MKP-1 expression and stabilization and cGK I{alpha} prevent diabetes- associated abnormalities in VSMC migration

Asha Jacob,1 Albert Smolenski,2 Suzanne M. Lohmann,2 and Najma Begum1,3

1Diabetes Research Laboratory, Winthrop University Hospital, Mineola 11501; 3School of Medicine, State University of New York, Stony Brook, New York 11794; and 2Institut fur Klinische Biochemie und Pathobiochemie, Medizinische Universitatsklinik, D-97080 Wurzburg, Germany

Submitted 3 November 2003 ; accepted in final form 14 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Diabetes mellitus is a major risk factor in the development of atherosclerosis and cardiovascular disease conditions, involving intimal injury and enhanced vascular smooth muscle cell (VSMC) migration. We report a mechanistic basis for divergences between insulin’s inhibitory effects on migration of aortic VSMC from control Wistar Kyoto (WKY) rats versus Goto-Kakizaki (GK) diabetic rats. In normal WKY VSMC, insulin increased MAPK phosphatase-1 (MKP-1) expression as well as MKP-1 phosphorylation, which stabilizes it, and inhibited PDGF-mediated MAPK phosphorylation and cell migration. In contrast, basal migration was elevated in GK diabetic VSMCs, and all of insulin’s effects on MKP-1 expression and phosphorylation, MAPK phosphorylation, and PDGF-stimulated migration were markedly inhibited. The critical importance of MKP-1 in insulin inhibition of VSMC migration was evident from several observations. MKP-1 small interfering RNA inhibited MKP-1 expression and abolished insulin inhibition of PDGF-induced VSMC migration. Conversely, adenoviral expression of MKP-1 decreased MAPK phosphorylation and basal migration rate and restored insulin's ability to inhibit PDGF-directed migration in GK diabetic VSMCs. Also, the proteasomal inhibitors lactacystin and MG132 partially restored MKP-1 protein levels in GK diabetic VSMCs and inhibited their migration. Furthermore, GK diabetic aortic VSMCs had reduced cGMP-dependent protein kinase I{alpha} (cGK I{alpha}) levels as well as insulin-dependent, but not sodium nitroprusside-dependent, stimulation of cGMP. Adenoviral expression of cGK I{alpha} enhanced MKP-1 inhibition of MAPK phosphorylation and VSMC migration. We conclude that enhanced VSMC migration in GK diabetic rats is due at least in part to a failure of insulin-stimulated cGMP/cGK I{alpha} signaling, MKP-1 expression, and stabilization and thus MAPK inactivation.

chemotaxis; proteasome inhibitors; mitogen-activated protein kinases; insulin; cGMP-dependent protein kinase {alpha}


DIABETES AND OBESITY ARE OFTEN associated with an increased risk of hypertension, atherosclerosis, and cardiovascular disease (12, 13, 30, 31). Numerous epidemiologic studies indicate that insulin resistance and hyperinsulinemia associated with Type 2 diabetes contribute largely to development of hypertension and atherosclerotic lesions (1, 12, 13, 25, 30, 31). However, the molecular basis of this association is not completely understood. Vascular smooth muscle cells (VSMCs) are the major constituents of blood vessel walls responsible for the maintenance of vascular tone. These cells also play an important role in the pathogenesis of Type 2 diabetes, hypertension, and cardiovascular diseases. The migration of VSMCs from the arterial media to the intima is a crucial event in the formation and progression of atherosclerosis and restenosis frequently observed in diabetes (2). A variety of growth factors, platelet-derived growth factor (PDGF), cytokines, and proteases mediate VSMC migration and are known to be present at sites of vascular injury (14).

We recently showed (7, 18) that insulin inhibits PDGF-directed migration in VSMCs isolated from control Wistar-Kyoto (WKY) rats partly by causing dephosphorylation and inactivation of MAPKs via the induction of MKP-1, a dual-specificity tyrosine/threonine-specific phosphatase that dephosphorylates MAPK family members (32). The NO/cGMP signaling pathway mediates insulin induction of MKP-1 expression via the activation of cGMP-dependent protein kinase I{alpha} (cGK I{alpha}), the downstream effector of NO/cGMP signaling (5, 18). Failure of insulin to activate NO/cGMP signaling and induce MKP-1 expression in the vasculature of diabetic rats may result in excessive VSMC growth and migration, which may contribute to enhanced atherosclerosis and restenosis observed in diabetes and hypertension.

MKP-1 and its family members are the products of immediate-early genes and therefore are under tight transcriptional control (9, 19, 20). In addition, recent evidence suggests that MKP-1 is also subjected to posttranscriptional regulation via proteasomal degradation. Thus MKP-1 is a labile protein with a half-life of 45 min and is degraded by ubiquitin-directed proteasome complex (9, 10). Furthermore, phosphorylation of MKP-1 on serine residues at the carboxy terminus is known to stabilize MKP-1 protein (9, 10). Whereas we have previously shown that insulin induces MKP-1 expression at the transcriptional level (4), it is unclear whether insulin also stabilizes MKP-1 protein.

Since diabetes is accompanied by excessive protein degradation due to defective insulin signaling, the present study was undertaken to investigate whether diabetes-related elevations in VSMC migration are mediated by impaired MKP-1 expression and stability. Furthermore, the role of cGK I{alpha} in insulin-induced MKP-1 expression/stability and in diabetes was investigated with VSMCs transduced with adenovirus expressing cGK I{alpha}.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animal procedures were performed according to the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Materials. PDGF-BB, cell culture reagents, fetal bovine serum (FBS), and antibiotics were purchased from Life Technologies (Grand Island, NY); [{gamma}-32P]ATP (specific activity 3,000 Ci/mmol) from Dupont-New England Nuclear (Boston, MA); 8-bromo-cGMP (8-BrcGMP), lactacystin, MG132, and E64 from Biomol Research (Plymouth Meeting, PA); electrophoresis and protein assay reagents from Bio-Rad (Richmond, CA); type I collagenase from Worthington Biochemical (Freehold, NJ); SDS-polyacrylamide gel electrophoresis and Western blot reagents from Bio-Rad (Hercules, CA); anti-ERK2 and MKP-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); and phospho-MAPK and MAPK antibodies from New England Biolabs (Beverly, MA). cGK I antibody was prepared as described previously (18). Protein A-Sepharose CL-4B, protease inhibitors, sodium orthovanadate, and all other reagents were from Sigma (St. Louis, MO). Porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN). siRNA Target Finder, Design tool, and the pSilencer siRNA expression system were purchased from Ambion (Austin, TX).

Culture of VSMCs and treatment. VSMCs were isolated by enzymatic digestion of the aortic media of male WKY rats and Goto-Kakizaki (GK) diabetic rats weighing 200–220 g, as described in our recent publications (6, 7, 18). A monoclonal antibody against smooth muscle {alpha}-actin was used to assess the purity (>99%) of the SMC 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 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 detailed in Figs. 110. In some experiments, VSMCs were pretreated with protease inhibitors for 6 h before insulin and subsequent exposure to PDGF, as detailed in Fig. 9, A and B.



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Fig. 1. Insulin fails to inhibit migration in Goto-Kakizaki (GK) diabetic vascular smooth muscle cells (VSMCs). Serum-starved, trypsinized VSMCs were exposed to 100 nM insulin for 30 min and transferred to Transwell chambers containing 10 ng/ml platelet-derived growth factor (PDGF)-BB with and without insulin. Cells migrated to the lower surface of the Transwell membrane were counted after 5 h. Results are expressed as fold increase ± SE (n = 4). *P < 0.05 vs. Wistar-Kyoto (WKY) basal; **P < 0.05 vs. WKY PDGF; ***P < 0.05 vs. basal and insulin treatments in WKY; ****P < 0.05 vs. WKY insulin -> PDGF.

 


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Fig. 10. GK diabetic VSMCs exhibit impaired insulin-mediated increase in MKP-1 phosphorylation. VSMCs were labeled with [32P]orthophosphate (0.5 mCi/ml) for 3 h and exposed to insulin for 0.5–3.0 h followed by immunoprecipitation of 1 mg (WKY) and 4 mg (GK) lysate proteins with anti-MKP-1 antibody. The immunoprecipitates were washed and separated by SDS-PAGE, and protein was transferred to PVDF membrane followed by autoradiography and subsequent Western blot analysis with MKP-1 antibody. A: representative autoradiogram. B: quantitation of MKP-1 phosphorylation by densitometric scanning.

 


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Fig. 9. A: proteasomal inhibitors restore defective MKP-1 protein levels in GK diabetic VSMCs. Serum-starved VSMCs were exposed to the proteasomal inhibitor MG132 (20 µM) or E64 (10 µM), a cystinyl protease inhibitor, for 6 h before exposure to insulin (100 nM) for 30 min followed by PDGF (10 ng/ml) for 10 min. MKP-1 expression was examined by Western blot analysis. Top: representative Western blot. Bottom: quantitation of MKP-1 protein by densitometry from 4 different experiments. Results are expressed as fold increases over WKY basal values, which were assigned a value of 1. The rest of the data are expressed relative to WKY basal levels. *P < 0.05 vs. respective treatments of vehicle-treated WKY VSMCs; **P < 005 vs. vehicle-treated GK diabetic VSMCs. B: proteasomal inhibitors attenuate diabetes-related elevations in basal VSMC migration and block PDGF-directed migration in WKY. VSMCs were trypsinized, treated with proteasomal inhibitors for 30 min as detailed in A, and transferred to Transwell chambers containing 10 ng/ml PDGF-BB with or without MG132 or E64. Cells migrated to the lower surface of the Transwell membrane were counted after 5 h. Results are expressed as fold increases ± SE (n = 4). *P < 0.05 vs. WKY basal; **P < 0.05 vs. PDGF-treated WKY VSMCs; ***P < 0.05 vs. WKY basal; #P < 0.05 vs. GK basal and PDGF-treated; ****P < 0.005 vs. MG132-treated GK diabetic VSMCs.

 
Cell migration assay. Migration assays were performed with 24-well cell culture inserts with 8.0-µm polyethylene terephthalate Cyclopore membranes (Falcon) as described by Lundberg et al. (21). The underside of the membrane was coated with 10 µl of rat tail collagen type I (50 µg/ml) for 18–20 h, washed, and air-dried before each experiment. Serum-starved VSMCs were trypsinized and resuspended in {alpha}-MEM, 2 x 104 VSMCs/250 µl were treated with 10–100 nM insulin for 30 min, and all were subsequently loaded into the cell culture inserts. The inserts were placed in the wells containing PDGF and/or insulin. In some experiments, VSMCs were exposed to proteasome inhibitors 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 x400 magnification, and five or six different fields were counted (1).

Immunoblot analysis of MKP-1 and cGK I{alpha}. Confluent serum-starved VSMCs were exposed to insulin (0–100 nM) for 10–30 min, and equal amounts of cell lysate proteins (100 µg) were analyzed by performing Western blot analysis using either the MKP-1 or cGK I{alpha} antibody.

Immunoblot analysis of MAPK phosphorylation status. Confluent, serum-starved VSMCs treated and extracted as described above were examined using Western blot analysis. Briefly, equal amounts of proteins (25–50 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, probed with phospho-p44/42 MAPK antibody or p44/42 MAPK antibody, and detected with horseradish peroxidase (HRP)-conjugated secondary antibody (18). Phospho-MAPK antibody recognizes both ERK1 and ERK2. ERK2 is the predominant isoform in VSMCs. Therefore, quantitation of the enhanced chemiluminescence (ECL) signals was performed on ERK2.

Construction of adenoviral vectors expressing MKP-1 and cGK I{alpha}. MKP-1-, {beta}-galactosidase-, and cGK I{alpha}-expressing adenoviruses were generated by cotransfection of plasmid pACCMVpLpA-MKP-1, pACCMVpLpA-{beta}-galactosidase, or pCMVI/cGKI{alpha} with pJM17 in HEK-293 cells as detailed in our recent publication (18). Each adenovirus was plaque purified, expanded, and titered after detection of visible plaques in a HEK-293 monolayer by agarose overlays.

Silencing of MKP-1 with siRNA strategy. Recent studies have shown that siRNA constructs targeting the 106–123 region of human MKP-1 cDNA successfully blocked the expression of MKP-1 protein in BT-474 breast carcinoma cells (27). We synthesized oligonucleotides after aligning those sequences with rat MKP-1 cDNA. These oligonucleotides with overhanging ApaI and EcoRI restriction sites were cloned into the siRNA expression vector pSilencer 3.1-U6 puro, and the resulting vector was transfected into VSMCs and selected with puromycin as described previously (18). Clones were screened by Western blotting to identify those that had lowest basal as well as FBS-induced MKP-1 protein levels. One of the sequences inhibited MKP-1 expression >90%. The corresponding oligonucleotides in rat MKP-1 were 5'-CGCCGGCCACATCGTGGGC. As controls, VSMCs were transfected with pSilencer 3.1U6 puro with cloned oligonucleotides that had a composition identical to the siRNA construct but a scrambled sequence (ss) that was not complementary to any known genes.

Analysis of MKP-1 phosphorylation status. VSMCs were labeled with [32P]orthophosphate (0.5 mCi/ml) for 3 h, exposed to insulin for 0.5–3.0 h, followed by immunoprecipitation of 1 mg (WKY) and 4 mg (GK diabetic) lysate protein with anti-MKP-1 antibody. The immunoprecipitates were washed and separated using SDS-PAGE, and protein was transferred PVDF membrane for analysis using MKP-1 antibody.

Protein assay. Protein in cellular lysates was quantitated by the bicinchoninic acid method (28).

cGMP assay. cGMP was extracted from VSMCs with 90% ethanol. The ethanol extracts were cleared by centrifugation and evaporated, followed by reconstitution with cGMP assay buffer. cGMP was measured by using a highly sensitive radioimmunoassay kit (Amersham).

Statistics. The results are presented as means ± SE. Analysis of variance (ANOVA) followed by Dunnett's test were performed to compare the mean values between various treatments as well as control vs. GK VSMCs. A P value of <0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of GK diabetic rats. As reported previously by us (3, 4) and others (17), the diabetic GK rat, a rat model of Type 2 diabetes, exhibits features that are characteristic of human Type 2 diabetes, including the frequent presence of atherosclerosis. We observed a twofold increase in postprandial plasma glucose and insulin levels compared with age-matched WKY controls. In contrast, body weight was comparable between the age-matched WKY control and GK diabetic rats. There was a twofold increase in the levels of fasting plasma glucose (14.1 ± 0.21 vs. 6.7 ± 0.1 mmol/l) and insulin (1,100 ± 65 vs. 675 ± 43 pmol/l) in this rat model, which was comparable to those reported earlier (23). Our earlier studies (24) showed that VSMCs isolated from GK diabetic rats maintain the phenotype in culture up to seven passages as evidenced by increased growth rate and elevated intracellular Ca2+.

VSMCs isolated from GK diabetic rats exhibit elevated basal migration rate: insulin fails to inhibit VSMC migration. Our recent studies (18) showed that physiological concentrations of insulin dose-dependently inhibit PDGF-directed migration of VSMCs isolated from WKY rats. To further understand the importance and pathophysiological relevance of this inhibitory effect of insulin, we examined insulin effects on basal and PDGF-directed migration in VSMCs isolated from GK diabetic rats. As shown in Fig. 1, GK rats exhibit a 1.8-fold increase in basal VSMC migratory rates compared with control WKY rats. In WKY, PDGF caused a 2.2-fold increase in migration, whereas migration was not further increased by PDGF in GK diabetic VSMCs. Insulin treatment blocked PDGF-directed VSMC migration in WKY, but not in GK diabetic VSMCs. Notably, PDGF caused no increase in VSMC migration in GK beyond the basal values. This may be due to cellular desensitization to PDGF.

Enhanced migration in GK diabetic VSMCs is accompanied by increased MAPK phosphorylation and its activation. Numerous studies, including ours (18), have shown that MAPK signaling plays a crucial role in VSMC migration (8, 15, 21, 22). Therefore, we examined whether the elevations in basal migration observed in GK diabetic VSMCs are associated with MAPK phosphorylation. As shown in Fig. 2A, MAPK is phosphorylated in the basal state in GK diabetic but not WKY VSMCs. In WKY, PDGF treatment for 10 min resulted in a 20-fold increase in MAPK phosphorylation, which was significantly inhibited by insulin. VSMCs isolated from GK diabetic rats exhibited a 40-fold increase in PDGF-induced MAPK phosphorylation relative to basal levels. Insulin failed to inhibit PDGF-induced MAPK phosphorylation in these GK diabetic VSMCs, in contrast to a 45% decrease observed in WKY VSMCs (Fig. 2B).



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Fig. 2. GK diabetic VSMCs exhibit elevated MAPK phosphorylation: insulin fails to dephosphorylate MAPK. Serum-starved VSMCs were exposed to insulin (100 nM for 30 min) followed by PDGF (10 ng/ml for 10 min). Equal amounts of cell lysate proteins were analyzed by Western blot with anti-phospho-p44/42MAPK (T202/Y204; pMAPK) and anti-MAPK antibodies, respectively. A: representative Western blot. Similar results were obtained in 5 separate experiments. B: linear enhanced chemiluminescence (ECL) pMAPK and MAPK signals from 5 different experiments were quantitated by densitometric analysis. To correct for variations in proteins, the signal intensity of pMAPK from each blot was divided by the corresponding MAPK protein signal intensity and expressed as pMAPK-to-MAPK ratio. *P < 0.05 vs. WKY basal; **P < 0.05 vs. PDGF-treated WKY; ***P < 0.05 vs. WKY VSMC; ****P < 0.05 vs. respective PDGF- and ins->PDGF-treated WKY.

 
Elevations in MAPK phosphorylation in GK diabetic VSMCs are accompanied by reductions in MKP-1 protein expression levels. Our earlier studies (5) showed that insulin rapidly induces MKP-1 protein expression in VSMCs isolated from control WKY rats via the phosphatidylinositol 3-kinase (PI3-kinase)/NO/cGMP signaling pathway. Thus we investigated whether the observed lack of insulin effect on MAPK phosphorylation in GK diabetic VSMCs is due to impaired induction of MKP-1 protein expression by insulin. As shown in Fig. 3, insulin caused a rapid dose-dependent induction of MKP-1 protein expression within 30 min in WKY. A maximal sixfold increase in MKP-1 protein expression was observed in 10 nM insulin. In contrast, GK diabetic VSMCs displayed a 50% decrease in basal MKP-1 protein levels, and insulin-stimulated MKP-1 expression barely exceeded the basal MKP-1 protein levels observed in WKY VSMCs.



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Fig. 3. Impaired insulin-induced MKP-1 expression in GK diabetic VSMCs. Confluent, serum-starved VSMCs were exposed to insulin (0–100 nM) for 30 min. Equal amounts of cell lysate proteins (100 µg) were analyzed by Western blot with MKP-1 antibody. A: representative blot from 4 separate experiments. B: linear ECL signal intensities from 4 different experiments were quantitated by densitometric scanning, and results are expressed as arbitrary densitometric units (ADU) ± SE. *P < 0.05 vs. respective MKP-1 expression levels in GK diabetic VSMCs at various insulin concentrations.

 
Adenoviral MKP-1 expression reduces MAPK phosphorylation and decreases basal migration and cellular insulin responsiveness in GK diabetic VSMCs. The results shown in Fig. 3 suggested that reduced expression of MKP-1 could result in sustained phosphorylation and activation of MAPK and might be responsible for the observed increase in the basal migratory rates of GK diabetic VSMCs. To further explore this possibility, GK diabetic VSMCs were infected with MKP-1 adenovirus and examined for PDGF-induced MAPK phosphorylation (Fig. 4). Infection with MKP-1 adenovirus did not affect the VSMC phenotype but caused >20-fold increase in MKP-1 protein levels in WKY and GK diabetic VSMCs (Fig. 4C). MKP-1 expression was accompanied by >90% decrease in PDGF-induced MAPK phosphorylation in WKY VSMCs (Fig. 4, A and D). Ad-MKP-1-infected GK diabetic VSMCs also exhibited decreased basal and PDGF-induced MAPK phosphorylation (Fig. 4, A and D); however, the latter was not completely decreased to the level found in Ad-MKP-1-infected WKY VSMCs. Coinfection of these VSMCs with cGK I{alpha} adenovirus further decreased PDGF-induced MAPK phosphorylation to levels observed in WKY and restored insulin's inhibitory effect on PDGF-stimulated MAPK phosphorylation (Fig. 5, A and E).



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Fig. 4. Adenoviral MKP-1 expression partially inhibited PDGF-stimulated MAPK phosphorylation in GK diabetic VSMCs and more completely inhibited it in WKY VSMCs. VSMCs isolated from WKY and GK diabetic rats were infected with adenoviral MKP-1 (Ad.MKP-1) at 20 pfu/cell for 24 h, exposed to insulin and PDGF, and examined for MAPK phosphorylation (A), MAPK proteins (B), and MKP-1 expression (C) as detailed in Fig. 2. D: quantitation of MAPK phosphorylation by densitometry. Linear ECL pMAPK and MAPK signals from 5 different experiments were quantitated by densitometric analysis. To correct for variations in proteins, the signal intensity of pMAPK from each blot was divided by the corresponding MAPK protein signal intensity and expressed as pMAPK-to-MAPK ratio. *P < 0.05 vs. WKY basal; **P < 0.05 vs. PDGF-treated WKY; ***P < 0.05 vs. respective treatments in WKY; ****P < 0.005 vs. uninfected PDGF- and ins->PDGF-treated WKY; #P < 0.05 vs. corresponding treatment of uninfected GK diabetic VSMCs.

 


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Fig. 5. Combined expression of MKP-1 and cGMP-dependent protein kinase I{alpha} (cGK I{alpha}) prevents PDGF-stimulated MAPK phosphorylation in GK diabetic VSMCs. VSMCs were infected first with Ad.MKP-1 as detailed in Fig. 4 followed by adenoviral cGK I{alpha} (Ad.cGK I{alpha}) at 1 x 1010 virus particles/ml for 2 h and then by addition of 10% fetal bovine serum (FBS) as detailed in MATERIALS AND METHODS. Serum-starved VSMCs were examined for MAPK phosphorylation (A), MAPK proteins (B), MKP-1 protein (C), and cGK I{alpha} expression (D). A representative composite picture is shown. Similar results were obtained in 3 or 4 separate experiments. E: signal intensities from different experiments were quantitated by densitometric scanning, normalized for MAPK proteins by dividing the pMAPK signal by the MAPK signal intensity, and expressed as pMAPK-to-MAPK ratio. *P < 0.05 vs. uninfected WKY basal; **P < 0.05 vs. uninfected, PDGF-treated WKY; ***P < 0.005 vs. corresponding treatments in uninfected WKY; ****P < 0.05 vs. uninfected WKY basal; #P < 0.05 vs. PDGF, insulin->PDGF treatment of uninfected WKY; ##P < 0.005 vs. respective treatments of uninfected GK diabetic VSMCs.

 
Reductions in MAPK phosphorylation by adenoviral MKP-1 were accompanied by a 25% decrease in basal migratory rates and a complete ablation of PDGF-induced migration in WKY (Fig. 6A). MKP-1 overexpression in GK diabetic VSMCs decreased basal migration rates to levels seen in MKP-1-infected WKY VSMCs, restored sensitivity to PDGF, which resulted in a twofold increase in migration over the basal values, and also restored sensitivity to insulin as evidenced by a 40% reduction in PDGF-directed migration, in contrast to uninfected VSMCs, in which insulin increased migration by 25% over the basal values when present along with PDGF (Fig. 6B). Coexpression of cGK I{alpha} along with MKP-1 prevented basal and PDGF-directed migration in VSMCs isolated from GK (Fig. 6B).



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Fig. 6. Expression of MKP-1 and cGK I{alpha} inhibits VSMC migration. VSMCs were infected with MKP-1 and cGK I{alpha} viruses separately or in combination and exposed to insulin, PDGF, and 8-bromo-cGMP (8-BrcGMP; 1 mM) as detailed in Fig. 5. VSMC migration was assayed as detailed in Fig. 1. Results are expressed as fold increase ± SE (n = 5). A: *P < 0.05 vs. uninfected WKY basal; **P < 0.05 vs. PDGF treatment of uninfected WKY; ***P < 0.05 vs. PDGF-treated uninfected WKY; ****P < 0.05 vs. PDGF-treated Ad.cGK I{alpha}-infected WKY; #P < 0.05 vs. uninfected WKY basal and PDGF treatment; ##P < 0.05 vs. uninfected WKY basal and PDGF, ins->PDGF, and 8-BrcGMP->PDGF treatment. B: &P < 0.05 vs. respective treatments of uninfected WKY; &&P < 0.05 vs. respective treatments of uninfected GK diabetic; &&&P < 0.005 vs. 8-BrcGMP-treated uninfected GK diabetic; +P < 0.05 vs. Ad.MKP-1-infected control GK diabetic; ++P < 0.05 vs. Ad.MKP-1-infected, PDGF-treated GK diabetic; &&&&P < 0.05 vs. corresponding treatments of Ad.cGK I{alpha}-treated GK diabetic.

 
cGK I{alpha} expression alone did not alter basal migratory rates in WKY but mimicked the effect of insulin by decreasing PDGF-directed migration by 85%. cGMP agonist, which activates cGK I{alpha}, further enhanced cGK I{alpha} by decreasing PDGF-directed migration to 60% of the basal values seen in uninfected WKY. In GK diabetic VSMCs, cGK I{alpha} expression decreased basal levels by 38%. cGMP agonist further enhanced the effect of cGK I{alpha} by decreasing migration to levels below the basal values of WKY VSMCs. Coexpression of MKP-1 and cGK I{alpha} decreased migration under all treatment conditions to below the basal values of uninfected WKY VSMCs (Fig. 6).

Inhibition of MKP-1 expression through siRNA causes resistance to insulin in WKY VSMCs. To further establish that it is indeed insulin-induced MKP-1 via cGMP/cGK I{alpha} that plays a major role in insulin inhibition of PDGF-directed migration under normal conditions, MKP-1 protein expression was silenced in VSMCs derived from WKY by transfection with pSilencer-MKP-1 siRNA construct. A stable pool of VSMCs expressing the lowest basal and FBS-induced MKP-1 levels were amplified and examined for insulin effect on PDGF-directed VSMC migration. Expression of MKP-1 siRNA decreased basal and insulin-, cGMP-, cGK I{alpha}-, and proteasome inhibitor-mediated increase in MKP-1 protein expression by >90% (Fig. 7A) and blocked insulin's inhibitory effect on PDGF-mediated migration (Fig. 7B). In addition, the proteasomal inhibitor MG132 failed to prevent PDGF-directed migration in VSMCs expressing MKP-1 siRNA (Fig. 7B). Similarly, cGMP agonist as well as cGK I{alpha} did not block PDGF-directed migration in VSMCs expressing MKP-1 siRNA.



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Fig. 7. Small interfering RNA (siRNA) silencing of MKP-1 expression reduces MKP-1 protein levels and abolishes insulin inhibition of PDGF-directed VSMC migration in WKY. VSMCs isolated from control WKY rats were transfected with pSilence vector encoding MKP-1 siRNA or scrambled sequence RNA (ssRNA). Puromycin-resistant clones expressing the lowest levels of inducible MKP-1 were amplified and examined for the effect of insulin, cGMP, cGK I{alpha}, and MG132 on MKP-1 levels (A) and insulin- and MG132-mediated inhibition of PDGF-stimulated VSMC migration (B). Results are means ± SE of 5 separate experiments performed in duplicate. *P < 0.05 vs. ssRNA basal; **P < 0.05 vs. ssRNA PDGF; ***P < 0.05 vs. PDGF+insulin-treated VSMCs expressing ssRNA.

 
Decreased insulin-stimulated cGMP generation and cGK I{alpha} protein expression is observed in GK diabetic VSMCs. Recent studies at our laboratory (18) showed that the NO/cGMP signaling pathway participates in insulin stimulation of MKP-1 expression and insulin inhibition of PDGF-directed migration in WKY VSMC. Therefore, we tested whether the abnormalities observed in MKP-1 expression, MAPK phosphorylation, and migration of GK diabetic VSMCs is associated with defective cGK I{alpha} protein expression and/or its activation by its upstream regulator, cGMP. We measured cellular cGMP after stimulation of VSMCs with insulin and also examined the levels of cellular cGK I{alpha} protein under the above conditions. As shown in Fig. 8, insulin caused a fourfold increase in cGMP levels in WKY, whereas GK diabetic VSMCs exhibited decreased levels of cGMP in the basal state and severely impaired insulin-stimulated cGMP generation. However, sodium nitroprusside (SNP)-induced generation of cGMP levels was comparable between WKY and GK rat VSMCs, suggesting that guanylyl cyclase (downstream effector of NO) signaling was intact in GK diabetic VSMCs. Analysis of cGK I{alpha} protein, the downstream effector of cGMP, also revealed a threefold decrease in GK diabetic VSMCs compared with WKY (Fig. 8), and this was accompanied by reductions in cGK I enzymatic activity (data not shown).



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Fig. 8. Diabetes results in decreased cGMP generation and cGK I{alpha} protein expression. VSMCs were treated with insulin (Ins) for 10 min and analyzed for cGMP levels by radioimmunoassay using a commercial kit from Amersham. cGK I{alpha} protein levels were measured by immunoblot analysis of equal amounts of VSMC lysate proteins. Results are the mean of 3 separate experiments performed in duplicate. A: *P < 0.05 vs. WKY basal; **P < 0.05 vs. WKY basal and insulin-treated; B: P < 0.05 vs. cGK I{alpha} levels in WKY under basal and insulin-treated conditions. SNP, sodium nitroprusside.

 
Proteasome inhibitors partially restore MKP-1 protein levels in GK diabetic VSMCs and inhibit basal VSMC migration. Recent evidence suggests that MKP-1 is a highly labile protein, as it is known to be degraded by proteasome complex (9, 10). Since the diabetic state is accompanied by excessive proteolysis, we tested whether the reductions observed in basal and insulin-stimulated MKP-1 protein levels in GK diabetic VSMCs is due to increased MKP-1 breakdown in addition to impaired MKP-1 expression. VSMCs were treated separately with specific proteasome inhibitors (MG132 and lactacystin) and cystinyl protease inhibitors (E64) for 6 h, exposed to insulin and PDGF, and examined for MKP-1 protein levels. As shown in Fig. 9A, treatment of WKY VSMCs with MG132 increased basal MKP-1 protein levels by four- to sixfold compared with insulin-stimulated VSMCs treated with vehicle alone and E64 (Fig. 9A, compare lanes 4–6 with lanes 1–3 and 7–8). GK diabetic VSMCs also exhibited elevations in MKP-1 protein content, but the levels were half of those of WKY VSMCs (Fig. 9A), suggesting that the MKP-1 reductions observed in GK diabetic VSMCs resulted not only from excessive degradation but also from impaired expression. Analysis of VSMC migration revealed that treatment with proteasome inhibitors completely abolished PDGF-directed migration in WKY VSMCs and decreased basal migration in VSMCs isolated from diabetic GK rats, whereas E64 had very little effect on VSMC migration (Fig. 9B).

Insulin increases MKP-1 phosphorylation in WKY but not GK diabetic VSMCs. Recent evidence suggests that phosphorylation of MKP-1 stabilizes MKP-1 protein (9, 10). To investigate whether insulin treatment differentially affects MKP-1 phosphorylation, cells were metabolically labeled with [32P]orthophosphate, treated with insulin for 30 min to 3 h, and examined for MKP-1 phosphorylation status. Because GK diabetic VSMCs exhibit impaired MKP-1 protein expression, a fourfold higher amount of cell lysate proteins was used for immunoprecipitation to match the amount of MKP-1 protein immunoprecipitated from WKY VSMCs. MKP-1 phosphorylation was quantitated after normalization for MKP-1 protein by dividing the intensity of phospho-signals by the protein signals. As shown in Fig. 10, insulin caused a 2.5-fold increase in MKP-1 phosphorylation at 30 min, which was sustained for 1 h and then gradually decreased to basal levels after 3 h of insulin exposure. GK diabetic VSMCs showed reductions in basal as well as insulin-stimulated MKP-1 phosphorylation (Fig. 10).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The results of this study indicate that reductions in cGK I{alpha} and MKP-1 protein levels observed in VSMCs isolated from GK diabetic rats lead to deficient MKP-1-mediated MAPK dephosphorylation and thereby increased/unbalanced MAPK phosphorylation and VSMC migration. Thus enforced expression of MKP-1 as well as stabilization of endogenous MKP-1 protein by proteasome inhibitors abolished the elevations in basal and PDGF-directed VSMC migration observed in GK diabetic VSMCs.

Several lines of evidence presented in this study suggest that diabetes in GK rats results in impaired basal and insulin-induced MKP-1 protein expression in VSMCs because of a combination of blunted NO/cGMP signaling and excessive proteasome degradation of the already reduced MKP-1 protein levels that allow excessive MAPK phosphorylation and VSMC migration. First, insulin-induced MKP-1 expression was markedly decreased in GK diabetic VSMCs at all concentrations of insulin tested compared with WKY VSMCs. Second, reductions in MKP-1 protein levels were accompanied by reductions in insulin-stimulated MKP-1 phosphorylation and MKP-1 enzymatic activity measured with 32P-labeled recombinant ERK2 as a substrate (data not shown). Third, decreased levels of cGK I{alpha} proteins as well as decreased cGMP generation in response to insulin were observed in GK diabetic VSMCs. Fourth, restoration of MKP-1 levels by adenoviral expression in GK diabetic VSMCs decreased basal VSMC migratory rates to values comparable to those in WKY and restored responsiveness to PDGF and insulin sensitivity. Fifth, treatment with proteasome inhibitors restored MKP-1 expression in GK diabetic VSMCs to levels observed in insulin-treated WKY VSMCs and reversed diabetes-related elevations in migration. Sixth, coexpression of MKP-1 and cGK I{alpha} decreased basal and PDGF-induced MAPK phosphorylation and completely inhibited VSMC migration in GK diabetic rats under all conditions tested. Finally, siRNA-directed cellular depletion of MKP-1 in WKY VSMCs caused resistance to insulin as evidenced by a lack of insulin-dependent increase in MKP-1 expression and decrease in PDGF-mediated MAPK phosphorylation as well as migration.

Previously, we showed (24) decreased insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation in VSMCs leading to reductions in PI3-kinase enzymatic activity, iNOS protein induction, NO generation, defective insulin-stimulated relaxation, and excessive growth in GK diabetic VSMCs. The current studies add a new dimension to these findings by documenting that insulin fails to inhibit MAPK phosphorylation and migration in these VSMCs. The fact that adenoviral expression of MKP-1 only partially inhibited PDGF-induced MAPK phosphorylation in GK diabetic VSMCs whereas it completely abolished MAPK phosphorylation in WKY suggests that factors in addition to upregulation of endogenous MKP-1 levels may be necessary for dephosphorylation of MAPK and inhibition of VSMC migration. Preliminary results suggest that coexpression of cGK I{alpha} along with MKP-1 indeed resulted in complete dephosphorylation and inactivation of MAPK in VSMCs isolated from GK diabetic rats. Given the knowledge that MKP-1 and its other family members associate with MAPKs and are activated by MAPKs (11, 34), it is plausible that cGK I{alpha} may facilitate the interaction between MKP-1 and MAPKs and enhance MKP-1 dephosphorylation of ERKs. Alternatively, upregulation of MKP-1 levels in GK may result in positive feedback regulation of Raf-1 and MEK as proposed by Shapiro and Ahn (26).

In addition to inducible transcription and further activation by association with MAPKs, MKP-1 is regulated by protein degradation via a ubiquitin-mediated proteasomal pathway. Moreover, phosphorylation of MKP-1 at the carboxy terminus is known to inhibit its degradation (9, 10). We did observe an increase in MKP-1 phosphorylation by insulin in WKY VSMCs, whereas those isolated from GK diabetic rats exhibited impaired insulin-mediated MKP-1 phosphorylation, which could contribute to excessive degradation of MKP-1. Thus MG132 inhibition of proteasomal degradation increased MKP-1 protein accumulation in both WKY and GK diabetic VSMCs, and caused complete reversal of migratory defects observed in GK diabetic VSMCs as well as attenuation of PDGF-directed VSMC migration in WKY. The protease inhibitor effect was specific to MKP-1, as cellular levels of cGK I{alpha} and MAPKs were not altered upon treatment with MG132. Further studies are warranted to understand the role of cGK I{alpha} in MKP-1 phosphorylation.

Collectively, these findings illustrate a complex control mechanism designed to limit undesirable long-term activation of MAPKs under normal conditions and further demonstrate the importance of regulated protein degradation in the control of cell growth and migration. In contrast, in diabetes, insulin fails to inactivate MAPKs because of reduced PI3-kinase/Akt-mediated inducible nitric oxide synthase (iNOS) induction and cGMP generation, which is needed to activate cGK I{alpha}, culminating in impaired MKP-1 expression. Furthermore, excessive production of growth factors observed in diabetes may interfere with insulin activation of PI3-kinase signaling and upregulate the Ras/Raf-1 signaling pathway, and thereby support increased MAPK activation and migration.

In summary, the results of the present study suggest that the diabetic condition leads to reduced cGMP and CGKK levels, leading to reduced MKP-1 expression as well as to excessive proteasomal MKP-1 degradation, all of which culminates in reduced MAPK dephosphorylation and increased VSMC migration.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by a research grant from the American Diabetes Association and Medical Education Funds from Winthrop-University Hospital, and in part by a grant from the Deutsche Forschungsgemeinschaft (to S. M. Lohmann).

This article was written in a personal capacity and does not necessarily represent the opinions or reflect the views of the National Institutes of Health, the Department of Health and Human Services, or the Federal Government.


    ACKNOWLEDGMENTS
 
We thank Dr. Robert V. Farese (Department of Veterans Affairs Hospital, Tampa, FL) for the generous gift of diabetic GK rats and Dr. Jeffrey Molkentin (University of Cincinnati, Cincinnati, OH) for providing the MKP-1 adenovirus.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Begum, National Institutes of Health, Bethesda, MD 20892 begumn2003{at}yahoo.com

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.


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