPKC DN). Compared with cells that were infected with AdNull, AdPKC dominant-negative–infected SMCs exhibited markedly reduced levels of Smad3 protein (Figure 3A). In correlation with the diminished Smad3 protein expression, the abundance of Smad3 mRNA was dramatically decreased in AdPKC DN-infected cells (Figure 3B). Furthermore, PKC null SMCs expressed markedly reduced levels of Smad3 protein and mRNA compared with their wild-type counterparts (Figure 3C and 3D). Based on these data, we postulated that inhibition of PKC impedes the ability of TGFß to upregulate fibronectin expression by diminishing Smad3 expression. Therefore, ectopic expression of Smad3 should rescue TGFß function by restoring the level of cellular Smad3. To this end, we infected A10 cells with AdSmad3 before rottlerin treatment. As shown, inhibition of PKC resulted in diminished TGFß-induced fibronectin production and levels of Smad3. More importantly, AdSmad3 rescues the ability of TGFß1 to upregulate fibronectin protein expression despite the presence of the PKC inhibitor rottlerin (Figure 4A and 4B). Similarly, ectopic expression of Smad3 restored the ability of TGFß to induce fibronectin gene expression in PKC null SMCs (Figure 4C and 4D). Together, these data suggest that PKC modulates fibronectin expression, at least in part, through regulating Smad3 expression.
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Discussion |
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Since the initial description in 1986,29 numerous authors have described TGFß’s ability to upregulate fibronectin synthesis in a host of different cell types. We began our studies by reconfirming that TGFß upregulates the level of both fibronectin protein and mRNA in A10 aortic SMCs. Our findings were in accordance with those of others who have shown that the induction of fibronectin is a general response of vascular SMCs to TGFß.
Although Smad proteins are well established as signal mediators downstream of TGFß receptors, whether TGFß stimulates fibronectin expression through a Smad-dependent pathway is controversial. Using various mutants of TGFß type I receptor, Itoh et al showed that TGFß-induced fibronectin expression requires activation of Smad proteins.30 Moreover, the authors demonstrated that the TGFß-mediated fibronectin induction is absent in a Smad4-deficient cell line, MDA-MB-468, and this TGFß dysfunction can be rescued by ectopic Smad4 expression. Here, in VSMCs, we showed that overexpression of Smad3 resulted in an increase in fibronectin protein and mRNA, whereas ectopic expression of the inhibitory Smad, Smad7, blocked the induction of fibronectin by TGFß. In further support of our findings, Isno et al demonstrated that cotransfection of Smad3 stimulated the activity of a fibronectin promoter reporter, whereas Smad7 or a dominant-negative Smad3 mutant inhibited TGFß-induced fibronectin promoter activity.31 Although these studies support a positive role of a Smad-dependent pathway in the regulation of fibronectin, opposite findings have also been previously reported. Also, using Smad4-deficient cell lines, including MDA-MB-468, Hocevar et al showed that Smad4 is dispensable in the regulation of fibronectin.32 Consistently, mouse fibroblasts lacking Smad2, and 3 also retained the ability to respond to TGFß with enhanced fibronectin expression.33,34 It is unclear what is underlying this discrepancy. Given the complexity of TGFß signaling, it is possible that TGFß’s regulation of fibronectin involves different signaling intermediates depending on the cell type and cellular status.
The pro-fibrotic role of Smad3 has been established in the regulation of other ECM proteins including type I collagen and PAI1. It has been demonstrated that Smad3 directly bind to a CAGAC element of the promoter of 
2(I)collagen or PAI1.35 Interestingly, the rat fibronectin gene also contains a CAGAC Smad binding element in the proximal region of its promoter.31 Further studies are still needed to clarify which transcription factors collaborate with Smads to activate the fibronectin gene in response to stimulation by TGFß.
Using pharmacological, molecular, and genetic approaches, we also demonstrated that, in addition to Smad3, TGFß requires PKC for regulation of fibronectin. Stimulation of A10 SMCs with TGFß led to a rapid activation of PKC. Additionally, inhibition of PKC activity by a selective chemical inhibitor, dominant-negative mutant, or gene deletion completely ablated the ability of TGFß to upregulate fibronectin, whereas overexpression of PKC enhanced TGFß’s effect. To our knowledge, this is the first demonstration of the direct involvement of PKC in regulation of smooth muscle cell fibronectin expression.
The involvement of PKC in TGFß-dependent regulation of extracellular matrix26,36,37 has been previously demonstrated in nonvascular cells. Using pharmacological inhibitors, Kucich et al showed that PKC, along with phosphatidylcholine-specific phospholipase C, and the MAP kinase p38, is required for TGFß induced fibronectin expression in human lung fibroblasts.38 Through the use of the chemical inhibitor rottlerin and a dominant-negative mutant, Jinnin et al demonstrated that PKC is involved in the regulation of the 2(I) collagen gene in the presence of TGFß in human dermal fibroblasts.39 Lastly, PKC and p38 MAPK have been shown to be required for TGFß-mediated elastin mRNA stabilization12 in cultured human fetal lung fibroblasts. These observations, together with our data, suggest that PKC is an important signaling component required for the profibrotic effects of TGFß.
Our data indicate that PKC mediates fibronectin expression, at least in part, through regulation of Smad3 expression. Emerging evidence supports the notion that PKC is a prominent mediator of Smad expression or activity. In agreement with our findings, PKC depletion caused by long-term phorbol ester treatment results in diminished Smad3 expression in mesangial cells.10 Additionally, PMA, which activates many PKC isotypes including PKC, is shown to induce Smad3 phosphorylation at Ser37 and Ser70,40 which decreased the ability of Smad3 to bind and activate its target genes. It remains to be determined whether PKC, in vascular SMCs, has additional effects on Smad3 expression, such as Smad3 protein phosphorylation as well as protein or mRNA stability.
In addition to PKC, other classical signaling cascades, such as the MAP kinases, are also found to play a role in regulation of fibronectin production. In particular, the MAP kinase JNK has been shown to mediate TGFß-induced fibronectin expression.41 Using the JNK inhibitor, SP600125, we also found that inhibition of JNK blocked TGFß’s induction of fibronectin (Liu and Kent, unpublished observation). Although our current data clearly showed an interaction between PKC and Smad3 expression, we do not exclude the possibility of other signaling proteins, such as MAP kinase, being involved in the regulation of fibronectin in vascular SMCs. In fact, our previous studies42 showed that PKC is a potent regulator of MAPK activity. It is possible that TGFß stimulated PKC activates members of MAP kinase family, which in turn stimulate the expression of Smad3.
In summary, we have demonstrated that in vascular SMCs, TGFß requires both PKC and Smad3 to induce fibronectin gene expression. Activated by the TGFß signal, PKC may function to maintain a sufficient cellular level of Smad3, which could directly interact with the fibronectin promoter. Although these findings demonstrate a complex mechanism underlying the regulation of matrix biosynthesis by TGFß, they also identify PKC, as well as Smad3, as important signaling components that contribute to the pathogenesis of extracellular matrix protein accumulation in neointimal hyperplasia.
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Acknowledgments |
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This work was supported by a National Heart, Lung, and Blood Institute grant HL-68673 (K.C.K., B.L.), American Heart Association Heritage Foundation grant-in-aid 0455859T (B.L.), and a National Institute of Health Training Grant T32 CA68971-07 (E.J.R.).
Received October 11, 2005; accepted January 24, 2006.
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