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Originally published In Press as doi:10.1074/jbc.M610452200 on May 11, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19808-19819, July 6, 2007
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Leukocyte Antigen-related Deficiency Enhances Insulin-like Growth Factor-1 Signaling in Vascular Smooth Muscle Cells and Promotes Neointima Formation in Response to Vascular Injury*

Xi-Lin Niu, Juxiang Li, Zeenat S. Hakim, Mauricio Rojas, Marschall S. Runge, and Nageswara R. Madamanchi1

From the Department of Medicine, Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599-7126

Received for publication, November 9, 2006 , and in revised form, April 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR-/- mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR-/- VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR-/- VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR-/- VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR-/- mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR-/- mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular smooth muscle cell (VSMC)2 proliferation, dedifferentiation, and migration in response to atherogenic stimuli are all governed by activation of key intracellular signaling pathways that in turn are regulated by the phosphorylation status of requisite tyrosine and serine residues of the integral proteins. Many growth factor receptors are protein-tyrosine kinases (PTKs) and undergo tyrosine phosphorylation and dephosphorylation in a concerted manner in response to a stimulus to initiate a signaling cascade that results in a physiological response (1). The characterization of protein-tyrosine phosphatases (PTPs), which reverse the activity of PTKs is essential for understanding the pathophysiological function of these kinases that underlies many cardiovascular diseases such as cardiac hypertrophy, ischemia/reperfusion injury, and atherogenesis and for the identification of therapeutic targets (2).

A total of 48 PTPs have been identified and it is estimated that up to 100 PTPs are present in human genome (3). Little is known, however, about which PTPs are expressed in vascular cells and blood vessels, the substrate specificity of those expressed and their role in vascular remodeling (4). Leukocyte antigen-related (LAR) protein-tyrosine phosphatase is a receptor-like tyrosine phosphatase with a broad tissue distribution including epithelial, neuronal, and cardiac cells (5). LAR is expressed on the cell surface as a complex of two noncovalently associated subunits of 150 and 85 kDa, which are generated by the action of an endogenous protease on the pro-protein. The 150-kDa fragment, representing the amino terminus of the protein, is exclusively extracellular and is modified by N-linked glycosylation. The 85-kDa C-terminal subunit contains a short ectodomain, a transmembrane domain, and two tandem phosphatase domains. The membrane-proximal phosphatase domain exhibits enzyme activity in vitro.

Many studies of LAR have focused on its role in regulating insulin signaling. Antisense suppression of LAR enhanced insulin-dependent insulin receptor (IR) phosphorylation and phosphatidylinositol 3-kinase (PI3K) activity in hepatoma cells (6). Furthermore, LAR expression was not only increased in obese subjects but also correlated with enhanced IR dephosphorylation and insulin resistance (7, 8). LAR physically interacts with IR in vivo and activation of IR enhances its binding to LAR that suggest that LAR regulates insulin action at the receptor level (9). LAR is likely important for other critical cellular functions. For instance, LAR plays a role in establishing and maintaining neuronal networks and deficiency of this PTP results in reduction in the size of basal forebrain cholinergic neurons and loss of cholinergic innervation of the dentate gyrus (10).

Insulin-like growth factor-1 (IGF-1) is secreted by VSMC and other vascular cells and plays an important role in multiple vascular pathologies (11). IGF-1 is a potent VSMC mitogen and antiapoptotic factor and a good stimulant for migration. Because of these functions, decrease in IGF-1 levels might be beneficial in early stages of atherosclerotic plaque formation characterized by VSMC hypertrophy and hyperplasia (12), but detrimental in advanced plaque conditions due to destabilization caused by the loss of VSMC (13). Consistent with this notion, enhanced IGF-1 binding was observed in injury-induced intimal hyperplasia (14). IGF-1 exerts most of its known physiological effects by binding and activating its receptor, IGF-1R (15). IGF-1R is highly homologous to IR (15) and the mitogenic effect of insulin is mediated through IGF-1R in VSMC (16). In addition, IGF-1R density is a critical determinant of VSMC growth and up-regulation of its expression or activation play an important role in the mitogenic effects of agonists such as fibroblast growth factor, angiotensin II, and thrombin (17, 18). Cross-talk between IGF-1R and other receptors also modulates the function of IGF-1. For example, blocking {alpha}Vbeta3 ligand occupancy leads to premature recruitment of tyrosine phosphatase SHP-2 to IGF-1R and attenuates IGF-1R signaling (19). This also suggests that PTPs are important modulators of IGF-1 signaling. However, there is no evidence for the role of LAR with either IGF-1 or IGF-1R-stimulated signaling in vascular cells.

In the present study, we investigated the effect of LAR on cellular phenotype and IGF-1-induced signaling pathways using aortic VSMC derived from LAR knock-out mice. Our results demonstrate that LAR interacts with IGF-1R and regulates IGF-1-induced signaling. Absence of LAR results in increased VSMC proliferation and migration in response to IGF-1 and enhanced neointima formation in response to arterial injury in mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Recombinant human IGF-I was a generous gift from Dr. Clemmons (University of North Carolina, Chapel Hill). LY294002 and U0126 were obtained from Calbiochem. [methyl-3H]Thymidine (70 Ci/mmol) was obtained from Amersham Biosciences. The antibodies used were anti-IGF-IR beta-subunit (C20), anti-PDGFR-beta (M20), anti-IRS-1, and anti-PI 3-kinase p85{alpha} (Santa Cruz Biotechnology), anti-phosphospecific ERK1/2 and anti-ERK1/2 (New England Biolabs), anti-phosphotyrosine (PY-20) and anti-GRB2 (BD Transduction Laboratories). The phosphospecific IGF-1R antibodies used were anti-IR/IGF1R (Tyr(P)972, the corresponding residue in IGF-1R is 950), anti-IR/IGF1R (Tyr(P)1158, the corresponding residue in IGF-1R is 1131), anti-IR/IGF1R (Tyr(P)1162/Tyr(P)1163, the corresponding residues in IGF-1R are 1135 and 1136) and anti-IR (Tyr(P)1334) (BIOSOURCE). Two different anti-LAR antibodies, anti-LAR monoclonal (catalog number 610350, BD Transduction Laboratories) and goat anti-LAR polyclonal (sc-1119, Santa Cruz Biotechnology), were used in this study. The monoclonal antibody was raised against an epitope corresponding to amino acids 24-194 of human LAR and recognizes the 150-kDa extracellular fragment of mouse LAR. The goat polyclonal antibody was raised against an epitope in the COOH-terminal cytoplasmic domain of rat LAR and recognizes the 85-kDa C-terminal subunit of human LAR, but not that of mouse LAR.

LAR-deficient Mice—LAR-deficient mice (supplied by Dr. Frank Longo, University of North Carolina, Chapel Hill, NC) were generated by the gene trap method (10). VSMC were isolated from mice that were backcrossed at least 8 times into the DBA/2J background.

Cell Culture—Aortic VSMC were isolated from 4-month-old male wild-type and LAR-/- mice as previously described by us (20). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) as described previously (21). All experiments were conducted using VSMC between passages 4 and 11 that were growth arrested by incubation in DMEM containing 0.1% FBS for 72 h.

Preparation of GST-C-LAR and Pull-down Assays—GST-human cytosolic LAR (GST-C-LAR) (amino acid 1247-1898) was constructed by PCR in bacterial expression vector PGEX-4AT-3 using sense primer (5'-GAGCCGGAGATGCTGTGGGTGACGGGT-3') and antisense primer (5'-TTACGTTGCATAGTGGTCAAAGCTGCCG-3'). GST or GST-C-LAR were expressed in Escherichia coli and purified by affinity chromatography on glutathione-Sepharose beads as described by Frangioni and Neel (22). Approximately 500 ng of GST or GST-C-LAR were immobilized on glutathione-Sepharose beads (Amersham Biosciences) and the beads were washed three times with ice-cold lysis buffer. The beads were then incubated with 1 ml of VSMC lysate at 4 °C for 2 h, and then washed 3 times with lysis buffer and the bound proteins were eluted by boiling in Laemmli sample buffer. GST-C-LAR bound proteins were visualized following Western analysis.

Construction of Recombinant Adenoviruses—Ad-LAR was constructed by subcloning full-length human LAR cDNA (generously provided by Robert A. Mooney, University of Rochester, Rochester, NY) into adenoviral shuttle vector, pShuttle-CMV. After homologous recombination by electroporation of pShuttle-CMV containing LAR cDNA into BJ5183 E. coli (Stratagene) containing the adenoviral backbone plasmid pADEasy-1, recombinants were selected. Recombinant (LE1/E3-deficient) adenoviruses were generated by transfection of human embryonic kidney 293 cells with the recombinant plasmid using Lipofectamine (Invitrogen), then the virus was serially amplified, purified on a CsCl density gradient by ultracentrifugation and titered (23). A control adenovirus consisting of the identical adenovirus backbone with beta-galactosidase cDNA insert (Ad-betagal) was provided by Dr. Huang (University of North Carolina).


Figure 1
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  		FIGURE 1.
LAR negatively regulates VSMC proliferation in standard growth medium and DNA synthesis induced by IGF-1 but not PDGF-BB. A, Western analysis of LAR expression in VSMC using anti-LAR monoclonal antibody (mAb). B, proliferation of wild-type and LAR-/- VSMC in standard growth medium. Cells were plated at equal density and cell counts were performed in triplicate. C, growth-arrested VSMC were treated with 100 ng/ml IGF-1 in the presence and absence of LY249002 or U0126 for 16 h and [3H]thymidine incorporation was measured during the last 3 h of treatment. Data presented are mean ± S.E. (n = 3) and representative of three separate experiments conducted using three different isolates of VSMC (*, p < 0.001 compared with the respective controls; **, p < 0.001 compared with IGF-1-treated wild-type VSMC; # and @, p < 0.001 compared with the respective IGF-1-treated VSMC). D, growth-arrested VSMC were treated with 10 ng/ml PDGF for 16 h and [3H]thymidine incorporation was measured during the last 3 h of treatment. Data presented are mean ± S.E. (n = 3) and representative of two separate experiments.

 
Adenovirus Infection—Adenoviral infection of nearly confluent VSMC was performed at a multiplicity of infection of 100 in DMEM containing 2% FBS. After 16 h incubation, the cells were quiescenced in DMEM containing 0.1% FBS for 72 h.

IGF-1R Dephosphorylation Assay—Growth-arrested LAR-/- VSMC were treated with 100 ng/ml IGF-1 for 10 min. Cells were then lysed, and IGF-1R was immunoprecipitated and immobilized on protein A-agarose beads. The beads were washed and incubated in the absence or presence of purified recombinant LAR (New England Biolabs, Ipswich, MA) at 30 °C. The reaction was stopped by adding Laemmli sample buffer and boiling for 5 min. Samples were analyzed by SDS-PAGE, and probed with either anti-phosphotyrosine or anti-IGF-1R antibody following Western analysis.

Specific Activity of LAR—The specific activity of LAR against IGF-1R was determined using the LAR tyrosine phosphatase assay kit (BIOMOL QuantizymeTM assay system). Initial attempts to use immunoprecipitated, activated-IGF-1R from VSMC as a substrate to study the dose-dependent activity of LAR were unsuccessful because the amount of free phosphate released from activated IGF-1R by LAR phosphatase was below the detection limit of the Quantizyme assay system. Therefore, we used phosphopeptide, TRDIpYETDpYpYRK (IGF-1R Tyr(P)1131, Tyr(P)1135, and Tyr(P)1136; corresponding to IR Tyr(P)1158, Tyr(P)1162, and Tyr(P)1163; Biomol) as the substrate. Dose-dependent activity of LAR was determined by incubating different doses of LAR with 150 µM phosphopeptide at 30 °C for 30 min. The amount of phosphate released was quantified using Biomol GreenTM reagent according to the manufacturer's protocol.

Blood Pressure—Blood pressure was measured in conscious animals by the tail-cuff method daily for 1 week. The animals were warmed in a heating chamber (32 °C) for 20-30 min and 20 consecutive pressures were recorded for 15 min.

Immunoprecipitation and Western Analysis—Cells were lysed either in RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05 mM sodium fluoride, 1 mM EDTA, 1% Igepal, 0.05% sodium deoxycholate, 0.1% SDS, and protease inhibitors) or Triton lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100) containing protease inhibitors. Immunoprecipitation (500 µg of protein) and Western analysis were performed as described previously (21). Cell lysates containing 50 µg of protein were analyzed in Western blotting experiments.

PI3K Activity—To measure IRS-1-associated PI3K activity, growth-arrested VSMC were treated with 100 ng/ml IGF-1 for 10 min and the cell lysates containing 500 µg of protein were immunoprecipitated with 5 µg of anti-IRS-1 antibodies. PI3K activity was measured as described previously (24).

[3H]Thymidine Incorporation Assay—Growth-arrested VSMC were treated with or without 100 ng/ml IGF-1 in the presence or absence of PI3K inhibitor, LY 249002 (10 µM) or MEK inhibitor, U0126 (1 µM), for 16 h. Cells were labeled with [methyl-3H]thymidine for 3 h, and its incorporation into DNA was measured as described previously (20).


Figure 2
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  		FIGURE 2.
Absence of LAR enhances IGF-1-induced VSMC migration toward vitronectin. A, haptotactic migration of wild-type and LAR-/- VSMC on various extracellular matrix proteins (*, p < 0.001 compared with bovine serum albumin (BSA)). B, IGF-1 increased VSMC migration on vitronectin in wild-type and LAR-/- VSMC. Basal and IGF-1-induced VSMC migration on vitronectin were inhibited by both LY249002 and U0126. Data presented are mean ± S.E. (n = 3) and representative of three separate experiments conducted using three different isolates of VSMC (*, p < 0.001 compared with the respective controls; **, p < 0.001 compared with IGF-1-treated wild-type VSMC; #, p < 0.001 compared with the respective IGF-1-treated cells).

 
Haptotactic Cell Migration Assay—Haptotactic cell migration assays were performed using modified Boyden chambers (6.5-mm polycarbonate Transwell filter inserts with 8-µm pores, Transwell; Costar). The bottom sides of the filters were coated with 10 µg/ml individual extracellular matrix protein or bovine serum albumin. Transwell filters were air dried for 1 h in tissue culture hood. The filters were washed with PBS, and blocked with 2% bovine serum albumin in PBS. Growth-arrested VSMC were trypsinized, centrifuged, and resuspended in DMEM without serum. VSMC were treated with or without IGF-1 for 30 min and 2 x 104 cells in 100 µl of DMEM were seeded onto the top of the Transwell filters (DMEM with or without 10 µM LY249002 or 1 µM U0126 in the bottom chamber). Cells were incubated at 37 °C in a humidified 95% air, 5% CO2 atmosphere for 12 h. The Transwells were washed twice with PBS, the cells from the top side of the filter were removed with a cotton swab, and cells on the bottom side of the filter were stained for 30 min with a Gill hematoxylin (Vector Laboratories). The number of stained cells was scored for six randomly selected high power fields (magnification, x400).

Plasma Assays—Plasma glucose was measured using a Blood Glucose Monitoring System (Thera-Sense, Inc.). Total cholesterol and triglyceride levels were measured using Vitros 250 Chemical Analyzer (Ortho-clinical Diagnostics) at the Animal Clinical Core Facility in the University of North Carolina, Chapel Hill. Insulin (Mercodia), IGF-1 (R & D Systems), adiponectin (LINCO Research), tumor necrosis factor-{alpha} (R&D Systems), and free fatty acids (Roche) were assayed using commercial kits according to the manufacturer's protocols.

Femoral Artery Injury, Immunohistochemistry, and Morphometry—Mouse transluminal femoral artery injury was performed as previously described (25). Briefly, a 0.25-mm angioplasty guidewire (Guidant Advanced Cardiovascular Systems) was inserted and then withdrawn into femoral artery up to the aortic bifurcation 4 times to denude the endothelium. Mice were sacrificed 4 weeks after arterial injury. Two 5-mm thick transverse segments were cut from each artery at the level of injury. Histological sections were cut from both segments (eight 5-µm sections were made at 200-µm intervals for the entire length of the segment) and stained with combined Masson trichrome elastic stain. Arterial specimens were analyzed by computerized morphometry (NIH Image 1.60 software) in a blinded fashion and the average intima, media, and intima-to-media ratio were calculated. Shamoperated mice underwent all maneuvers (dissection, vascular clamping, arteriotomy, and ligation) except the passage of the guidewire. All animal procedures were in compliance with the University of North Carolina Institutional Animal Care and Use Committee guidelines.

Immunohistochemistry—Representative sections of femoral artery were immunohistochemically stained using either polyclonal rabbit anti-phospho-IGF-1R antibody (GeneTex, Inc., San Antonio, TX) or anti-human smooth muscle actin/horseradish peroxidase staining kit (DakoCytomation, Inc., Denmark). Formalin-fixed, paraffin-embedded sections were deparafinized, rehydrated, and blocked with 3% hydrogen peroxide. Immunoperoxidase staining was carried out using Vector DAB substrate kit for phosphor-IGF-1R, VIP substrate kit for {alpha}-actin (Vector Laboratories, Inc., Burlingame, CA) and Vectastain Elite ABC kit following the instructions of the manufacturer. The sections were counterstained with Vector hematoxylin and permanently mounted with VectaMount mounting medium.


Figure 3
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  		FIGURE 3.
Effect of LAR expression on IGF-1R autophosphorylation. A, growth-arrested VSMC were treated with 100 ng/ml IGF-1 for the indicated times and cell lysates were prepared. Cell lysates with equal amounts of protein were immunoprecipitated (IP) with anti-IGF1Rbeta antibody and Western (WB) analysis was performed with anti-phosphotyrosine antibody (4G10). B, the same membrane was reprobed with anti-IGF-1Rbeta antibody. C, densitometric analysis of IGF-1R autophosphorylation (mean ± S.E., n = 3; *, p < 0.001; **, p < 0.001; and ***, p < 0.05 compared with wild-type VSMC treated with IGF-1 at respective the time points).

 
Statistical Analysis—Data were analyzed with one-way and two-way analysis of variance and in the case of one-way analysis of variance, post hoc analysis was performed using Newman-Keuls test. Student's t test was used to compare the stimulation of signaling proteins in wild-type and LAR-/- VSMC treated with IGF-1. Statistical significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LAR Deficiency Decreases Insulin and IGF-1 Levels in Mice—Ren et al. (26) reported increased insulin sensitivity in the basal state and insulin resistance by clamp studies in adult LAR knock-out mice generated by insertional mutagenesis. However, in this study LAR-/- mice of mixed C57BL/6 and 129 genetic backgrounds were compared with an unrelated outbred strain of Swiss-Webster mice, making it difficult to conclude whether the reported differences in insulin levels, body weight, and glucose homeostasis resulted form LAR deficiency or strain background. To clarify the role of LAR in regulating insulin/IGF-1 action, we determined the phenotype of LAR-/- mice (Table 1). LAR-/- mice were grossly normal in appearance. However, a small (13.3%) but significant (p < 0.05) decrease in their body weight was observed compared with the wild-type mice. LAR-/- mice also had 86% lower fasting plasma insulin levels (p < 0.01) and 32% lower fasting IGF-1 (p < 0.01) levels compared with the wild-type mice. However, no significant difference was observed in glucose (fasting or postprandial), cholesterol, triglyceride, free fatty acid, tumor necrosis factor-{alpha}, or adiponectin levels of LAR-/- and wild-type mice. Absence of LAR also had no significant effect on systolic or mean blood pressure of mice. These data indicate that LAR deficiency enhances insulin/IGF-1 sensitivity in mice.


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  		TABLE 1
Plasma metabolite, hormone, and cytokine levels and blood pressure in wild-type and LAR–/– mice at 12 weeks of age

All data were obtained from fasting animals except for postprandial glucose and blood pressure. Data are mean ± S.D. (n = 7 for wild-type and n = 8 for LAR–/– mice).

 
Serum- and IGF-1-induced Proliferation and IGF-1-induced Migration Are Enhanced in VSMC Lacking LAR—Because the "gene trap" method used to generate LAR-deficient mice may form "leaky" mutations resulting in a low level expression of the target gene product (24), we first examined LAR expression in VSMC from wild-type and LAR-deficient mice. LAR was highly expressed in wild-type VSMC as determined by Western analysis (Fig. 1A). Similar to lung and liver tissue (27), VSMC from LAR-deficient mice had no detectable expression of this protein. Based on these data, we considered VSMC from these mice to be deficient in LAR. To investigate the effects of LAR deficiency on VSMC function, we compared the phenotypes of wild-type and LAR-/- VSMC. Determination of cell counts under normal growth conditions showed a significant increase in cell proliferation with time in both the genotypes (p < 0.0001) (Fig. 1B). However, cell proliferation was significantly greater (p = 0.0003) in LAR-/- VSMC compared with wild-type VSMC.

The expression of insulin receptor is either very low or absent in VSMC, and the effects of both insulin and IGF-1 are mediated via IGF-1 receptor (28). IGF-1 stimulates VSMC proliferation and migration, two critical determinants of the extent and character of neointima formation following arterial injury (29). Therefore to understand the effect of IGF-1 on the phenotype of LAR-deficient VSMC, we then measured DNA synthesis and cell migration in wild-type and LAR-/- VSMC. As shown in Fig. 1C, physiological concentration of IGF-1 (100 ng/ml) (30, 31) induced a 3.52- and a 6.71-fold increase in DNA synthesis as measured by thymidine uptake in wild-type and LAR-/- VSMC compared with their respective controls (p < 0.001). Thymidine uptake was also significantly higher (p < 0.001) in LAR-/- VSMC compared with wild-type VSMC following IGF-1 treatment. In contrast, thymidine uptake was not significantly different between wild-type and LAR-/- VSMC following treatment with PDGF-BB (Fig. 1D). IGF-1-induced thymidine uptake in both wild-type and LAR-/- VSMC was significantly inhibited by LY294002 and U0126 (p < 0.001 for each compared with the respective IGF-1-treated cells), inhibitors of PI3K and MEK (MAPK/ERK kinase), respectively (Fig. 1C). These inhibitors per se had no significant effect on DNA synthesis.


Figure 4
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  		FIGURE 4.
LAR associates with IGF-1R. A, equal amounts of GST or GST-C-LAR fusion protein were immobilized on glutathione-Sepharose beads and then incubated with VSMC lysates containing an equal amount of protein. GST or GST-C-LAR-associated proteins were analyzed by Western blotting using anti-IGF-1Rbeta antibody. B, the same membrane was reprobed with anti-GST antibody. C, mouse aortic VSMC were infected with Ad-LAR or Ad-betagal. Growth-arrested cells were stimulated with IGF-1 (100 ng/ml) for 10 min and cell lysates containing equal amounts of protein were immunoprecipitated with anti-IGF-1Rbeta antibody and Western (WB) analysis was performed using anti-LAR polyclonal antibody that recognizes cytosolic LAR. D, the same membrane was re-probed with anti-IGF-1Rbeta antibody. E, Western analysis of adenovirus-infected VSMC lysates with anti-LAR polyclonal antibody. F, human aortic VSMC were stimulated with IGF-1 (100 ng/ml) for 10 min and cell lysates containing equal amounts of protein were immunoprecipitated with anti-IGF-1Rbeta antibody and Western analysis was performed using anti-LAR antibody that recognizes cytosolic LAR (upper panel). The same membrane was re-probed with anti-IGF-1Rbeta antibody (lower panel).

 
Because the phenotype and function of VSMC are influenced by their interaction with extracellular matrix (32), we determined the haptotactic migration of these cells on various extracellular matrix proteins. Both wild-type and LAR-/- VSMC showed greater migration toward vitronectin, laminin, and extracellular matrix gel compared with fibronectin and collagen (p < 0.001 compared with bovine serum albumin, Fig. 2A). We then tested the effect of IGF-1 on haptotactic migration of VSMC toward vitronectin (Fig. 2B). IGF-1 significantly enhanced vitronectin-induced migration of both wild-type and LAR-/- VSMC (p < 0.001), but the migration was greater in LAR-/- VSMC compared with the wild-type cells (p < 0.001). Similar to their effect on thymidine uptake, LY294002 and U0126 significantly attenuated IGF-1-induced haptotactic migration toward vitronectin in both wild-type and LAR-/- VSMC (p < 0.001 for each with the respective IGF-1-treated VSMC). Thus, our results show that absence of LAR PTP leads to increased VSMC proliferation in response to serum and IGF-1 treatment and IGF-1-induced haptotactic migration of VSMC. These results also suggest that the same signaling pathways are involved in IGF-1-induced VSMC proliferation and migration.

IGF-1R Activation Is Enhanced in LAR-/- VSMC Treated with IGF-1—To gain insight into the biochemical mechanisms that underlie enhanced proliferation and migration in LAR-/- VSMC, we measured IGF-1-stimulated IGF-1R autophosphorylation by immunoprecipitation/immunoblotting of IGF-1Rbeta (Fig. 3, A-C). Little or no basal tyrosine phosphorylation of the IGF-1R was present in either wild-type or LAR-/- VSMC. Tyrosine phosphorylation of the IGF-1R increased following IGF-1 treatment in both the cell types. However, the peak and time course of IGF-1R tyrosine phosphorylation was affected in the absence of LAR. IGF-1-induced IGF-1R phosphorylation was 2.0- (p < 0.001), 2.2- (p < 0.001), and 2.6-fold (p < 0.05) higher in LAR-/- VSMC than in wild-type VSMC at 10, 30, and 60 min, respectively. IGF-1-induced IGF-1R phosphorylation was observed at 120 min in LAR-/- VSMC, but not in wild-type VSMC. Thus, the elevated tyrosine phosphorylation of IGF-1R in the absence of LAR may contribute to the enhanced proliferative and migratory phenotype of LAR-/- VSMC.


Figure 5
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  		FIGURE 5.
LAR dephosphorylates activated IGF-1R, but not activated PDGF receptor (PDGFR-beta). A, IGF-1R was immunoprecipitated from lysates of VSMC treated with IGF-1 for 10 min, immobilized on protein A-agarose beads, and incubated with 50 units/ml recombinant LAR. Western (WB) analysis of IGF-1R immunoprecipitates (IP) was performed with either anti-phosphotyrosine antibody or anti-IGF-1R antibody. B, IGF-1R from IGF-1-treated VSMC was immunoprecipitated, immobilized on protein A-agarose beads, and incubated with 50 units/ml LAR at 30 °C for 10 min. Western analysis was performed with the indicated site-specific, anti-phosphotyrosine IR/IGF-1R antibodies. The total IGF-1R levels were analyzed by Western blotting with anti-IGF-1Rbeta antibody. C, PDGFR-beta immunoprecipitated from lysates of VSMC treated with 10 ng/ml PDGF-BB was immobilized on protein A-agarose beads and incubated with 50 units/ml recombinant LAR. Western analysis of PDGFR-beta immunoprecipitates was performed with either anti-phosphotyrosine antibody or anti-PDGFR-beta antibody. All the experiments in the figure were repeated three times with similar results.

 
LAR Binds to and Dephosphorylates Activated IGF-1R—Based on the findings that phosphorylation of IR, an IGF-1R homologue, increases in the absence of LAR and LAR binds directly to IR (9), we hypothesized that LAR also binds directly to IGF-1R. However, it was not possible to utilize standard immunoprecipitation/Western blot experiments to test this hypothesis because anti-LAR COOH-terminal antibody (raised against an epitope mapping within a COOH-terminal cytoplasmic domain of LAR of rat origin) does not recognize mouse LAR (and we were unsuccessful in two attempts to generate mouse anti-LAR antibodies). As an alternative approach, we examined whether IGF-1R in lysates from VSMC treated with or without IGF-1 binds to GST-C-LAR in vitro. As shown in Fig. 4, A and B, Western analysis revealed trace association between C-LAR and IGF-1R in untreated wild-type and LAR-/- VSMC lysates. IGF-1 treatment enhanced the affinity of LAR to IGF-1R and this association was stronger in LAR-/- VSMC than in wild-type VSMC. To confirm the specificity of LAR-IGF-1R binding, we also examined potential interaction between LAR and the PDGF receptor (PDGFR-beta). Lysates of VSMC treated with or without PDGF-BB were incubated with GST-C-LAR and there was no evidence of an association between LAR and the PDGFR-beta by Western analysis (data not shown). We corroborated the binding of LAR to IGF-1R by overexpressing human LAR in mouse VSMC by adenoviral infection (Fig. 4, C-E). Immunoprecipitation with anti-IGF-1R antibody followed by Western analysis with anti-LAR COOH-terminal antibody revealed association of LAR with IGF-1R in basal conditions and enhanced binding in response to IGF-1 treatment. Furthermore, enhanced binding of LAR with IGF-1R was observed in human aortic VSMC treated with IGF-1 (Fig. 4F). Together, these results indicate that LAR associates preferentially and directly with IGF-1R and therefore, IGF-1R is a possible substrate of LAR in vivo.

Next, we examined whether LAR dephosphorylates IGF-1R in vitro. LAR-/- VSMC were treated with IGF-1 for 10 min and IGF-1R in the lysates was immunoprecipitated with anti-IGF-1R antibody and incubated with purified recombinant LAR (New England Biolabs) or vehicle. As shown in Fig. 5A, Western analysis of immunoprecipitates with anti-PY20 antibody indicated that LAR dephosphorylated IGF-1R in a time-dependent manner. IGF-1R tyrosine dephosphorylation was inhibited in the presence of sodium vanadate, a potent inhibitor of tyrosine phosphatases. Next, we assessed the specific activity of LAR against IGF-1R by determining the dose-dependent dephosphorylation of an IGF-1R phosphopeptide (residues 1127-1138). LAR had a specific activity of 116 nmol of Pi/min/mg of protein. We also examined whether dephosphorylated IGF-1R tyrosine residues are homologous to those dephosphorylated in IR using phosphospecific anti-IR/IGF-1R antibodies (Fig. 5B). As reported for 1146 (33) and 1161 tyrosine residues (34) in IR, LAR showed a high specificity in dephosphorylating IGF-1-stimulated homologous tyrosine residues at 1131 and 1135/1136 in IGF-1R. Additionally, LAR strongly dephosphorylated another insulin-stimulated tyrosine residue at 950 in IGF-1R. In contrast to IGF-1R, recombinant LAR did not dephosphorylate PDGFR-beta immunoprecipitated from PDGF-BB-treated LAR-/- VSMC lysates (Fig. 5C). These results support the argument that activated IGF-1R is a possible direct target of LAR in vivo.

LAR Deficiency Enhances IGF-1-induced Cell Signaling Distal to IGF-1R—The intrinsic tyrosine kinase activity of the IGF-1R and subsequent phosphorylation of various intracellular substrates, including IRSs, is initiated by its autophosphorylation upon binding of IGF-1 (15, 36). To determine whether enhanced IGF-1-induced IGF-1R activation in the absence of LAR results in increased downstream signaling, we examined activation of IRS-1 and its association with PI3K and the adaptor molecule Grb2. Immunoprecipitation/Western analysis demonstrated that IGF-1 treatment results in tyrosine phosphorylation of IRS-1 in both wild-type and LAR-/- VSMC. Notably, the increase in IRS-1 phosphorylation was significantly higher in LAR-/- than in wild-type VSMC (p < 0.05) (Fig. 6, A and B). Because increased tyrosine phosphorylation of IRS-1 results in enhanced association with PI3K via its p85 regulatory subunit (37), we examined the presence of PI3K in IRS-1 immunoprecipitates using anti-p85 antibody (Fig. 6, C and D). PI3K-IRS-1 association was increased by 99% in LAR-/- VSMC compared with wild-type cells in response to IGF-1 treatment (p < 0.05). The increased tyrosine phosphorylation of IRS-1 following IGF-1 treatment also led to a 90% increase in association with Grb2 in LAR-/- VSMC compared with the wild-type cells (p < 0.05) (Fig. 6, E and F). We confirmed equal amounts of protein in immunoprecipitates by Western analysis with IRS-1 antibody (Fig. 6G).


Figure 6
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  		FIGURE 6.
LAR expression affects IGF-1-induced insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and its association with PI3K and Grb2. A, growth-arrested VSMC were treated with 100 ng/ml IGF-1 for 10 min and cell lysates were prepared. Cell lysates with equal amounts of protein were immunoprecipitated (IP) with anti-IRS-1 antibody and Western analysis was performed with anti-phosphotyrosine antibody. B, densitometric analysis of IRS-1 phosphorylation (mean ± S.E., n = 3; *, p < 0.05 compared with wild-type VSMC treated with IGF-1). C, anti-IRS-1 immunoprecipitates from VSMC lysates were analyzed by Western blotting with anti-PI3K p85{alpha} antibody. D, densitometric analysis of IRS-1-PI3K p85 complex (mean ± S.E., n = 3; *, p < 0.05 compared with wild-type VSMC treated with IGF-1). E, anti-IRS-1 immunoprecipitates from VSMC lysates were analyzed by Western blotting with anti-Grb2 antibody. F, densitometric analysis of IRS-1-Grb2 complex (mean ± S.E., n = 3; *, p < 0.05 compared with wild-type VSMC treated with IGF-1). G, Western (WB) analysis of IRS-1 immunoprecipitates with anti-IRS-1 antibody.

 
To test whether the increased association of PI3K with tyrosyl-phosphorylated IRS-1 following IGF-1 treatment resulted in increased PI3K activity, we measured the activity of this kinase in IRS-1 immunoprecipitates. PI3K activity was 130% higher in LAR-/- VSMC compared with wild-type cells (p < 0.05; Fig. 7, A and B). Grb2 links tyrosine kinase receptors to mitogen-activated protein kinases (MAPK) via the activation of p21ras (36). To assess the role of enhanced Grb2 binding with IRS-1 in LAR-dependent IGF-1-mediated signaling, we next measured ERK1/2 phosphorylation. ERK1/2 phosphorylation increased significantly in both wild-type and LAR-/- VSMC in response to IGF-1 treatment. As predicted, phosphorylation of these MAPKs was significantly higher in LAR-/- VSMC than in wild-type cells (p < 0.05) (Fig. 7, C and D). Enhanced ERK1/2 activation in LAR-/- VSMC in response to IGF-1 treatment appears specific as no significant difference in the phosphorylation of these kinases was observed between wild-type and LAR-/- VSMC treated with PDGF-BB (Fig. 7, E and F). Together, these results indicate that absence of LAR leads to activation of at least two divergent signaling pathways in VSMC treated with IGF-1.

Mice Lacking LAR Show Enhanced Neointimal Hyperplasia in Response to Injury—We next examined the role of LAR in vivo in the pathogenesis of neointimal hyperplasia. The femoral arteries of male mice (wild-type and LAR-/- mice on DBA/2J background; n = 7 in each group) were injured using a guidewire. We chose to study LAR deficiency on DBA/2J background because these mice are more prone to injury-induced neointimal hyperplasia than C57BL/6J mice (38). Histopathological examination of arterial cross-sections 28 days after injury revealed markedly enhanced neointimal thickening in LAR-/- mice compared with wild-type mice (p < 0.05) (Fig. 8, A-D). Mean intima/media ratios were significantly higher (p < 0.05) in LAR-/- arteries than in wild-type arteries (Fig. 8E). Both neointimal and medial cells were positive for {alpha}-smooth muscle actin in immunohistological staining (Fig. 8, F and G) indicating that the intima was composed of SMC. These results suggest that LAR deficiency increases VSMC proliferation in vivo under pathophysiological conditions.

IGF-1R Phosphorylation Is Increased in the Neointima of LAR-/- Mice—To ascertain the functional relevance of enhanced IGF-1 signaling observed in LAR-/- VSMC in cell culture, we examined IGF-1Rbeta phosphorylation in arterial cross-sections of guidewire-injured femoral arteries (Fig. 9). Markedly enhanced phosphorylation of IGF-1Rbeta was observed in the neointima of LAR-/- mice compared with wild-type mice. These results further support the notion that LAR plays an important role in IGF-1-mediated regulation of cell proliferation and migration of VSMC.


Figure 7
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  		FIGURE 7.
LAR expression affects IGF-1-induced PI3K activity and ERK1/2 phosphorylation. A, growth-arrested VSMC were treated with 100 ng/ml IGF-1 for 10 min and cell lysates were prepared. Cell lysates with equal amounts of protein were immunoprecipitated with anti-IRS-1 antibody and the PI3K activity in the immunocomplexes was measured by immunocomplex kinase assay using phosphatidylinositol as a substrate. Phospholipids were separated by thin layer chromatography and [32P]phosphate incorporation into phosphatidylinositol 1,4,5-trisphosphate (PIP) was visualized by autoradiography. B, densitometric analysis of PIP levels (mean ± S.E., n = 3; *, p < 0.05 compared with wild-type VSMC treated with IGF-1). C, after IGF-1 treatment, VSMC lysates were analyzed by Western blotting with either anti-phosphospecific ERK1/2 or anti-ERK1/2 antibody. D, densitometric analysis of normalized ERK1/2 phosphorylation (mean ± S.E., n = 3; *, p < 0.05 compared with wild-type VSMC treated with IGF-1). E, after treatment with PDGF-BB (10 ng/ml), VSMC lysates were analyzed by Western blotting with either anti-phosphospecific ERK1/2 or anti-ERK1/2 antibody. F, densitometric analysis of normalized ERK1/2 phosphorylation (mean ± S.E., n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
An enormous emphasis has been placed on the therapeutic potential of inhibiting abnormal protein phosphorylation for a variety of disease processes. Nearly all of these studies have focused solely on the function of PTKs (39). The potential role of PTPs, the endogenous inhibitors of PTKs, has yet to be investigated, particularly for vascular disease. In the present report, we characterize the in vitro and in vivo phenotype of VSMC derived from mice that are deficient in LAR PTP. Our data indicate that this receptor PTP plays an important role in modulating VSMC proliferation and migration in response to IGF-1 treatment and vascular injury. Whereas other studies have emphasized the importance of PTKs in these processes (40), the present data underline the importance of PTPs in modulation of VSMC phenotype.

LAR is known to have a direct impact on insulin signaling. It is widely expressed in insulin-sensitive tissues (7) and increased association of LAR with IR occurs after insulin treatment (9). Antisense knockdown of LAR enhanced insulin signaling in rat hepatoma cells (6), whereas overexpression of this PTP in skeletal muscle suppressed insulin receptor signaling and induced insulin resistance in mice (41). In contrast, down-regulation of LAR by small interfering RNA induced postreceptor insulin resistance in HEK293 cells (42). However, strong evidence exists for the role of LAR in the pathogenesis of insulin resistance, 1) increased LAR expression and activity were observed in the adipose tissue (7) and skeletal muscle (8) of obese humans; and 2) improved sensitivity to insulin following weight loss in obese subjects is accompanied by decreased expression and activity of LAR (43). Consistent with this, LAR-/- mice in the present study had a significant decrease in body weight, fasting plasma insulin, and IGF-1 levels compared with wild-type mice. Our results show that association of LAR with IGF-1R is enhanced in response to IGF-1 treatment and IGF-1R is a substrate for LAR in vitro. Furthermore, LAR preferentially dephosphorylates IGF-1R tyrosine residues homologous to those dephosphorylated by LAR in IR (33, 34) and absence of LAR enhances IGF-1R tyrosine phosphorylation, which suggest that LAR is a negative regulator of IGF-1 signaling in VSMC.

Our data also indicate that LAR is an important regulator of VSMC proliferation and migration. Consistent with published reports (44), enhanced IGF-1R phosphorylation in response to IGF-1 treatment in LAR-/- VSMC led to increased phosphorylation of adaptor protein, IRS-1, and its association with another docking protein, Grb2. It is well documented that binding of tyrosyl phosphorylated IRS-1 to the SH2 domain of p85 activates PI3K (37) and formation of IRS-1-Grb2 complex leads to the stimulation of MAP kinases (36). Our data are in agreement with these reports as we have observed increased activation of PI3K and ERK1/2 in both wild-type and LAR-/- VSMC treated with IGF-1. Selective inhibitors of PI3K and MEK (an upstream kinase of ERK1/2) inhibited IGF-1-induced thymidine uptake and significantly attenuated IGF-1-induced haptotactic migration of VSMC toward vitronectin in both wild-type and LAR-/- VSMC (Fig. 1, C and E). These results indicate that both PI3K and ERK1/2 are involved in IGF-1-mediated effects on growth and migration in VSMC. Furthermore, IGF-1-induced PI3K and ERK1/2 activation are significantly higher in LAR-/- VSMC than in wild-type VSMC and these increases are positively correlated with higher thymidine uptake and migration of LAR-/- VSMC compared with wild-type cells following IGF-1 treatment. These results indicate that protein tyrosine dephosphorylation by LAR, which terminates IGF-IR activation induced by IGF-1, regulates downstream signaling pathways that have pathophysiological significance.


Figure 8
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  		FIGURE 8.
Absence of LAR enhances neointima formation in response to vascular injury. A-D, representative cross-sections of femoral arteries from wild-type and LAR-/- mice, 28 days after femoral arterial injury, were stained with Masson trichrome-elastic stain. E, morphometric analysis of femoral artery sections (mean ± S.E., n = 7, *, p < 0.001 compared with wild-type injured; @, p < 0.001 compared with the respective control). F and G, cross-sections were stained for smooth muscle {alpha}-actin. Magnification for A-G, x200.

 
Enhanced IGF-1-induced PI3K activation, in the absence of LAR, may lead to activation of downstream effectors such as the PKB/Akt pathway and phosphorylation of glycogen synthase kinase-3beta (45). Phosphorylation of glycogen synthase kinase-3beta inhibits its activity and results in the accumulation of beta-catenin in the cytosol and its subsequent translocation to the nucleus where it forms a complex with T-cell transcription factor/lymphoid-enhancer binding factor and activation of Wnt target genes involved in VSMC proliferation (46). It has been proposed that tyrosine phosphorylation of the cadherin-catenin complex regulates the stability of adherens junctions (47). Increased tyrosine phosphorylation of beta-catenin results in the inhibition of cadherin-mediated adhesion (48) and may result in increased migration. This is further supported by the observation that beta-catenin is a substrate for LAR in vitro and that ectopic expression of LAR inhibits cell migration induced by growth factors via negative regulation of beta-catenin tyrosine phosphorylation (49). In addition, tyrosine phosphorylation of beta-catenin may disrupt the interaction of the E-cadherin complex with the actin cytoskeleton (50, 51). The subsequent increase in free cytoplasmic levels of beta-catenin may lead to nuclear translocation and activation of beta-catenin responsive genes and increase in cell proliferation (51). The notion that tyrosine phosphorylation of beta-catenin may cause increased growth and motility is supported by the observations that IGF-1 causes nuclear translocation of beta-catenin (52), its translocation is enhanced in LAR-/- VSMC compared with wild-type cells in response to IGF-1 treatment,3 and beta-catenin is involved in growth factor-induced VSMC proliferation (46).


Figure 9
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  		FIGURE 9.
Absence of LAR enhances IGF-1Rbeta activation in neointima. Representative cross-sections of femoral arteries from wild-type and LAR-/- mice, 28 days after femoral arterial injury, were stained for the presence of phospho-IGF-1Rbeta (upper panel). PBS was substituted for the primary antibody in the negative control (lower panel).

 
Based on our data with regards to IGF-1-induced proliferation and migration of VSMC from LAR-/- mice, we hypothesized that absence of LAR might modulate medial VSMC proliferation after arterial injury. Vascular injury causes the release of many growth factors and cytokines including IGF-1, which cause pathological vascular growth (54). Moreover, overexpression of IGF-1 in mouse VSMC was shown to enhance neointima formation in response to arterial injury (53). It was reported that overexpression of PTEN, a lipid phosphatase that shares sequence homology but not functional homology with PTPs and is an endogenous inhibitor of PI3K in VSMC, also inhibited neointimal hyperplasia (35). Our current data that absence of LAR, a negative regulator of IGF-1 signaling, results in enhanced neointima formation in the femoral artery in response to guidewire injury (Fig. 8) is consistent with these observations. Similar to increased IGF-induced signaling in LAR-/- VSMC compared with wild-type cells, increased IGF-1R phosphorylation was observed in injury-induced neointima of LAR-/- mice compared with that in wild-type mice (Fig. 9). In addition, our preliminary observation that LAR is down-regulated in human atherectomy arteries with restenosis (data not shown) further suggests that LAR is a critical regulator of VSMC growth.

In conclusion, this is the first demonstration that LAR negatively regulates IGF-1-mediated migration and proliferation of VSMC and that deficiency in LAR expression markedly increases the development of neointima. Thus, whereas expression of LAR in VSMC protects against restenosis, enhanced expression of this PTP in adipose and skeletal muscle tissue contributes to the pathogenesis of insulin resistance. PTP inhibitor development is an active area of research by many pharmaceutical companies for treatment of obesity and diabetes. Our data underscore the complex and distinct roles individual PTPs can play in regulating multiple signaling pathways simultaneously in vivo and the opposing effects PTP inhibitors could have on insulin resistance and restenosis.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant HL-57352. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 7312B Medical Biomolecular Research Bldg., University of North Carolina, Chapel Hill, NC 27599-7126. Tel.: 919-843-4584; Fax: 919-966-1012; E-mail: nrmadama{at}med.unc.edu.

2 The abbreviations used are: VSMC, vascular smooth muscle cell; PTK, protein-tyrosine kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PTP, protein-tyrosine phosphatase; LAR, leukocyte antigen-related protein; IR, insulin receptor; PI3K, phosphatidylinositol 3-kinase; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GST, glutathione S-transferase; PBS, phosphate-buffered saline. Back

3 J. Li, B. S. Mandavilli, M. S. Runge, and N. R. Madamanchi, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Vishram Kedar, University of North Carolina at Chapel Hill, for providing GST-C-LAR construct.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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