Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs

Asha Jacob1, Jeffery D. Molkentin3, Albert Smolenski4, Suzanne M. Lohmann4, and Najma Begum1,2

1 The Diabetes Research Laboratory, Winthrop University Hospital, Mineola 11501 and 2 School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794; 3 Division of Molecular Cardiovascular Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 4 Institut für Klinische Biochemie und Pathobiochemie, Medizinische Universitätsklinik, D97080 Würzburg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the role of insulin in the control of vascular smooth muscle cell (VSMC) migration in the normal vasculature. Platelet-derived growth factor (PDGF) increased VSMC migration, which was inhibited by pretreatment with insulin in a dose-dependent manner. Insulin also caused a 60% decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK) phosphorylation and activation. Insulin inhibition of MAPK was accompanied by a rapid induction of MAPK phosphatase (MKP-1), which inactivates MAPKs by dephosphorylation. Pretreatment with inhibitors of the nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase Ialpha (cGK Ialpha ), the downstream effector of cGMP signaling, blocked the activation of MAPK and prevented PDGF-directed VSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPK phosphorylation. We conclude that insulin inhibition of VSMC migration may be mediated in part by NO/cGMP/cGK Ialpha induction of MKP-1 and consequent inactivation of MAPKs.

nitric oxide; guanosine 3',5'-cyclic monophosphate; cGMP-dependent protein kinase Ialpha ; platelet-derived growth factor; hypertension


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN PROFOUNDLY INFLUENCES the function of the vasculature in both normal and pathological conditions (1, 21, 26-28). In normal individuals, physiological concentrations of insulin stimulate nitric oxide (NO) generation by the endothelium, which promotes vasodilation and increases blood flow (26, 28). The vasorelaxant effects of insulin are impaired in individuals who are also resistant to metabolic actions of insulin (29). Although associations between vascular disease and insulin-resistant states of obesity, non-insulin-dependent diabetes mellitus, and essential hypertension have been firmly established, the molecular basis of this association is unclear.

Using vascular smooth muscle cells (VSMCs) isolated from control rats, we (7) and others (32) have recently demonstrated that acute exposure to physiological concentrations of insulin causes a very small transient effect on mitogen-activated protein kinase (MAPK) activity with a rapid return to basal levels within 30 min. This was accompanied by a rapid increase in induction of MAPK phosphatase-1 (MKP-1) expression, which inactivates MAPKs by dephosphorylation (5, 7, 32). In contrast, insulin resistance associated with hypertension and diabetes is accompanied by impaired induction of MKP-1 expression by insulin and elevated insulin-stimulated MAPK activation and cell proliferation (5, 7). Furthermore, chronic treatment with insulin or high glucose stimulates MAPKs and promotes VSMC growth that is accompanied by impaired inducible NO synthase (iNOS) and MKP-1 expression (4, 32).

The migration of VSMCs from the arterial media to the intima is a crucial pathogenic event in the formation and progression of atherosclerosis and restenosis (25). A variety of growth factors [for example, platelet-derived growth factor (PDGF)], cytokines, and proteases mediate VSMC migration and are known to be present at sites of vascular injury (2, 9, 10, 14, 17, 24, 34). However, although many chemoattractants have been identified in the vasculature, relatively little is known about the intracellular pathways involved in cell movement and directed migration under normal conditions as well as those stimulated by hyperinsulinemia.

Several investigators (12, 16, 22) have studied the effects of insulin on VSMC migration, but the results are controversial. Grotendorst et al. (16) reported that 150 nM insulin does not affect the migration of cultured VSMCs from bovine aorta. Nakao et al. (22) reported that physiological concentrations of insulin do not affect the acute migration of cultured rat aortic VSMCs. However, if the cells are pretreated with insulin for 5 days, the migration of cells stimulated by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid is increased. Gockerman et al. (12) reported that supraphysiological concentrations of insulin (300 nM) stimulate the migration of porcine aortic smooth muscle cells. This concentration of insulin can stimulate insulin-like growth factor-1 receptors, which are known to mediate VSMC migration (8). Studies by Kahn et al. (18) have shown that low concentrations of insulin can inhibit basal migration of canine aortic VSMCs only when iNOS is induced by lipopolysaccharide and interleukin (IL)-1beta . Whether insulin antagonizes the effect of mitogens on migration was not studied.

Because physiological concentrations of insulin are known to limit cell proliferation and promote relaxation of VSMCs isolated from control rats via NO/cGMP (3, 7), in this study our goal was to test whether insulin antagonizes PDGF-directed VSMC migration via NO/cGMP-dependent mechanism by inactivating MAPKs that are implicated in PDGF-directed migration.

We found that insulin inhibits PDGF-directed migration of control VSMCs partly by inactivating MAPKs via the induction of MKP-1 expression. The NO/cGMP signaling pathway mediates insulin inhibition of VSMC migration via MKP-1 expression. Inhibition of NO/cGMP signaling as well as expression of MKP-1 antisense RNA blocks insulin-induced MKP-1 expression and abolishes the inhibitory effects of insulin on MAPK activation and VSMC migration, whereas adenoviral expression of MKP-1 or cGMP-dependent protein kinase Ialpha (cGK Ialpha ), the downstream effector of the NO/cGMP signaling pathway, enhances the inhibitory effects of insulin on PDGF-directed VSMC migration.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. PDGF-BB, cell culture reagents, fetal bovine serum, and antibiotics were purchased from Life Technologies (Grand Island, NY). [gamma -32P]ATP (specific activity >= 3,000 Ci/mmol) was from NEN (Boston, MA). The Rp diastereomer of 8-bromoguanosine 3',5'-cyclic monophosphothioate (Rp-8-BrcGMPS), 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), and NG-monomethyl-L-arginine (L-NMMA) were from Biomol Research (Plymouth Meeting, PA). Electrophoresis and protein assay reagents were from Bio-Rad (Richmond, CA). Okadaic acid was from Moana Bioproducts (Honolulu, Hawaii). Type 1 collagenase was from Worthington Biochemical (Freehold, NJ). SDS/polyacrylamide gel electrophoresis and Western blot reagents were from Bio-Rad (Hercules, CA). Anti-extracellular signal-regulated kinase 2 (ERK2) and MKP-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-MAPK and MAPK antibodies were from New England Biolabs (Beverly, MA). Protein A-Sepharose CL-4B, protease inhibitors, sodium orthovanadate, and all other reagents were from Sigma Chemical (St. Louis, MO). Porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN).

Culture of VSMCs and treatment. VSMCs were isolated by enzymatic digestion of the aortic media of male Wistar Kyoto rats weighing 200-220 g, as described in our recent publications (4, 5, 7). A monoclonal antibody against smooth muscle alpha -actin was used to assess the purity (>99%) of the smooth muscle cell cultures. Unless otherwise indicated, primary cultures of VSMCs were maintained in alpha -MEM containing 10% FBS and 1% antibiotic/antimycotic mixture. All experiments on MAPK activation, MKP-1 expression, and cell migration were performed on confluent cells (5-7 days in culture) at passage 5. Before each experiment, cells were serum-starved for 24 h in alpha -MEM containing 5.5 mM glucose and 1% antibiotics. The next day, cells were exposed to PDGF-BB (0-10 ng/ml), insulin (0-100 nM), or insulin followed by PDGF as indicated. In some experiments, VSMCs were pretreated with various inhibitors for 30 min before insulin and subsequent exposure to PDGF, as indicated.

Cell migration assay. Migration assays were performed by using 24-well cell culture inserts with 8.0-µm polyethylene terephthalate Cyclopore membranes (Falcon) as detailed in Lundberg et al. (19). The underside of the membrane was coated with 10 µl of rat tail collagen type I (50 µg/ml) for 18-20 h and was washed and air-dried before each experiment. Serum-starved VSMCs were trypsinized and resuspended in alpha -MEM. Then 2 × 104 VSMCs/250 µl were treated with 10-100 nM insulin or 100 µM 8-BrcGMP for 30 min, and all was subsequently loaded into the cell culture inserts. The inserts were then added to the wells of 24-well plates, which were filled with PDGF diluted in alpha -MEM with 0.1% BSA, and insulin or 8-BrcGMP was also added to this medium below the inserts. In some experiments, VSMCs were exposed to 1 mM L-NMMA and 100 µM Rp-8-BrcGMPS for 30 min before insulin exposure. The inhibitors and insulin were added to the lower chamber as well. The chambers were then incubated at 37°C for 5 h for cell migration. Afterward, cells were completely removed from the upper side of the membrane with a cotton swab and cells migrated to the underside of the membrane were fixed and stained with Diff-Quik solution (Dade Behring). The inserts were then examined under ×400 magnification, and five to six different fields were counted.

Immunoprecipitation and assay of MAPK activity. Confluent, serum-starved VSMCs were exposed to insulin (1-100 nM) for 10-30 min followed by PDGF (1-10 ng/ml) for 10 min. In some experiments, cells were pretreated with inhibitors before treatment with insulin. The cells were then rinsed in ice-cold PBS containing 1 mM sodium orthovanadate and extracted with 50 mM HEPES buffer (pH 7.5) containing 250 mM sucrose, 4 mM EDTA, 2 mM dithiothreitol, 0.5% Triton X-100, and a mixture of phosphatase and protease inhibitors as detailed in our recent publication (7). Cell lysates were prepared by sonication and centrifuged at 16,000 g at 4°C for 20 min. Equal amounts of precleared lysate proteins (100 µg) were immunoprecipitated with 5 µg of anti-ERK2 antibody. The immunoprecipitates were assayed for MAPK activity by using myelin basic protein (MBP) as a substrate (7). Because ERK2 isoform is abundant in VSMCs, antibody specific for ERK2 was used in these experiments.

Immunoblot analysis of MAPK phosphorylation status. Confluent, serum-starved VSMCs treated and extracted as described in Immunoprecipitation and assay of MAPK activity were analyzed by Western Blot. Briefly, equal amounts of protein (25-50 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed with phospho-p44/42 MAPK antibody or p44/42 MAPK antibody, and detected using horseradish peroxidase-conjugated secondary antibody (7).

Retrovirus-mediated expression of MKP-1 in VSMCs. Plasmids carrying MKP-1 were subcloned into the EcoRI site of the retroviral vector pBabe, carrying a puromycin resistance marker. The resulting constructs with MKP-1 in the sense and antisense orientation were verified by restriction enzyme analyses as well as by automated DNA sequencing. pBabe-MKP-1 sense and pBabe-MKP-1 antisense constructs were introduced into a retroviral packaging cell line, LE (a derivative of Bosc23; kindly given by Dr. G. Hannon, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY) by transfection using Lipofectamine Plus reagent according to the manufacturer's instructions. The next day, cells were fed with complete growth medium containing 5 mM sodium butyrate and 1 µM dexamethasone and then incubated at 32°C. Culture supernatants containing the retroviral recombinant virus particles expressing MKP-1 sense and MKP-1 antisense RNA were collected 48 h posttransfection and used for infection of VSMCs.

Briefly, overnight cultures of VSMCs (2 × 104 cells/well at passage 3) were infected with filtered retroviral supernatant and polybrene (8 µg/ml) in 2 ml of growth medium. The culture plates were centrifuged at 480 g in a Beckman table top centrifuge (Allegra 6) for 1 h at room temperature, incubated overnight at 32°C, and then transferred to 37°C. At the end of 48 h, VSMCs were trypsinized and plated into five 100-mm dishes containing 2 µg/ml puromycin to generate stably expressing clones. A pool of stable clones overexpressing MKP-1 and antisense MKP-1 RNA were amplified and used for functional assays to examine the impact of MKP-1 overexpression and MKP-1 depletion on MAPK activation status and agonist-stimulated VSMC migration.

Construction of adenoviral vectors expressing cGK Ialpha and MKP-1. The adenoviral vector for expressing cGK Ialpha was constructed by first cloning the cDNA of human cGK Ialpha into the multiple cloning site of the adenoviral transfer plasmid pCMVI/Delta E1sp1A, itself generated by cloning the expression cassette of the pCI expression vector (Promega, Madison, WI) into the plasmid pDelta E1sp1A containing NH2-terminal E1-deleted sequences of Ad5 (Microbix Biosystems, Toronto, Canada). The resulting plasmid pCMVI/cGK Ialpha was then cotransfected with pJM17, a plasmid containing the full-length genome of replication-deficient type 5 adenovirus (Microbix), into the Ad5-transformed human embryonic kidney cell line HEK-293. E1A-deficient recombinant virus (Ad5-cGK Ialpha ) was generated via homologous recombination between pCMVI/cGK Ialpha and pJM17, screened for by using the polymerase chain reaction, and then recovered and plaque-purified as described previously for Ad5-cGK Ibeta (33). MKP-1- and beta -galactosidase-expressing adenoviruses were generated by cotransfection of plasmid pACCMVpLpA-MKP-1 or pACCMVpLpA-beta -galactosidase with pJM17 in HEK-293 cells as detailed recently (15). Each adenovirus was plaque-purified, expanded, and titered after detection of visible plaques in a HEK-293 monolayer by agarose overlays.

Protein assay. Protein in cellular lysates was quantitated by the bicinchoninic acid method (30).

Statistics. Results are presented as means ± SE. Analysis of variance was used to compare the mean values between various treatments. A P value of <0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin inhibits PDGF-directed migration. Exposure of confluent serum-starved VSMCs to PDGF-BB (10 ng/ml) resulted in a twofold increase in VSMC migration compared with untreated controls (Fig. 1A). An exposure to 100 nM insulin for 30 min before exposure to PDGF-BB caused marked reductions in PDGF-directed VSMC migration. Insulin treatment alone did not affect VSMC migration (Fig. 1A). PDGF-directed VSMC migration was dose dependent with a half-maximal effect observed at 0.25 ng/ml PDGF and a maximal effect at 10 ng/ml PDGF (Fig. 1B). Insulin concentrations of 10 and 100 nM caused 74 and 82% decreases, respectively, in VSMC migration stimulated by 10 ng/ml PDGF. A lower concentration of insulin (1 nM) also decreased PDGF-directed migration (data not shown).


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Fig. 1.   Insulin inhibits platelet-derived growth factor (PDGF)-directed migration of vascular smooth muscle cells (VSMCs). A: confluent serum-starved VSMCs were trypsinized and incubated for 30 min at 37°C with or without 100 nM insulin and then added to the transwell chambers in 24-well tissue culture plates at a density of 2 × 104 cells/well. The lower chamber contained 0.75 ml of alpha -MEM-0.1% BSA (control) with 10 ng/ml PDGF-BB (PDGF) alone, 100 nM insulin (insulin) alone, or 100 nM insulin plus 10 ng/ml PDGF (Insright-arrowPDGF). Incubation was continued for 5 h at 37°C. The number of cells that migrated to the lower surface of the transwell membrane was counted from 5 to 6 different high-power fields at ×400 magnification. Results are shown as the mean degree of increase + SE (n = 5). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF. B: insulin-induced inhibition of PDGF-directed VSMC migration is dose dependent. VSMCs were pretreated with 10 or 100 nM insulin and exposed to various concentrations of PDGF in the cell migration assay. Results are shown as mean degree of increase + SE (n = 3) of basal value. Treatment with 1 nM insulin also inhibited PDGF (10 ng/ml)-directed migration by 30%.

Insulin inhibits PDGF-induced MAPK phosphorylation and activation. Activation of MAPK pathway is considered critical for agonist-induced VSMC migration (2, 14) because inhibition of MAPKs by PD-98059 prevents PDGF-directed VSMC migration as well as growth (20, 23). Therefore, we next examined whether insulin affects PDGF-stimulated MAPK activation to cause inhibition of PDGF-directed VSMC migration. In these experiments, serum-starved VSMCs were exposed to insulin (1-100 nM) for 30 min, followed by treatment with and without PDGF (10 ng/ml ) for 10 min. VSMC lysates were examined for MAPK phosphorylation and enzymatic activity as detailed in MATERIALS AND METHODS. Treatment with 10 ng/ml PDGF for 10 min increased MAPK phosphorylation ninefold (Fig. 2A, top and bottom). Preexposure to 100 nM insulin for 30 min caused a 70% decrease in PDGF-induced MAPK phosphorylation. Insulin treatment alone for 30 min did not affect basal MAPK phosphorylation (Fig. 2A). Half-maximal inhibition of PDGF-induced MAPK phosphorylation was observed at 1 nM insulin and maximal inhibition at 100 nM insulin (Fig. 2B, top and bottom). The increase in MAPK phosphorylation in PDGF-treated VSMCs was accompanied by an eightfold increase in MAPK enzymatic activity assayed in ERK2 immunoprecipitates (Fig. 2C). As also observed in Fig. 2, A and B, prior exposure to insulin resulted in a >60% decrease in PDGF-induced MAPK activation (Fig. 2C). Insulin alone had very little effect on MAPK enzymatic activity (Fig. 2C). Kinetic analyses revealed that a 10-min preincubation with 100 nM insulin effectively reduced PDGF-induced MAPK phosphorylation and enzymatic activity, and the inhibitory effect of insulin was sustained for the 30-min period tested (data not shown). The inhibition of MAPK by insulin was accompanied by rapid induction of MKP-1 expression in a time- and dose-dependent manner (Fig. 2D).


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Fig. 2.   Insulin inhibits PDGF-induced mitogen-activated protein kinase (MAPK) phosphorylation and activation. A: confluent, serum-starved VSMCs were exposed to 100 nM insulin for 30 min followed by 10 ng/ml PDGF for 10 min. Equal amounts of cell lysate proteins were analyzed by Western blot using phospho-p44/42MAPK antibody (T202/Y204; pMAPK) and MAPK antibody (MAPK). A representative autoradiogram from 5 experiments is shown (top). Autoradiograms from independent experiments were scanned, and the ratio between MAPK phosphorylation and MAPK protein was determined and plotted (bottom). Results are means + SE (n = 5). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF. B: VSMCs were exposed to 1, 10, and 100 nM insulin for 30 min followed by 10 ng/ml PDGF for 10 min. A representative autoradiogram (top) and the ratio between MAPK phosphorylation and MAPK (bottom) are shown. Results are means + SE (n = 3). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF. C: an equal amount of precleared cell lysate proteins (100 µg) of samples from A were immunoprecipitated with anti-extracellular signal-regulated kinase 2 (ERK2) antibody, and MAPK activity was assayed using myelin basic protein (MBP) as a substrate. Results are means + SE (n = 5). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF. D: insulin inactivation of MAPK was accompanied by induction of MAPK phosphatase (MKP)-1 expression. Equal amounts of cell lysate protein (25 µg) were subjected to Western blot analysis with anti-MKP-1 antibody. A representative autoradiogram from 4 experiments is shown.

The NO/cGMP pathway mediates the inhibitory effects of insulin on MAPK phosphorylation and VSMC migration via MKP-1 expression. We have previously reported that insulin rapidly induces iNOS expression and cGMP generation in VSMCs (5). Furthermore, blocking the NO/cGMP pathway with L-NMMA (1 mM) or Rp-8-BrcGMPS (100 µM) inhibits insulin-induced MKP-1 mRNA expression (5). Therefore, we tested the effect of these inhibitors on insulin-mediated inhibition of PDGF-induced MAPK phosphorylation, ERK2 activation, MKP-1 expression, and VSMC migration. Preexposure to L-NMMA and Rp-8-BrcGMPS for 30 min before insulin treatment completely abolished insulin inhibition of MAPK phosphorylation [Fig. 3A, top (compare lanes 6 and 8 with lane 4) and bottom] and restored MAPK phosphorylation levels to those observed when cells were stimulated with PDGF alone [Fig. 3A, top (compare lanes 6 and with lane 2) and bottom]. In contrast, 8-BrcGMP, a cGMP agonist, mimicked the effect of insulin and abolished PDGF-induced MAPK phosphorylation [Fig. 3A, top (lane 9) and bottom]. Inhibitors when added alone had very little effect on MAPK phosphorylation [Fig. 3A, top (lanes and 7) and bottom]. Analyses of MAPK enzymatic activity in ERK2 immunoprecipitates revealed that L-NMMA and Rp-8-BrcGMPS prevented insulin-mediated inactivation of PDGF-induced MAPK enzymatic activity and restored PDGF-induced MAPK activation (Fig. 3B). In contrast, 8-BrcGMP mimicked insulin's effect of inhibiting PDGF-induced MAPK activation (Fig. 3B). The observed effects of NO/cGMP inhibitors on restoration of PDGF-stimulated MAPK phosphorylation and enzymatic activity were accompanied by inhibition of insulin-induced MKP-1 protein expression by these inhibitors (Fig. 3C).


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Fig. 3.   Nitric oxide (NO)/cGMP signaling pathway mediates insulin-induced MKP-1 expression as well as insulin inhibition of PDGF-induced MAPK phosphorylation and MAPK enzymatic activity. A: phospho-MAPK and MAPK expression were examined by Western blot analyses of serum-starved VSMCs treated with and without inhibitors of cGMP (100 µM Rp-8-BrcGMPS) and NO synthase [1 mM NG-monomethyl-L-arginine (L-NMMA)] for 30 min, followed by insulin for 10 min and subsequent exposure to PDGF for 10 min, or treated with 8-BrcGMP (100 µM) for 30 min followed by PDGF for 10 min (top). The ratio between MAPK phosphorylation and MAPK protein is shown (bottom). Results are means + SE (n = 3). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF; *** P < 0.05 vs. Insright-arrowPDGF. B: equal amounts of cell lysate proteins (100 µg) from A were immunoprecipitated with anti-ERK2 antibody, and MAPK activity was assayed using MBP as a substrate. Results are means + SE (n = 3). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF; *** P < 0.05 vs. Insright-arrowPDGF. C: inhibition of NO/cGMP signaling pathway prevented insulin-induced MKP-1 expression. Equal amounts of proteins (25 µg) from A were examined for MKP-1 expression by Western blot analysis with MKP-1 antibody.

We next examined the effect of inhibitors of the NO/cGMP signaling pathway on VSMC migration. As shown in Fig. 4, a 30-min preincubation with L-NMMA or Rp-8-BrcGMPS before exposure to insulin abolished insulin inhibition of PDGF-directed VSMC migration. In contrast, pretreatment with 8-BrcGMP prevented PDGF-directed VSMC migration. The magnitude of inhibition of VSMC migration by 8-BrcGMP was comparable to that of insulin (Fig. 4).


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Fig. 4.   NO/cGMP pathway mediates insulin inhibition of PDGF-stimulated VSMC migration. Confluent serum-starved VSMCs were trypsinized, resuspended in alpha -MEM, counted, and used in cell migration assays as detailed in Fig. 1. In some experiments, cells were pretreated with inhibitors of cGMP (100 µM Rp-8-BrcGMPS) and L-NMMA (1 mM) for 30 min before treatment with 100 nM insulin for 30 min. Results are mean degree of increase over control + SE (n = 5). * P < 0.05 vs. control; ** P < 0.05 vs. PDGF; *** P < 0.05 vs. Insright-arrowPDGF.

Expression of MKP-1 inhibits PDGF-induced MAPK activation and VSMC migration. To clearly understand the role of MKP-1 in insulin inactivation of PDGF-stimulated MAPKs and migration, we infected VSMCs with adenovirus carrying either beta -galactosidase (Ad.beta -gal) or MKP-1 (Ad.MKP-1) at a multiplicity of infection (MOI) of 5-25 plaque-forming units (pfu)/cell. Infection with Ad.MKP-1 caused a dose-dependent increase in MKP-1 protein expression that was maximal at an MOI of 15-20 pfu/cell compared with uninfected VSMCs (Fig. 5A). Adenovirus-mediated MKP-1 expression caused a dose-dependent decrease in PDGF-induced MAPK phosphorylation (Fig. 5B, top and bottom). Ad.MKP-1 at an MOI of 10 pfu/cell caused a 45% decrease in PDGF-induced MAPK phosphorylation. A 73% inhibition of PDGF-induced MAPK phosphorylation was observed at an MOI of 20 pfu/cell. All further experiments on MKP-1 expression were performed at an MOI of 20 pfu/cell. Adenoviral expression of MKP-1 decreased basal rates of VSMC migration by 25% and abolished PDGF-directed VSMC migration (Fig. 6) in contrast to 73% inhibition of MAPK phosphorylation. Similar results were observed with stable pools of MKP-1-expressing VSMCs generated using retrovirus-mediated gene transfer of MKP-1 (data not shown).


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Fig. 5.   PDGF-stimulated MAPK phosphorylation is inhibited by MKP-1 expression. Confluent VSMCs uninfected or infected with Ad.MKP-1 at various plaque-forming units (pfu)/cell for 24 h at 37°C were serum-starved and treated with 10 ng/ml PDGF for 10 min. Top: protein lysate proteins (50 µg) were analyzed by Western blot using MKP-1 antibody (A), phospho-MAPK (B), and MAPK antibody (C). Bottom: the ratio between MAPK phosphorylation and MAPK protein is shown as mean + SE from triplicate experiments.



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Fig. 6.   MKP-1 inhibits PDGF-directed migration of VSMCs. Confluent VSMCs were infected with 20 pfu/cell Ad.beta -gal or Ad.MKP-1 as detailed in Fig. 3. Serum-starved VSMCs were then trypsinized, resuspended in alpha -MEM, counted, treated with and without insulin and PDGF, and used in cell migration assays. Results are means + SE (n = 4). * P < 0.05 vs. Ad.beta -gal control; ** P < 0.05 vs. Ad.beta -gal PDGF; *** P < 0.05 vs. respective treatment of Ad.beta -gal.

Ad.cGK Ialpha enhances insulin effect on MKP-1 expression and inhibits PDGF-induced MAPK activation and VSMC migration. cGMP-dependent protein kinase (cGK) is a major downstream effector of NO/cGMP signaling pathway. To directly demonstrate the role of the NO/cGMP signaling pathway in insulin inhibition of PDGF-directed VSMC migration, we infected VSMCs with an adenovirus carrying a wild-type cGK Ialpha , a major isoform of cGK I found in VSMCs. The infected VSMCs were serum-starved 24 h later and examined for insulin effect on MKP-1 protein expression and PDGF-induced MAPK activation. As shown in Fig. 7A, cGK Ialpha infection of VSMCs was accompanied by an increase in cGK Ialpha protein expression 10-fold higher than that of uninfected cells. Furthermore, cGK Ialpha expression increased insulin and 8-BrcGMP-induced MKP-1 expression more than fourfold compared with uninfected VSMCs (Fig. 7B). This was accompanied by a 50% decrease in PDGF-stimulated MAPK phosphorylation in VSMCs infected with cGK Ialpha (Fig. 7C) compared with that in noninfected VSMCs (Fig. 7C, lane 2). Most importantly, insulin and 8-BrcGMP, which activate cGK Ialpha (6), decreased PDGF-induced MAPK phosphorylation by 80 and 90%, respectively, in cGK Ialpha -infected cells (Fig. 7C, lanes 6 and 7), whereas in noninfected VSMCs, insulin caused only a 63% decrease in PDGF-induced MAPK phosphorylation (Fig. 7C, lane 3). The observed changes in MAPK phosphorylation were not due to variations in MAPK protein expression (Fig. 7D) but, rather, to an increase in basal as well as insulin/cGMP-mediated expression of MKP-1 protein (Fig. 7B). Thus cGK Ialpha expression enhances insulin-mediated inhibition of MAPK phosphorylation in PDGF-stimulated cells via MKP-1 expression.


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Fig. 7.   cGMP-dependent protein kinase Ialpha (cGK Ialpha ) enhances insulin-mediated inhibition of PDGF-stimulated MAPK phosphorylation. VSMCs were uninfected or infected with adenovirus carrying cGK Ialpha (Ad5.cGK Ialpha ; 1 × 1010 virus/ml) for 2 h followed by addition of 10% FBS. After 24 h, VSMCs were serum-starved, stimulated with various agonists, and examined for cGK Ialpha (A), MKP-1 (B), phospho-MAPK (C), and MAPK expression (D). Autoradiograms show results representative of those obtained in 4 different experiments.

We also examined the effect of cGK Ialpha expression on PDGF-directed VSMC migration. As shown in Fig. 8, MKP-1 expression alone or in combination with cGK Ialpha decreased basal VSMC migration by 20 and 30%, respectively, compared with VSMCs infected with pBabe and completely abolished PDGF-induced migration in contrast to a 2.3-fold increase observed in pBabe. In VSMCs expressing recombinant cGK Ialpha and MKP-1, treatment with insulin or 8-BrcGMP followed by PDGF caused a 40 and 75% decrease, respectively, in basal migration rates compared with basal values of pBabe. Thus elevation of cGK Ialpha levels in cells overexpressing MKP-1 results in reduction of basal migration rates and enhances insulin- and cGMP-mediated inhibitory effects on PDGF-directed VSMC migration.


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Fig. 8.   cGK Ialpha enhances MKP-1-mediated inhibition of PDGF-directed VSMC migration. Cell migration assay was performed in cells infected with retrovirus pBabe, pBabe-MKP-1, Ad5.cGK Ialpha (1 × 1010 virus/ml), or previously infected with pBabe-MKP-1 and then subsequently infected with Ad5.cGK Ialpha (see MATERIALS AND METHODS). Results are means + SE of 3 separate experiments shown as degree of increase vs. basal migration for pBabe. * P < 0.05 vs. pBabe and Ad5.cGK Ialpha ; ** P < 0.05 vs. pBabe, *** P < 0.05 vs. pBabe, Ad5.cGK Ialpha , and pBabe-MKP-1; **** P < 0.05 vs. pBabe, Ad5.cGK Ialpha , and pBabe-MKP-1.

Expression of antisense MKP-1 RNA prevents insulin-mediated inactivation of MAPK phosphorylation by PDGF and restores PDGF-directed VSMC migration. To further confirm that induction of MKP-1 expression by insulin indeed plays a major role in insulin inhibition of PDGF-directed migration via MAPK inactivation, we infected VSMCs with retrovirus particles carrying antisense MKP-1 RNA. A stable pool of VSMCs expressing antisense MKP-1 RNA was amplified, stimulated with and without insulin (100 nM for 30 min) followed by PDGF (10 ng/ml for 10 min), and examined for MKP-1 expression (Fig. 9A), MAPK phosphorylation status (Fig. 9B), and its impact on VSMC migration (Fig. 9C). Compared with VSMCs infected with control pBabe virus, cells infected with a virus carrying antisense MKP-1 RNA exhibited an ~70% inhibition of basal and insulin-stimulated MKP-1 expression (Fig. 9A). When we examined the activation state of MAPKs, we observed that the inhibition of PDGF-stimulated MAPK phosphorylation exerted by insulin in pBabe (Fig. 9B, lane 4) was reverted in MKP-1-depleted cells (Fig. 9B, compare lane 8 with lane 4), suggesting a role for MKP-1 as a mediator of the inhibitory effect of insulin on MAPK phosphorylation and activation. The fact that MKP-1 depletion did not appreciably affect basal MAPK phosphorylation suggests that basal MAPK phosphorylation and activity escapes MKP-1 regulation. Alternatively, another phosphatase (for example, PP-2A) may be responsible for regulation of basal MAPK phosphorylation. The lack of insulin's inhibitory effect on MAPK phosphorylation was accompanied by restoration of PDGF-directed VSMC migration in MKP-1-depleted cells (Fig. 9C) compared with pBabe, which exhibited inhibition of PDGF-directed VSMC migration by insulin.


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Fig. 9.   Depletion of endogenous MKP-1 by antisense RNA prevents insulin-mediated inactivation of MAPKs and restores PDGF-directed VSMC migration. VSMCs were infected with a retrovirus carrying antisense MKP-1RNA. A stable pool of VSMCs expressing antisense MKP-1 was exposed to insulin, followed by treatment with and without PDGF as detailed in Fig. 2, and examined for insulin-induced MKP-1 expression (A), PDGF-stimulated MAPK phosphorylation (B), and PDGF-directed VSMC migration (C). In A and B, a representative autoradiogram is shown. Similar results were obtained in 3 separate experiments. In C, results are expressed as degree of increase over pBabe basal + SE of 4 separate experiments. * P < 0.05 vs. control; ** P < 0.05 vs. pBabe PDGF; *** P < 0.05 vs. pBabe Insright-arrowPDGF.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in this study indicate that insulin dose-dependently inhibits PDGF-directed migration in VSMCs isolated from control rats by inactivating MAPKs, one of the major intracellular signaling pathways that is critical for VSMC migration and proliferation. Our results suggest that insulin may dephosphorylate and inactivate PDGF-stimulated MAPKs by rapidly inducing the expression of MKP-1 because depletion of endogenous MKP-1 levels by antisense RNA strategy prevented insulin inactivation of MAPKs and restored PDGF-directed VSMC migration.

Several lines of evidence presented in this study indicate that insulin's inhibition of PDGF-directed VSMC migration by inactivating MAPKs via MKP-1 induction is mediated by the NO/cGMP signaling pathway. First, inhibition of NO/cGMP signaling with L-NMMA, an NOS inhibitor, as well as Rp-8-BrcGMPS, a cGMP antagonist, inhibited the effect of insulin on MKP-1 protein expression, abolished the inhibitory effect of insulin on PDGF-stimulated MAPK phosphorylation and ERK2 enzymatic activity, and restored PDGF-directed VSMC migration in a fashion similar to that observed in MKP-1-depleted cells. In contrast, treatment with a cGMP agonist, 8-BrcGMP, induced MKP-1 expression and mimicked the inhibitory effect of insulin on MAPK phosphorylation, ERK2 enzymatic activity, and PDGF-directed VSMC migration. Second, adenoviral expression of cGK Ialpha , a major downstream effector of the NO/cGMP signaling pathway, enhanced insulin's inhibitory effect on PDGF-induced MAPK phosphorylation and prevented PDGF-directed VSMC migration by increasing MKP-1 expression. Third, constitutive MKP-1 expression in VSMCs by adenovirus-mediated MKP-1 gene transfer caused 80% inhibition of PDGF-induced MAPK phosphorylation, decreased basal VSMC migration, and completely abolished PDGF-directed VSMC migration. Furthermore, infection of MKP-1-expressing VSMCs with cGK Ialpha adenovirus reduced basal VSMC migration rates, abolished the stimulatory effects of PDGF on VSMC migration, and enhanced the inhibitory effects of insulin and 8-BrcGMP on PDGF-directed VSMC migration, which was reduced to 60 and 25%, respectively, of basal values in VSMCs infected with control virus. 8-BrcGMP, which activates cGK Ialpha directly, was more potent than insulin in inhibiting VSMC migration in cGK Ialpha /MKP-1-expressing VSMCs. This observation suggests that, in addition to cGK Ialpha , cGMP generation is a major determinant of insulin-mediated inhibition of VSMC migration via MKP-1 expression. It should be noted that cGK Ialpha and MKP-1 expression was more effective in inhibiting migration than MAPK phosphorylation. Thus it appears that although a threshold of 10-fold increase in MAPK phosphorylation is necessary for PDGF-directed migration, a 60% decrease is sufficient to completely abolish PDGF-directed migration. Therefore, it is plausible that additional unknown signaling pathways may be involved in cGK Ialpha and/or MKP-1 inhibition of PDGF migration.

To our knowledge, this is the first study to demonstrate that insulin's inhibition of PDGF-directed VSMC migration is mediated partly via MAPK inactivation by MKP-1, via the NO/cGMP signaling pathway. Other studies have reported inhibition of migration by 8-BrcGMP (11, 18) and troglitazone, the peroxisome proliferator-activated receptor-gamma agonist (13, 35). An earlier study by Sugimoto et al. (31) showed that atrial natriuretic peptide increases MKP-1 mRNA and protein expression by activating guanylyl cyclase. Our results indicate that insulin-mediated generation of NO/cGMP activates cGK Ialpha , which in turn induces the expression of MKP-1, leading to MAPK dephosphorylation and inhibition of VSMC migration. Furthermore, insulin appears to upregulate the NO/cGMP pathway because it caused a twofold increase in cGK I activity above basal in VSMCs infected with cGK Ialpha (6), which may be related to an increase in cellular cGMP levels caused by insulin's rapid induction of iNOS (5). In contrast, hypertension in spontaneous hypertensive rats and hyperglycemia in diabetic Goto-Kakizaki rats, as well as chronic treatment with insulin, result in impaired MKP-1 expression due to defective NOS signaling, leading to sustained MAPK activation, accelerated VSMC growth (5, 7, 32), and migration. Studies in progress indicate that diabetes is accompanied by a threefold increase in PDGF-directed VSMC migration, MAPK phosphorylation, inhibition of MKP-1 expression, and lack of insulin's inhibitory effect on migration.

Taken together, our findings provide evidence for upregulation of NO/cGMP/cGK signaling by insulin to limit VSMC migration that is impaired in states of pathology resulting from an abnormal insulin pathway.

In summary, the results of this study indicate that insulin inhibits PDGF-directed VSMC migration partly by inactivating MAPKs via dephosphorylation mediated by MKP-1. Furthermore, insulin's effects including increased MKP-1 expression appear to be dependent on NO/cGMP/cGK Ialpha signaling.


    ACKNOWLEDGEMENTS

This work was supported by an American Diabetes Association Research Grant, medical education funds from Winthrop-University Hospital, and funds from Deutsche Forschungsgemeinschaft.


    FOOTNOTES

Address for reprint requests and other correspondence: N. Begum, Diabetes Research Laboratory, Winthrop Univ. Hospital, 222 Station Plaza North, Suite 511-B, Mineola, NY 11501 (E-mail: nbegum{at}winthrop.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 8, 2002;10.1152/ajpcell.00110.2002

Received 12 March 2002; accepted in final form 25 April 2002.


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