Published online before print July 28, 2008, doi: 10.1161/CIRCULATIONAHA.107.761932
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2008;118:764-772.)
© 2008 American Heart Association, Inc.
Vascular Medicine |
Suppression of c-Cbl Tyrosine Phosphorylation Inhibits Neointimal Formation in Balloon-Injured Rat Arteries
From the Institute of Cardiovascular Science and Key Laboratory for Molecular Cardiovascular Sciences, Ministry of Education, Peking University, Beijing, China (Z.T., Y.W., Y.F., Y.Z., N.W.), and Departments of Bioengineering and Medicine, University of California San Diego, La Jolla (S.C.).
Correspondence to Nanping Wang, MD, PhD, Institute of Cardiovascular Science, Peking University Health Science Center, Beijing 100083, China (e-mail npwang{at}bjmu.edu.cn); or Shu Chien, MD, PhD, Department of Bioengineering, University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093–0412 (e-mail shuchien@ucsd.edu).
Received December 28, 2007; accepted June 10, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
Background— c-Cbl, a ubiquitously expressed protooncogene, is tyrosine phosphorylated in response to a variety of stimuli, including growth factors such as platelet-derived growth factor (PDGF), and consequently activates signaling proteins such as phosphatidylinositol-3 kinase (PI3K) and Akt. In the present study, we examined the role of c-Cbl tyrosine phosphorylation in vascular injury.
Methods and Results— Western blotting showed that the tyrosine phosphorylation of c-Cbl was increased in balloon-injured rat carotid arteries and in cultured smooth muscle cells on stimulation by PDGF-BB, followed by the activations of Akt and the mammalian target of rapamycin. Adenovirus-mediated overexpression of a c-Cbl mutant that ablates the major tyrosine phosphorylation sites attenuated the Akt and the mammalian target of rapamycin activation and decreased the proliferation and migration of smooth muscle cells in response to PDGF-BB or fibroblast growth factor. These effects could be reversed by constitutively active PI3K or Akt, suggesting that c-Cbl phosphorylation promotes the PDGF-BB–induced proliferation and migration of smooth muscle cells through the PI3K/Akt pathways. In addition, overexpression of c-Cbl-m increased the ubiquitination of the PDGF and fibroblast growth factor receptors. Importantly, in balloon-injured rat carotid arteries, local delivery of c-Cbl-m reduced the phosphorylation of Akt and the mammalian target of rapamycin, inhibited the migration and proliferation of smooth muscle cells, and prevented neointimal hyperplasia.
Conclusions— Our results demonstrate a novel role of c-Cbl in vascular remodeling after injury and suggest that modulation of c-Cbl tyrosine phosphorylation may be a therapeutic approach to treat vascular neointimal hyperplasia such as restenosis after angioplasty.
Key Words: angioplasty • gene therapy • restenosis • signal transduction • vessels
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
Growth factors released during the arterial injury caused by therapeutic coronary interventions can induce intimal hyperplasia and hence clinically significant restenosis.1,2 Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and their receptors (PDGFR and FGFR, respectively) play a central role in the pathogenesis of intimal hyperplasia and mediate a number of important processes, including the proliferation, migration, and survival of vascular smooth muscle cells (SMCs).3–5 The molecular mechanisms controlling growth factor–induced neointimal hyperplasia remain to be elucidated.
Clinical Perspective p 772
c-Cbl is the cellular homolog of retroviral oncogenic protein v-Cbl cloned from the Cas NS-1 retrovirus, which induces Casitas B-lineage lymphoma.6 It is an adaptor protein and E3 ubiquitin-protein ligase that can either positively or negatively regulate signal transduction cascades.7,8 c-Cbl is known to become tyrosine phosphorylated in response to a number of cellular stimuli, including ligation of T-cell and B-cell receptors, activation of integrins, and activation of receptor tyrosine kinases such as PDGFR and FGFR. On stimulation, c-Cbl is rapidly phosphorylated on the tyrosine residues localized within its C terminus, leading to its interaction with a plethora of SH2-containing proteins such as the p85 subunit of phosphatidylinositol 3-kinase (PI3K), the Crk adaptor proteins, and Src family tyrosine kinases.7,9 c-Cbl also can associate with activated receptor tyrosine kinases, including epidermal growth factor receptor and PDGFR, via its tyrosine kinase–binding domain and ubiquitinylate these receptors to result in their downregulation.7,8,10 c-Cbl is expressed in virtually all cell types primarily as a cytosolic protein. Emerging evidence has revealed essential roles of c-Cbl in multiple cellular processes, including immune response,11 cell growth and transformation,12 bone development,13 insulin signaling14 and platelet activation.15 In vascular endothelial cells, c-Cbl mediates the endothelial signaling elicited by shear stress and VEGF.16,17 Recently, c-Cbl also has been shown to be involved in angiogenesis.18 However, a functional role of c-Cbl in vascular SMCs and the injury response of vessel wall remain largely unknown. In the present study, we aimed to determine the pathophysiological role of c-Cbl in vascular injury response in SMCs in vitro and balloon-injured arteries in vivo.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
Cell Culture and Reagents
Rat aortic SMCs were isolated and grown in DMEM supplemented with 10% FBS and antibiotics. Antibodies against c-Cbl, ubiquitin, FGFR, and PDGFRβ were from Santa Cruz Biotechnology (Santa Cruz, Calif). Antibodies against phospho-Tyr731-c-Cbl, phospho-Thr308-Akt, Akt, the mammalian target of rapamycin (mTOR), and phospho-Ser2448-mTOR were from Cell Signaling Technology (Danvers, Mass). LY294002, basic FGF (bFGF), and BrdU were from Sigma-Aldrich (St Louis, Mo). Other reagents were from Sigma-Aldrich unless specified.
Adenoviral Vectors
To generate Ad-c-Cbl-m, the cDNA fragment encoding HA epitope–tagged c-Cbl mutant, in which tyrosines were mutated into phenylalanine at positions 700, 731, and 774,16,19 was subcloned into pAdlox and recombined with an E1- and E3-deleted adenovirus-5 genome DNA. The expression of the inserted gene was driven by a 7x tet operon/minimal cytomegalovirus promoter, which was further under the control of tetracycline-responsive transactivator (tTA). The adenoviruses were plaque purified and titrated in 293 cells. Ad-CA-PI3K expresses a constitutively activated PI3K catalytic domain fused with the binding domain of p85. Ad-CA-Akt expressing a constitutively active Akt (rendered by the addition of the Src myristoylation signal), Ad-tTA, and Ad-LacZ were described previously.20
Northern Blotting, Immunofluorescence, Immunoblotting, and Immunoprecipitation
Total RNA was isolated from SMCs for Northern blotting, which was performed as previously described.21 Proteins were extracted from SMCs or vessel segments and subjected to immunoblotting and immunoprecipitation.22 Immunofluorescence staining was performed with primary antibodies against c-Cbl and HA tag and detected with fluorescein-conjugated secondary antibodies. Experimental procedures are described in the online Data Supplement.
|
Cell Proliferation, Migration, and Cell Cycle |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
Experimental procedures were performed as previously published23 and are described in the online Data Supplement.
Balloon Injury and Adenoviral Gene Transfer
Animal protocols were approved by the Animal Research Committee of the Peking University Health Science Center. Balloon injury was performed as described previously.24 Briefly, rats weighing 350 to 400 g were anesthetized with sodium pentobarbital (60 mg/kg IP). The left common carotid artery was injured with a 2F Fogarty balloon catheter inserted from the external carotid artery. For adenoviral infection, a segment of common carotid artery 
1 cm long was isolated by placement of vascular clamps on the proximal common carotid and proximal internal carotid arteries. Adenoviral solution (20 µL) containing Ad-LacZ (used as a vector control for in vivo study) or Ad-c-Cbl-m, together with Ad-tTA (2x109 plaque-forming units, combined) in PBS, was introduced into the injured common carotid through a syringe placed in the external carotid artery and incubated for 10 minutes. After the viral solutions were withdrawn, the external carotid artery was ligated, and blood flow through the common and internal carotid arteries was restored.
Immunohistochemistry and Morphometry
The arteries were perfusion fixed with 4% paraformaldehyde in PBS under a pressure of 100 mm Hg and snap-frozen in optical coherence tomography embedding compound. Immunohistochemical staining was performed as previously described.19 The sections were reacted with appropriate primary antibodies and a horseradish peroxidase–conjugated secondary antibody. Diaminobenzidine tetrahydrochloride was used for the color reaction. Negative controls were prepared with the use of species- and isotype-matched IgG. The sections were counterstained with hematoxylin and eosin for morphological observation. Neointimal formation was evaluated from the intimal area and the intima-to-media ratio using Image-Pro Plus software.
To detect the vascular cell replication rate in vivo, rats were intraperitoneally injected with BrdU (50 mg/kg) at 18 and 2 hours before euthanasia. The cryosections of arterial segments were denatured with 1.5N HCl, reacted with anti-BrdU antibody, and detected with horseradish peroxidase–conjugated antibody. Nuclei were counterstained with hematoxylin.
Statistical Analyses
All values are expressed as mean±SEM. Student’s t test (paired groups) or 2-way ANOVA, followed by Bonferroni’s posttest (multigroup comparisons), was used to analyze the statistical significance of differences. Values of P<0.05 were considered significant.
The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
c-Cbl Tyrosine Phosphorylation Is Increased in Response to Vascular Injury In Vivo and PDGF In Vitro
To investigate the potential role of c-Cbl in vascular injury, we first determined whether phosphorylation of c-Cbl is altered in injured arteries. Rat carotid arteries were injured by balloon angioplasty. Proteins were extracted and subjected to Western blotting 1, 2, and 3 days after the balloon injury. As shown in Figure 1A, balloon injury resulted in a marked increase in phosphorylation at tyrosine 731, a major tyrosine site for c-Cbl phosphorylation, without affecting the c-Cbl protein expression level. These results indicate that arterial injury leads to tyrosine phosphorylation of c-Cbl.
|
Denudation of endothelial cells after balloon angioplasty results in the release of PDGF. Because PDGF, particularly PDGF-BB, plays an important role in restenosis after vascular injury, we studied the effect of PDGF-BB on c-Cbl tyrosine phosphorylation in cultured SMCs using Western blotting (Figure 1B). PDGF-BB induced a rapid c-Cbl tyrosine phosphorylation, which is known to recruit PI3K to activate Akt. Akt can specifically phosphorylate the mTOR on Ser2448 and is a downstream kinase implicated in SMC proliferation and migration. We found that PDGF-BB triggered a rapid and sustained phosphorylation of Akt on Thr308 and mTOR on Ser2448 (Figure 1B). Pharmacological inhibitors of PI3K and mTOR were used to further clarify their hierarchical relationship. The results showed that LY294002 abolished the phosphorylation of Akt and attenuated mTOR activation but had little effect on c-Cbl phosphorylation; rapamycin inhibited mTOR phosphorylation without suppressing c-Cbl and Akt phosphorylation (Figure 1C), suggesting the involvement of c-Cbl tyrosine phosphorylation in a hierarchical signaling cascade from c-Cbl to PI3K, Akt, and mTOR.
Ablation of c-Cbl Tyrosine Phosphorylation Suppresses Vascular SMC Proliferation and Migration
Tyrosine phosphorylation of c-Cbl provides a docking site for p85, the regulatory subunit of PI3K, and recruits PI3K to activate its effector Akt. To investigate the functional role of c-Cbl tyrosine phosphorylation in vascular SMCs, we constructed a recombinant adenovirus expressing c-Cbl-m in which tyrosine was mutated into phenylalanine at positions 700, 731, and 774, which are the major tyrosine phosphorylation sites for c-Cbl (Figure 2A).19 SMCs were coinfected with the adenovirus encoding c-Cbl-m (Ad-c-Cbl-m) and the virus expressing tTA (Ad-tTA). Conditional expression of c-Cbl-m in SMCs was confirmed at both the mRNA and protein levels (Figure 2B). Notably, expression of c-Cbl-m, as detected by anti-HA antibody, was suppressed in the presence of tetracycline. In addition, immunofluorescent staining demonstrated that the cytoplasmic distribution of c-Cbl-m in SMCs is similar to that of the endogenous c-Cbl (Figure 2C).
|
Because PDGF-BB is known to potently stimulate SMC migration and proliferation, which are the central events in neointimal hyperplasia after angioplasty,3,4 we examined the effect of inhibition of c-Cbl tyrosine phosphorylation on these PDGF-induced cellular processes. As shown in Figure 3A, the PDGF-BB–induced SMC proliferation was significantly suppressed by overexpression of c-Cbl-m but not by the sham infection. Wound closure assay also showed that c-Cbl-m significantly inhibited the PDGF-stimulated migration of SMCs (Figure 3B). In addition, flow cytometric analyses demonstrated that the overexpression of c-Cbl-m significantly restricted G1/S progression (Figure 3C).
|
P<0.05, Because agents such as FGF also may play important roles in SMC proliferation and stimulate tyrosine phosphorylation of c-Cbl,8,10 we examined the effect of c-Cbl-m on SMC proliferation stimulated by FGF or serum in the absence of PDGF. The results showed that the c-Cbl mutant strongly suppressed the proliferative response induced by FGF (Figure 3D), suggesting that c-Cbl tyrosine phosphorylation is a critical event in mediating the proliferation of SMCs in response to multiple mitogens.
c-Cbl Tyrosine Phosphorylation Mediates PI3K/Akt/mTOR Activation in SMCs
Because the primary binding site for p85 (Tyr731 in c-Cbl) has been removed in c-Cbl-m,25 we asked whether c-Cbl-m could inhibit the phosphorylation of Akt and mTOR. As shown in Figure 4, in the SMCs overexpressing c-Cbl-m, the phosphorylation of Akt and mTOR induced by PDGF-BB, bFGF, or serum was attenuated compared with those with sham infection. These results indicate that the inhibitory effects of the c-Cbl mutant on SMC proliferation and migration may result from suppression of Akt and mTOR phosphorylation.
|
To further define the functional involvement of the c-Cbl, PI3K, and Akt signaling pathways, we used the adenoviruses expressing constitutively activated PI3K or Akt. SMCs were coinfected with Ad-c-Cbl-m, together with Ad-CA-PI3K, Ad-CA-Akt, or Ad–green fluorescent protein (GFP) (as a control), and their proliferation and migration in response to PDGF-BB were measured. The overexpression of either CA-PI3K or CA-Akt can attenuate the suppressive effects of c-Cbl-m compared with the GFP control (Figure 4B). These results provide further support that the effects of c-Cbl-m on SMC proliferation and migration are mediated by the inhibition of PI3K and Akt pathways.
Overexpression of c-Cbl-m Increased Ubiquitination of PDGF and FGF Receptors
Besides being an adaptor protein to mediate the activation of the downstream kinase signaling cascade, c-Cbl also is known to be a negative regulator of receptor and nonreceptor tyrosine kinases, which is due largely to its E3 ubiquitin ligase activity on the RING finger domain.26 Therefore, we examined the effect of c-Cbl-m on the ubiquitination of PDGFRβ and FGFR in SMCs. Immunoprecipitation assays demonstrated that overexpression of c-Cbl-m promoted the ubiquitination of both receptors, shown as increased large-molecular-weight ubiquitin smears. Subsequently, the abundance of the receptors for FGF and PDGF was decreased in SMCs expressing c-Cbl-m (Figure 5). These results suggest that the ubiquitination-mediated downregulation of the surface receptors for FGF and PDGF also may contribute to the diminution of their signaling by c-Cbl mutant.
|
P<0.05 vs lane 3.
Overexpression c-Cbl-m Inhibits SMC Proliferation, Migration, and Intimal Hyperplasia In Vivo
To ascertain the physiological role of the c-Cbl tyrosine phosphorylation in intimal hyperplasia in vivo, rat carotid arteries were balloon injured and infected with Ad-c-Cbl-m or Ad-LacZ. Adenoviral infection at the dose used did not cause significant cell loss compared with the balloon injury without viral infection (Figure I of the Data Supplement). Gene expression of c-Cbl mutant to the injured arteries was confirmed by immunohistochemical staining 72 hours after injury, showing that most of the medial cells were positive for the HA-tagged c-Cbl mutant. Immunoblotting with antibody against the HA tag also detected the overexpression of c-Cbl mutant in the vessel wall (Figure 6A). Tyrosine phosphorylation of c-Cbl in the injured vessels was decreased by overexpression of the c-Cbl mutant compared with the LacZ control. In parallel, the c-Cbl mutant reduced the phosphorylation of Akt and mTOR (Figure 6B), suggesting that c-Cbl tyrosine phosphorylation serves as an upstream signal to activate Akt and mTOR cascade in vivo.
|
To examine the effect of mutant c-Cbl on SMC proliferation in vivo, in vivo labeling of replicating SMCs with BrdU was performed 7 days after injury. Immunohistochemical studies demonstrated that the c-Cbl mutant effectively suppressed SMC proliferation in neointima (Figure 6C). Consistent with the previous studies,24 examination at 4 and 14 days after injury showed rare BrdU labeling within intima (data not shown). To investigate whether SMC migration from the media to the intima was suppressed, we measured the number of SMCs in the intima at 4 days after injury when the cells first migrate in and before they have time to replicate.27 The decrease in cells in the neointima, although not a direct measurement of the migratory activity, suggests that the mutant c-Cbl inhibited SMC migration into intima in vivo, thus corroborating the suppressive effect of c-Cbl-m on SMC migration in vitro (Figure 6D).
Fourteen days after the balloon injury and gene transfer, we evaluated the effect of c-Cbl-m on intimal hyperplasia and restenosis. As shown in Figure 6E, gene transfer of c-Cbl-m significantly decreased the neointimal area and intima-to-media ratio compared with the control group. Immunohistochemical study using an antibody against von Willebrand factor did not find an effect of the c-Cbl mutant on reendothelialization in the injured arteries (supplementary Figure II). These results demonstrated that c-Cbl tyrosine phosphorylation plays a critical role in neointimal hyperplasia.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
Proliferation and migration of SMCs in the arterial wall play pivotal roles in atherosclerosis and restenosis.1,28 In the present study, we demonstrate for the first time that balloon injury in arteries and PDGF in cultured SMCs stimulate the tyrosine phosphorylation of c-Cbl. We also show that the c-Cbl mutant, which is deficient in the major tyrosine phosphorylation sites, attenuates the activation of the Akt/mTOR pathway and inhibits SMC migration and proliferation in response to PDGF, FGF, and serum. Finally, we demonstrate that in vivo gene transfer of the c-Cbl mutant inhibits SMC proliferation and migration in vivo and prevents neointimal hyperplasia after balloon injury.
c-Cbl undergoes tyrosine phosphorylation in response to a multitude of stimuli, including activated growth factor receptor protein tyrosine kinases (PTKs; such as epidermal growth factor receptor, FGFR, and PDGFR), cytokines, hormones, and mechanical stimuli (such as shear stress). We describe here that c-Cbl undergoes tyrosine phosphorylation in the arterial wall after balloon injury and in cultured aortic SMCs after PDGF-BB stimulation. Given that PDGF-BB and FGF, released as a result of endothelial denudation, are the most potent SMC chemoattractant and mitogen, respectively, they may account for the c-Cbl tyrosine hyperphosphorylation in the balloon-injured vessel. However, stimulation from other growth factors such as EGF could not be excluded. To the best of our knowledge, this study is the first to show the functional significance of c-Cbl tyrosine phosphorylation in terms of vascular SMC proliferation and migration both in vitro and in vivo. Most important, gene transfer of a tyrosine phosphorylation–deficient c-Cbl mutant potently suppressed neointimal formation in the well-characterized rat carotid artery balloon injury model. These findings point toward a previously unrecognized role of c-Cbl in pathological vascular remodeling. From a therapeutic standpoint, our study provides the first proof of concept that the modulation of c-Cbl phosphorylation may be a fruitful approach to the treatment of vascular diseases such as atherosclerosis, transplant vasculopathy, and restenosis after angioplasty.
A key finding of the present study is the identification of the molecular mechanisms by which the c-Cbl mutant exerts its vascular protective effect. Phosphorylation of the tyrosine residues on the C-terminus by receptor or nonreceptor PTKs enables c-Cbl to act as a multivalent adaptor for a plethora of SH-containing molecules to positively regulate these signal pathways. Constitutive tyrosine phosphorylation of c-Cbl has been found in various specimens and cell lines from cancers.29 Tyrosine 700, 731, and 774 have been identified as major phosphorylation sites by receptor and nonreceptor PTKs.19 Phosphorylation of tyrosine 731 provides a docking site for p85-PI3K, which generates specific inositol phospholipids and recruits Akt to the cell membrane for its activation.9,30,31 Akt promotes cell-cycle progression of SMCs and plays an essential role in injury-induced neointimal hyperplasia32 and in-stent restenosis.33 By phosphorylating and inactivating the tuberous sclerosis complex (TSC-2), Akt activates mTOR. Activated mTOR further phosphorylates p70S6 kinase (S6K) and 4E binding protein-1 (4EBP-1), which are engaged in ribosome biogenesis and initiation of translation, thus promoting cell growth.34 A role of mTOR in pathological vascular remodeling is highlighted by the clinical effectiveness of rapamycin (sirolimus)-coated stents in reducing restenosis and the observation that mTOR is activated in neointimal SMCs, exhibiting increased phosphorylation.35 In agreement with the previous observations, we found that the phosphorylation of Akt and mTOR is increased in balloon-injured arteries and in cultured SMCs stimulated by PDGF-BB, bFGF, or serum. In this study, disrupting the major tyrosine phosphorylation sites of c-Cbl suppresses activation of Akt and mTOR in vitro and in vivo. We postulate that this suppressive effect is due to the blockade of PI3K/Akt/mTOR cascade because the mutation of the primary binding site for p85 (Tyr731 in c-Cbl) disrupts the p85 association with c-Cbl. This notion is reinforced by the finding that these suppressive effects are overridden by overexpression of constitutive activation of either PI3K or Akt. c-Cbl also is a negative regulator of many signal pathways. The c-Cbl RING finger has intrinsic E3 ligase activity and can recruit E2s for the transfer of ubiquitin to substrates.36,37 c-Cbl promotes ligand-induced ubiquitination of PDGF receptors 
and β in fibroblasts and FGFR in HeLa cells.10,26,38 Although the major tyrosine residues responsible for docking the SH2-containing proteins have been removed in c-Cbl-m, the tyrosine residue implicated in the E3 activity of c-Cbl located between tyrosine kinase–binding and RING (Tyr-371 and Tyr-368)39 remains intact, and this preserves the negative regulatory activity (Figure 5). Therefore, overexpression of the c-Cbl mutant may inhibit neointimal hyperplasia via a mechanism involving 3 effects: negations of the PI3K/Akt/mTOR activation by nullifying its ability as an adaptor, downregulation of the growth factor receptors by enhancing its ability as an E3 ligase, and inhibition of the phosphorylation of the endogenous c-Cbl (Figure 7). Although we have demonstrated PI3K/Akt to be a major pathway responsible for the suppression of growth factor–induced proliferation and migration by c-Cbl-m in SMCs, many other kinase pathways such as Src, Cin85, Crk, and PLC
also depend on the adaptor function of c-Cbl,40–43 and their possible roles deserve further assessments.
|
|
Conclusion |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
The present study has demonstrated a novel role of c-Cbl in vascular remodeling after injury and suggests that modulation of c-Cbl phosphorylation may have potential implications in the treatment of pathological vascular remodeling such as restenosis after angioplasty.
|
Acknowledgments |
|---|
We thank Dr Ruiping Xiao for providing the Akt construct.
Sources of Funding
This study was supported by the grants from the National Science Foundation of China (30470810 and 30670848); the Ministry of Science and Technology, China (2006CB503906); and the National Institutes of Health, Bethesda, Md (HL085159 and HL080518).
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsCell Proliferation, Migration,...ResultsDiscussionConclusionReferences |
|---|
1. Lovqvist A, Emanuelsson H, Nilsson J, Lundqvist H, Carlsson J. Pathophysiological mechanisms for restenosis following coronary angioplasty: possible preventive alternatives. J Intern Med. 1993; 233: 215–226.[Medline] [Order article via Infotrieve]
2. Schwartz SM, deBlois D, O'Brien ER. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995; 77: 445–465.
3. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991; 253: 1129–1132.
4. Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992; 89: 507–511.[Medline] [Order article via Infotrieve]
5. Agrotis A, Kanellakis P, Kostolias G, Di Vitto G, Wei C, Hannan R, Jennings G, Bobik A. Proliferation of neointimal smooth muscle cells after arterial injury: dependence on interactions between fibroblast growth factor receptor-2 and fibroblast growth factor-9. J Biol Chem. 2004; 279: 42221–42229.
6. Blake TJ, Shapiro M, Morse HC III, Langdon WY. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene. 1991; 6: 653–657.[Medline] [Order article via Infotrieve]
7. Thien CB, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol. 2001; 2: 294–307.[CrossRef][Medline] [Order article via Infotrieve]
8. Swaminathan G, Tsygankov AY. The Cbl family proteins: ring leaders in regulation of cell signaling. J Cell Physiol. 2006; 209: 21–43.[CrossRef][Medline] [Order article via Infotrieve]
9. Hartley D, Meisner H, Corvera S. Specific association of the beta isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl. J Biol Chem. 1995; 270: 18260–18263.
10. Wong A, Lamothe B, Lee A, Schlessinger J, Lax I. FRS2 alpha attenuates FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl. Proc Natl Acad Sci U S A. 2002; 99: 6684–6689.
11. Rao N, Dodge I, Band H. The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system. J Leukoc Biol. 2002; 71: 753–763.
12. Feng Q, Baird D, Peng X, Wang J, Ly T, Guan JL, Cerione RA. Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth. Nat Cell Biol. 2006; 8: 945–956.[CrossRef][Medline] [Order article via Infotrieve]
13. Chiusaroli R, Sanjay A, Henriksen K, Engsig MT, Horne WC, Gu H, Baron R. Deletion of the gene encoding c-Cbl alters the ability of osteoclasts to migrate, delaying resorption and ossification of cartilage during the development of long bones. Dev Biol. 2003; 261: 537–547.[CrossRef][Medline] [Order article via Infotrieve]
14. Molero JC, Turner N, Thien CB, Langdon WY, James DE, Cooney GJ. Genetic ablation of the c-Cbl ubiquitin ligase domain results in increased energy expenditure and improved insulin action. Diabetes. 2006; 55: 3411–3417.
15. Saci A, Pain S, Rendu F, Bachelot-Loza C. Fc receptor-mediated platelet activation is dependent on phosphatidylinositol 3-kinase activation and involves p120(Cbl). J Biol Chem. 1999; 274: 1898–1904.
16. Miao H, Yuan S, Wang Y, Tsygankov A, Chien S. Role of Cbl in shear-activation of PI 3-kinase and JNK in endothelial cells. Biochem Biophys Res Commun. 2002; 292: 892–899.[CrossRef][Medline] [Order article via Infotrieve]
17. Wang Y, Chang J, Li YC, Li YS, Shyy JY, Chien S. Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway. Am J Physiol Heart Circ Physiol. 2004; 286: H685–H692.
18. Singh AJ, Meyer RD, Navruzbekov G, Shelke R, Duan L, Band H, Leeman SE, Rahimi N. A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLC1 activation and angiogenesis. Proc Natl Acad Sci U S A. 2007; 104: 5413–5418.
19. Feshchenko EA, Langdon WY, Tsygankov AY. Fyn, Yes, and Syk phosphorylation sites in c-Cbl map to the same tyrosine residues that become phosphorylated in activated T cells. J Biol Chem. 1998; 273: 8323–8331.
20. Wang N, Verna L, Liao H, Ballard A, Zhu Y, Stemerman MB. Adenovirus-mediated overexpression of dominant-negative mutant of c-Jun prevents intercellular adhesion molecule-1 induction by LDL: a critical role for activator protein-1 in endothelial activation. Arterioscler Thromb Vasc Biol. 2001; 21: 1414–1420.
21. Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM, Stemerman MB. Constitutive activation of peroxisome proliferator-activated receptor-gamma suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem. 2002; 277: 34176–34181.
22. Qin X, Tian J, Zhang P, Fan Y, Chen L, Guan Y, Fu Y, Zhu Y, Chien S, Wang N. Laminar shear stress up-regulates the expression of stearoyl-CoA desaturase-1 in vascular endothelial cells. Cardiovasc Res. 2007; 74: 506–514.
23. Hsu PP, Li S, Li YS, Usami S, Ratcliffe A, Wang X, Chien S. Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation. Biochem Biophys Res Commun. 2001; 285: 751–759.[CrossRef][Medline] [Order article via Infotrieve]
24. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49: 327–333.[Medline] [Order article via Infotrieve]
25. Feshchenko EA, Shore SK, Tsygankov AY. Tyrosine phosphorylation of c-Cbl facilitates adhesion and spreading while suppressing anchorage-independent growth of V-Abl-transformed NIH3T3 fibroblasts. Oncogene. 1999; 18: 3703–3715.[CrossRef][Medline] [Order article via Infotrieve]
26. Miyake S, Mullane-Robinson KP, Lill NL, Douillard P, Band H. Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation: a critical role for Cbl tyrosine kinase-binding domain. J Biol Chem. 1999; 274: 16619–16628.
27. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994; 75: 539–545.
28. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia. Circulation. 1989; 79: 1374–1387.
29. Kamei T, Machida K, Nimura Y, Senga T, Yamada I, Yoshii S, Matsuda S, Hamaguchi M. c-Cbl protein in human cancer tissues is frequently tyrosine phosphorylated in a tumor-specific manner. Int J Oncol. 2000; 17: 335–339.[Medline] [Order article via Infotrieve]
30. Fukazawa T, Reedquist KA, Trub T, Soltoff S, Panchamoorthy G, Druker B, Cantley L, Shoelson SE, Band H. The SH3 domain-binding T cell tyrosyl phosphoprotein p120: demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase. J Biol Chem. 1995; 270: 19141–19150.
31. Meisner H, Conway BR, Hartley D, Czech MP. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol Cell Biol. 1995; 15: 3571–3578.[Abstract]
32. Stabile E, Zhou YF, Saji M, Castagna M, Shou M, Kinnaird TD, Baffour R, Ringel MD, Epstein SE, Fuchs S. Akt controls vascular smooth muscle cell proliferation in vitro and in vivo by delaying G1/S exit. Circ Res. 2003; 93: 1059–1065.
33. Zhou RH, Lee TS, Tsou TC, Rannou F, Li YS, Chien S, Shyy JY. Stent implantation activates Akt in the vessel wall: role of mechanical stretch in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003; 23: 2015–2020.
34. Inoki K, Guan KL. Complexity of the TOR signaling network. Trends Cell Biol. 2006; 16: 206–212.[CrossRef][Medline] [Order article via Infotrieve]
35. Zohlnhofer D, Nuhrenberg TG, Neumann FJ, Richter T, May AE, Schmidt R, Denker K, Clauss MA, Schomig A, Baeuerle PA. Rapamycin effects transcriptional programs in smooth muscle cells controlling proliferative and inflammatory properties. Mol Pharmacol. 2004; 65: 880–889.
36. Joazeiro CA, Wing SS, Huang H, Leverson JD, Hunter T, Liu YC. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science. 1999; 286: 309–312.
37. Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, Langdon WY, Beguinot L, Geiger B, Yarden Y. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 1998; 12: 3663–3674.
38. Miyake S, Lupher ML Jr, Druker B, Band H. The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proc Natl Acad Sci U S A. 1998; 95: 7927–7932.
39. Levkowitz G, Waterman H, Ettenberg SA, Katz M, Tsygankov AY, Alroy I, Lavi S, Iwai K, Reiss Y, Ciechanover A, Lipkowitz S, Yarden Y. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell. 1999; 4: 1029–1040.[CrossRef][Medline] [Order article via Infotrieve]
40. Tvorogov D, Carpenter G. EGF-dependent association of phospholipase C-gamma1 with c-Cbl. Exp Cell Res. 2002; 277: 86–94.[CrossRef][Medline] [Order article via Infotrieve]
41. Sanjay A, Houghton A, Neff L, DiDomenico E, Bardelay C, Antoine E, Levy J, Gailit J, Bowtell D, Horne WC, Baron R. Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v) beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility. J Cell Biol. 2001; 152: 181–195.
42. Sawasdikosol S, Chang JH, Pratt JC, Wolf G, Shoelson SE, Burakoff SJ. Tyrosine-phosphorylated Cbl binds to Crk after T cell activation. J Immunol. 1996; 157: 110–116.[Abstract]
43. Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I. Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature. 2002; 416: 183–187.[CrossRef][Medline] [Order article via Infotrieve]
|
Footnotes |
|---|
The online Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.761932/DC1.
Related Article:
Circulation 2008 118: 697-698.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||