Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension

Najma Begum, Young Song, Jennifer Rienzie, and Louis Ragolia

The Diabetes Research Laboratory, Winthrop University Hospital, Mineola 11501; and Department of Medicine, Health Science Center, State University of New York at Stony Brook, Stony Brook, New York 11574

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Hyperinsulinemia (HI) and insulin resistance (IR) are frequently associated with hypertension and atherosclerosis. However, the exact roles of HI and IR in the development of hypertension are unclear. Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, we examined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolated from aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). VSMCs were grown to confluence in culture, serum starved, and examined for DNA synthesis {using [3H]thymidine (TDR), immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1) induction}. Basal rate of TDR incorporation into DNA was twofold higher in SHR compared with WKY (P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did not abolish mitogenesis mediated by 10-100 nM insulin, suggesting that insulin effect is mediated via its own receptors. Insulin had a small mitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects on MAPK activity in WKY. In contrast, serum-stimulated MAPK activation was comparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059, completely blocked insulin's effect on MAPK activation and mitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin's effects on MAPK activation and mitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapid induction of MKP-1, the dual-specificity MAPK phosphatase. In contrast, VSMCs from SHR were resistant to insulin with respect to MPK-1 expression. We conclude that insulin is mitogenic in SHR, and the effect appears to be mediated by sustained MAPK activation due to impaired insulin-mediated MKP-1 mRNA expression, which may act as an inhibitory feedback loop in attenuating MAPK signaling.

mitogen-activated protein kinase phosphatase

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

VASCULAR SMOOTH MUSCLE cells (VSMCs) are one of the major constituents of blood vessel wall responsible for the maintenance of vascular tone (32). These cells also play an important role in the pathogenesis of several disease processes, for example, hypertension, non-insulin-dependent diabetes mellitus, atherosclerosis, cardiovascular disease, and dyslipidemia (syndrome X) (4, 22). Insulin resistance and hyperinsulinemia are closely associated with the above syndromes (4, 22, 23, 29). However, the pathogenetic role of insulin resistance and/or hyperinsulinemia in the development of hypertension and cardiovascular disease is still not clear. Increased VSMC growth and migration are major abnormalities observed in the arteries of hypertensive animal models (24). Although insulin at high concentrations is mitogenic in vascular tissue, whether this effect is mediated via the insulin receptor or the insulin-like growth factor I (IGF-I) receptor is still unclear. Targeted disruption of the insulin receptor gene did not cause growth retardation in utero (13); mice rapidly developed diabetic ketoacidosis along with marked postnatal growth retardation and died within 7 days after birth. IGF-I administration did not prevent death due to diabetic ketoacidosis in insulin receptor knockout mice. Thus the molecular basis of insulin's effect on growth and metabolism in various cell types, including VSMCs and its association with hypertension, still remains unclear. Analyses of arteries from hypertensive animal models have demonstrated hyperplasia and hypertrophy of VSMCs (24). From the therapeutic point of view, effective treatment and control of these diseases can only be achieved by an advancement of our understanding of the molecular mechanisms of abnormal VSMC growth.

Mitogen-activated protein kinase (MAPK) family members are well-characterized intracellular mediators that are activated in response to a variety of external signals, including growth factors, tumor promoters, cytokines, osmotic shock, and stress (6, 12, 19, 21). Three major subclasses of MAPKs have been identified recently and comprise the extracellular-regulated protein kinase (ERK), SAPK/JNK, and p38/HOG families (25). Full activation of MAPK requires phosphorylation on critical tyrosine and threonine residues. Several upstream dual-specificity kinases catalyzing this modification have been identified (6, 12, 19, 21, 25). Once activated, these kinases are responsible for the activation and phosphorylation of additional kinases as well as a battery of regulatory proteins, including transcription factors required for the expression of genes involved in cell growth and/or differentiation.

MAPK phosphorylation and activation are reversible processes, indicating that protein phosphatases play a critical role in controlling enzyme activity. Recent studies indicate that inactivation or attenuation of MAPK signaling is mediated by a class of dual-specificity protein phosphatases, like MKP-1 (also known as CL100, Erp, and hVH-1), which is encoded by the murine gene, 3CH134 (30). MKP-1 has dual-catalytic activity toward phosphotyrosine- and phosphothreonine-containing proteins and is known to inactivate ERK in vivo as well as in vitro (30). MKP-1 and the other family members are principally regulated at the transcriptional level as evidenced by very low to undetectable mRNA expression in quiescent cells and a rapid induction on treatment of cells with growth factors and any agents that cause oxidative stress and heat shock (30).

Sustained activation of ERKs leads to excessive cell growth (7). However, little is known about their physiological role or relevance to pathological conditions in humans or animal models of hypertension. The intracellular signaling pathways that regulate the termination of MAPK activity are largely undefined. The spontaneous hypertensive rat (SHR) is the best characterized animal model of essential hypertension (8, 18). Primary cultures of the aortic smooth muscle cells derived from SHR and normotensive Wistar-Kyoto rats (WKY) offer a good model system (18) to investigate the role of insulin in cell proliferation and the mechanism of insulin-stimulated mitogenesis.

In the present study, we have examined the effect of insulin on mitogenesis in highly confluent, growth-arrested primary VSMCs derived from WKY and SHR and examined the role and the mechanism of regulation of MAPK cascade in insulin-stimulated growth effects. The results indicate that insulin acting via its own receptors causes a rapid and sustained activation of MAPKs, leading to increased DNA synthesis in highly confluent VSMCs from SHR. The observed increase in MAPK activation in SHR appears to be due to an increase in tyrosine phosphorylation status of MAPK because of impaired dephosphorylation resulting from impaired insulin-mediated MKP-1 gene expression that attenuates MAPK signaling by feedback inhibition.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. DMEM, fetal bovine serum (FBS), antibiotics, trypsin, L-glutamine, freezing medium, erbstatin-A, and myelin basic protein (MBP) were obtained from Life Technologies; [3H]thymidine, [gamma -32P]ATP (specific activity, 3,000 Ci/mmol), and 125I-labeled protein A were purchased from DuPont NEN. Type-1 collagenase was from Worthington Biochemical. Antibodies against phosphotyrosine and p42/p44 MAPKs (ERKs) were purchased from Zymed. Protein A/G-agarose and anti-IGF-I antibodies were from Oncogene Science. Wortmannin, PD-98059, and calfostin-A were from Biomol. SDS-PAGE reagents were from Bio-Rad. Rat MKP-1 cDNA was a kind gift from Dr. Jyotirmoy Kusari (Tulane University, New Orleans, LA). All other chemicals and reagents were purchased from Sigma Chemical.

Isolation of VSMCs. VSMCs in primary culture were obtained by enzymatic digestion of the aortic media of male normotensive WKY and SHR of body weight 200-220 g, as described previously (31). Briefly, rats were deeply anesthetized with pentobarbital sodium and killed by an overdose of the anesthetic. The aortas were removed to sterile dishes containing medium and stripped of adventitia with a sterile forcep. The aortic media were dispersed into single cells by incubation with collagenase (1 mg/ml) and elastase (10 U/ml) for 60-90 min with shaking. Cells were sedimented and plated on 60-mm tissue culture dishes (Falcon Primaria) and were grown in DMEM containing 5 mM glucose, 10% FBS, and antibiotics. The growth medium was changed every 2 days. The subcultures of VSMCs from passages 3-5 were used in all the experiments. VSMCs prepared from the two strains of rats were not contaminated with fibroblasts or endothelial cells as evidenced by >99% positive immunostaining of smooth muscle alpha -actin with FITC-conjugated alpha -actin antibody (Sigma Chemical). All the experiments on mitogenesis and the MAPK assays were performed on highly confluent cells (9-11 days in culture) of identical passages. Before each experiment, cells were serum starved for 24-48 h in serum-free DMEM containing antibiotics.

[3H]thymidine incorporation. VSMCs from SHR and WKY were grown to confluence (9-11 days in culture) in 24-well plates and were serum starved in DMEM containing 5 mM glucose and antibiotics for 24 h. Insulin (0-1,000 nM) or other agents were added to triplicate wells in fresh DMEM, and cells were incubated for 6-48 h. The cells were pulse labeled with [3H]thymidine (1 µCi/ml) 3 h before the completion of the incubation period (13). At the end of the incubation, the medium was removed, and the cell monolayers were washed sequentially with ice-cold PBS (3 times) and once with 10% ice-cold TCA. The cells were solubilized by the addition of 200 µl of 0.1% SDS-0.1 N NaOH. The solubilized cell lysates (100 µl) were added to 5 ml of scintillation fluid (Instagel, Packard), and the incorporation of [3H]thymidine into DNA was determined by liquid scintillation spectrometry (14). Incorporation was expressed as dpm per milligram protein.

Immunoprecipitation and assay of MAPK activity. Confluent, serum-starved VSMCs in 35-mm dishes were exposed to insulin (0-1,000 nM) for 0-120 min or 6 h. In some experiments, cells were pretreated with various inhibitors or vehicle (0.1% DMSO) alone for 1 h before the addition of insulin. The incubation was stopped by removing the medium. The cells were rinsed with ice-cold PBS containing 1 mM sodium orthovanadate (a nonspecific tyrosine phosphatase inhibitor). The dishes were frozen quickly in liquid nitrogen after the addition of 300 µl of lysis buffer [20 mM HEPES, 80 mM beta -glycerophosphate, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 10 µM orthovanadate, and 0.5% Triton X-100 along with a cocktail of protease inhibitors (leupeptin, aprotinin, antipain, soy trypsin inhibitor, pepstatin A, each at a concentration of 10 µg/ml), 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride]. The dishes were thawed on ice, scraped, lysate sonicated, and centrifuged at 16,000 g at 4°C for 20 min. Equal amounts of proteins (50 µg) from the resulting supernatants were immunoprecipitated with rabbit polyclonal anti-MAPK antibody (2 µg) for 2 h at 4°C, followed by the addition of 50 µl of protein A Sepharose (50% vol/vol). The anti-MAPK antibody recognizes both ERK-1 and ERK-2 isoforms of MAPK. The incubation was continued for an additional 1 h with constant mixing on the rocker at 4°C. The immunoprecipitates were washed three times with 1 ml of lysis buffer and twice with the kinase buffer. The immunoprecipitates were suspended in 45 µl of kinase buffer containing MBP as a substrate. In some experiments, the assay buffer contained 2 µM chelerythrine HCl, a protein kinase C (PKC) inhibitor, and 2 µM protein kinase A (PKA) inhibitor to examine the contribution of these two kinases to MAPK activation. The reaction was initiated by the addition of 5 µl of [gamma -32P]ATP (1 µCi/assay). After 20 min at 30°C, the reaction was stopped by transferring an aliquot of the reaction mixture to Whatman p81 paper, which was dropped in ice-cold phosphoric acid (180 mM). The paper was washed five times with phosphoric acid and once with ethanol, and radioactivity incorporated into MBP was counted. Details are given in our earlier study (1). MAPK enzymatic activity was normalized to the amount of enzyme immunoprecipitated from different samples.

Immunoblot analysis of MAPK and tyrosine phosphorylated proteins. Immunodetection of MAPK and tyrosine phosphorylated proteins in control and insulin-treated VSMCs was performed as described in our recent study (1). Briefly, 20-50 µg of cell lysate proteins or MAPK immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (16). The membranes were probed with anti-MAPK antibody and antiphosphotyrosine antibody according to the manufacturers' protocols. Visualization of the primary antibody was performed with 125I-labeled protein A followed by autoradiography. In some experiments, immunoprecipitated MAPK was subjected to SDS-PAGE, followed by Western blot analysis with anti-phosphotyrosine antibody and anti-MAPK antibody.

Northern blot analysis of MKP-1 mRNA expression. Confluent serum-starved VSMCs from WKY and SHR were treated with and without insulin (100 nM) or IGF-I (10-8 M) for 30 min and then followed by extraction and isolation of RNA with guanidinium isothiocyanate using Qiagen RNAeasy kit. RNA was quantitated by measuring the absorbance at 260/280 nm. Equal amounts of RNA (10 µg/lane) were separated on a 1.2% agarose-formaldehyde denaturing gel, transferred overnight to nitrocellulose membrane, and hybridized with 32P-labeled MKP-1 cDNA according to the standard protocols, followed by detection by autoradiography. The blot was stripped by boiling at 100°C for 5 min in 1% SDS and reprobed with beta -actin. The MKP-1 mRNA and beta -actin expressions were quantitated by densitometric analyses of the autoradiograms. The MKP-1 mRNA was normalized with respect to beta -actin.

Protein assay. Proteins in the cellular extracts and lysates were quantitated by bicinchoninic acid (27) or by the Bradford technique (2).

Statistics. The results are presented as means ± SE of four to six independent experiments each performed in triplicate at different times. Student's t-test or ANOVA was used to compare the mean values between WKY and SHR. A P value of <0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Basal and insulin-stimulated DNA synthesis in VSMCs. In the initial studies, basal and insulin-stimulated mitogenesis was examined in highly confluent (9-11 days in culture), growth-arrested primary cultures of VSMCs isolated from normotensive Sprague-Dawley rats, normotensive WKY, and SHR. VSMCs from SHR exhibit greater than a twofold increase in the basal rate of [3H]thymidine incorporation into DNA compared with WKY and Sprague-Dawley rats (275 ± 25 vs. 130 ± 15 or 122 ± 10 dpm/mg protein, respectively, P < 0.005). Treatment of serum-starved VSMCs with 100 nM insulin for 12 h resulted in a 150% increase in [3H]thymidine incorporation over the basal values in SHR (Fig. 1). The insulin effect was sustained for 24 h with a decline toward basal levels after 36 h (Fig. 1). In contrast to SHR, insulin caused only a 30% increase in [3H]thymidine incorporation in VSMCs isolated from WKY (Fig. 1) and Sprague-Dawley rats (data not shown). The insulin effect in SHR was dose dependent with an EC50 of ~10 nM insulin and a maximum effect at 100 nM (Fig. 2). VSMCs from SHR examined at subconfluent densities also exhibited greater than twofold elevation in mitogenic responses to insulin compared with WKY (data not shown). Pretreatment of VSMCs from SHR with IGF-I receptor antibody, clone alpha -IR3 (10 µg/well), followed by insulin (1-100 nM) had very little effect on insulin-stimulated mitogenesis (Fig. 2). At higher insulin concentrations of 1,000 nM, alpha -IR3 partially inhibited insulin's effect on mitogenesis (Fig. 2). In control experiments, IGF-I-induced mitogenesis, studied with 100 nM peptide was completely blocked by alpha -IR3 (760 ± 30 vs. 301 ± 10 dpm/mg protein). These observations suggest that insulin's growth-promoting effects at 10-100 nM peptide are mediated via its own receptor rather than via the IGF-I receptor. Hence, subsequent experiments on MAPK activation, MKP-1 expression, and mitogenesis were performed with 100 nM insulin.


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Fig. 1.   Kinetics of insulin-stimulated [3H]thymidine incorporation in vascular smooth muscle cells (VSMCs). Highly confluent VSMCs (9-11 days in culture) isolated from spontaneous hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were serum starved for 24 h followed by treatment with insulin (100 nM) for the indicated times. [3H]thymidine (1 µCi/ml) was added during the last 4 h of insulin treatment. Cells were rinsed 3 times with ice-cold PBS, once with 10% TCA, washed once with H2O, and solubilized in 0.1 N NaOH containing 0.1% SDS, and radioactivity was counted. Results are means ± SE of 4 independent experiments each performed in triplicate. * P < 0.05 vs. WKY.


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Fig. 2.   Dose response of insulin-stimulated mitogenesis in VSMCs. Serum-starved VSMCs from SHR and WKY were treated with various doses of insulin (0-1,000 nM) for 12 h. [3H]thymidine was added during the last 4 h and the incorporation of the label into TCA insoluble material was measured as detailed in Fig. 1. Results are means ± SE of 3 independent experiments performed in triplicate and are expressed as % activation. * P < 0.05 vs. WKY. To determine the contribution of insulin-like growth factor I (IGF-I) receptor, cells were pretreated with IGF-I receptor antibody (IGFR-Ab; clone IR3, 10 µg/ml) for 2 h before insulin exposure. In control experiments, alpha -IR3 completely blocked DNA synthesis induced by 100 nM IGF-I (301 ± 10 vs. 760 ± 30 dpm/mg protein). alpha -IR3 alone did not affect the basal levels of [3H]thymidine incorporation.

Differential effects of insulin on MAPK activation in VSMCs from SHR and WKY. A large number of studies indicate that the ERK family of MAPKs plays an important role in integrating receptor tyrosine kinase-initiated mitogenic signaling events in many cell types including VSMCs (6, 12, 19, 21). To examine whether the mitogenic effects of insulin in SHR are mediated via increased MAPK expression and/or activation, we measured the protein contents of MAPKs by immunoblot analyses of cell extracts and MAPK enzymatic activities in the ERK-1/ERK-2 immunoprecipitates.

In confluent serum-starved VSMCs from WKY, insulin had a small effect (30-40% increase over basal values) on MAPK activation measured over a range of insulin concentrations (Fig. 3A). In contrast, VSMCs from SHR exhibited greater than a twofold increase in MAPK activity in response to insulin. Maximal stimulation of MAPK was observed at a concentration of 100 nM insulin, whereas higher concentrations of insulin appear to reduce the extent of MAPK stimulation (Fig. 3A). The effect of insulin on MAPK was rapid, occurring within 10 min (Fig. 3B), and was sustained above basal levels up to 6 h in SHR in contrast to VSMCs from WKY, which exhibited a rapid return to basal levels within 30 min. Pretreatment with IGF-I receptor antibody did not abolish insulin's effect on MAPK activation (data not shown). The presence of chelerythrine HCl (a PKC inhibitor) and heat-stable PKA inhibitor during the assay decreased basal MAPK activities in the immunoprecipitates by 15%. The insulin-stimulated MAPK activities were not affected by the presence of these two inhibitors.


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Fig. 3.   Insulin stimulates mitogen-activated protein kinase (MAPK) activation in VSMCs from SHR. A: serum-starved WKY and SHR VSMCs were pulsed with insulin (0-1,000 nM) for 5 min, and MAPK activity was assayed in the immunoprecipitates using myelin basic protein (MBP) as a substrate and normalized to the amount of MAPK protein present in the immunoprecipitates. B: kinetics of insulin-stimulated MAPK activation. Serum-starved VSMCs were treated with 100 nM insulin for indicated times followed by the assay of MAPK activity in the immunoprecipitates. Details are given in METHODS. Results are means of 2-3 independent experiments each performed in duplicate. * P < 0.05 vs. WKY.

To further confirm that the differential effect of insulin on MAPK activation observed in SHR was due to phenotypic differences in responsiveness to insulin between the two cell types rather than due to loss of responsiveness of WKY because of culture conditions, we examined the effect of growth factors present in FBS on MAPK activation. As seen in Fig. 4, the extent of stimulation of MAPK by growth factors present in the serum was comparable between SHR and WKY. This observation further supports the notion that VSMCs from SHR are more responsive to insulin than WKY.


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Fig. 4.   Serum stimulation of MAPK is identical between SHR and WKY. Confluent, serum-starved VSMCs were stimulated with medium containing 10% fetal bovine serum (FBS) for 5 min, followed by immunoprecipitation of equal amounts of proteins with anti-extracellular-regulated protein kinase (ERK)-1/ERK-2 antibody (Zymed labs, 1 µg/sample). Immunoprecipitates were resuspended in kinase buffer and assayed for MAPK activity as detailed under METHODS. Results are means of 3 independent experiments each performed in duplicate. * P < 0.05 vs. control.

Inhibition of MEK blocks insulin-mediated MAPK activation and DNA synthesis in SHR. The above results provided correlative evidence that the MAPK pathway may play a significant role in insulin-stimulated mitogenesis in SHR. To further confirm that MAPK activation indeed plays a dominant role in insulin's effect on DNA synthesis, we used the recently developed MAPK-ERK kinase (MEK) inhibitor, PD-98059 (9, 26). This inhibitor selectively blocks the activity of the MAPK pathway at the level of MEK (9, 26). Pretreatment of VSMCs with 50 µM PD-98059 for 1 h completely blocked insulin-stimulated MAPK activation (Fig. 5) in SHR. Western blot analysis of immunoprecipitated MAPK with anti-phosphotyrosine antibodies revealed that insulin-induced MAPK activation in SHR was accompanied by increased tyrosine phosphorylation of MAPK (Fig. 6A). PD-98059 blocked the insulin-mediated increase in the tyrosine phosphorylation status of ERKs (Fig. 6A). The increase in MAPK activity observed in SHR was not due to an increase in the amount of MAPK protein (Fig. 6B, compare lanes 1-4 vs. lanes 5-8) but rather due to an increase in the activation of MAPK as evidenced by an increase in the tyrosine phosphorylation of the immunoprecipitated MAPK in SHR (Fig. 6A). Insulin stimulated tyrosine phosphorylation of several proteins, including three major bands of apparent molecular masses of ~90, 120, and 160 kDa in WKY and SHR (Fig. 6C). The identity of these bands is currently under investigation. PD-98059 did not alter the tyrosine phosphorylation status of other cellular proteins in SHR (Fig. 6C). Inhibition of insulin-induced MAPK activation by PD-98059 was accompanied by attenuation of insulin's effect on mitogenesis in SHR (Fig. 7).


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Fig. 5.   PD-98059 blocks insulin-stimulated MAPK activation. Serum-starved VSMCs were treated with 50 µM PD-98059 (MEK inhibitor) for 1 h before stimulation with insulin. Equal amounts of proteins were immunoprecipitated with anti-MAPK antibody and MAPK activity assayed in the immunoprecipitates. Results are means ± SE of 3 independent experiments, each performed in duplicate. * P < 0.05 vs. control; ** P < 0.05 vs. insulin. MAPK activation is expressed as % stimulation by insulin after normalization for the amount of MAPK protein in the immunoprecipitates. Amount of MAPK protein in the immunoprecipitates was assessed by Western blot analyses with an anti-MAPK antibody.


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Fig. 6.   A: insulin effect on MAPK activation is accompanied by increases in tyrosine phosphorylation of MAPK. PD-98059 specifically blocks tyrosine phosphorylation of MAPK. Equal amounts of cell lysate proteins from the VSMCs of WKY and SHR were immunoprecipitated (IP) with anti-MAPK antibody, and the immunoprecipitates were separated by SDS-PAGE, followed by Western blot analysis with anti-phosphotyrosine (pTYR) antibody. A representative autoradiogram is shown. Similar results were obtained in 3 different experiments. B: increased MAPK activation in SHR is not due to altered MAPK protein expression. Insulin treatment did not alter the contents of MAPK in VSMCs from SHR and WKY. Cell lysates with equal amounts of protein were separated by SDS-PAGE, followed by Western blot analysis with anti-MAPK antibody. Primary antibody was detected by using 125I-labeled protein A, followed by autoradiography. A representative autoradiogram is shown. C: treatment with PD-98059 did not alter tyrosine phosphorylation of the other cellular proteins. Cell lysates with equal amounts of proteins were separated by SDS-PAGE, followed by Western blot analysis with anti-phosphotyrosine antibody and detection with 125I-labeled protein A. A representative autoradiogram is shown.


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Fig. 7.   Inhibition of MAPK activation by PD-98059 is accompanied by blockade of insulin's effect on mitogenesis. Serum-starved VSMCs from SHR were pretreated with PD-98059 (50 µM) for 30 min before addition of insulin (100 nM) for 24 h. [3H]thymidine was added during the last 4 h. Results are means of 4 experiments each performed in triplicate. * P < 0.05 vs. control; ** P < 0.05 vs. insulin.

Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3-kinase) signaling with wortmannin completely blocked insulin's effect on MAPK activation and mitogenesis (Fig. 8, A and B).


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Fig. 8.   Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) blocks insulin's effect on MAPK activation and mitogenesis. Serum-starved VSMCs from SHR were treated with and without wortmannin (100 nM) for 30 min, followed by treatment with insulin (100 nM) for MAPK activation (A) and DNA synthesis (B). Results are means ± SE of 3-4 independent experiments each performed in duplicate. * P < 0.05 vs. control; ** P < 0.05 vs. insulin.

Differential effect of insulin on MKP-1 gene expression in WKY and SHR. It is well known that activation of all three family members of MAPKs is mediated by dual phosphorylation on tyrosine and threonine residues (6, 12, 19, 21, 25). Inactivation or attenuation of MAPK signaling is mediated by a class of dual-specificity protein phosphatases, for example, MKP-1. These phosphatases are transcriptionally regulated and are induced on treatment of cells with growth factors (25). To further understand the mechanism of abnormal MAPK activation by insulin in SHR, we examined the extent of MKP-1 induction in response to insulin. As seen in Fig. 9, low levels of MKP-1 mRNA are expressed in VSMCs in the basal state of both WKY and SHR. Insulin treatment resulted in a rapid increase (>2-fold over basal levels with 100 nM insulin) in MKP-1 mRNA induction in WKY in 30 min and a return to basal levels in 60 min (data not shown). The magnitude of induction of MKP-1 mRNA by insulin is comparable with that observed previously with ANG II (10). In contrast, VSMCs from SHR exhibited a 50-60% reduction in MKP-1 mRNA levels in the basal state (Fig. 9). Moreover, insulin as well as IGF-I caused very little induction of MKP-1 mRNA expression in SHR compared with WKY (Fig. 9). However, the effect of FBS on MKP-1 gene expression was comparable between SHR and WKY (Fig. 9). Studies are in progress to understand the exact signaling pathway utilized by insulin for MKP-1 induction in VSMCs.


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Fig. 9.   Increased insulin-stimulated MAPK activation in SHR is accompanied by impaired induction of MKP-1 mRNA by insulin and IGF-I. Serum-starved VSMCs from WKY and SHR were treated with insulin (100 nM), IGF-I (10 ng/ml), or 10% FBS for 30 min followed by extraction of RNA. Equal amount of RNA (10 µg/well) was separated on agarose-formaldehyde gel and followed by overnight transfer to nitrocellulose membrane. Membranes were hybridized with [32P]-labeled MKP-1 cDNA probe, stripped and reprobed with a beta -actin probe. Top: a representative autoradiogram. Bottom: quantitation of MKP-1 mRNA levels from multiple experiments by densitometric analyses. Relative mRNA levels were determined by laser densitometric scanning of the autoradiograms. To correct for variations in RNA loading, intensity of MKP-1 signal was divided by the intensity of beta -actin signal. Results are expressed as arbitrary densitometric units. To compare results from different experiments, mRNA from WKY controls were assigned a value of 1 and the rest of the data was calculated relative to WKY control. Results are means ± SE of 4 separate RNA blots from different experiments. * and ** P < 0.05 vs. WKY control; *** vs. insulin/IGF-I-treated WKY.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Primary cultures of VSMCs from SHR offer a good model system to examine the molecular basis of excessive cell growth commonly observed in hypertension, arteriosclerosis, and restenosis following balloon angioplasty. We have recently observed that the primary cultures of VSMCs isolated from SHR retain the phenotypic characteristics (excessive cell growth in response to mitogens and elevated intracellular calcium levels) in tissue culture for up to five passages that we have analyzed thus far (28).

The results presented in this study indicate that insulin differentially stimulates mitogenesis in highly confluent quiescent primary cultures of VSMCs derived from SHR, whereas VSMCs from WKY grown under identical conditions barely increase DNA synthesis in response to insulin. Studies by Mikhail et al. (18) also reported elevated insulin effect on cell growth in VSMCs derived from SHR.

On the basis of the results of insulin dose-response data and the inhibition by PD-98059, a synthetic inhibitor of MEK, the effects of insulin on mitogenesis appear to be mediated via the activation of the MAPK cascade, which in turn phosphorylates and activates other kinases and a battery of transcription factors, leading to increased DNA and protein synthesis. Thus MAPK activation may directly contribute to pathological changes such as smooth muscle cell hypertrophy/proliferation associated with large artery remodeling that occurs in response to hypertension. The observed effects of insulin on DNA synthesis and MAPK activation were mediated through an insulin receptor rather than the IGF-I receptor, as pretreatment with IGF-I receptor antibody did not abolish insulin's effects on MAPK activation and mitogenesis; however, inhibition of tyrosine kinase activity by treatment with erbstatin A as well as inhibition of PI3-kinase signaling with wortmannin completely blocked insulin's effects on MAPK activation and DNA synthesis.

MAPK activation by insulin was accompanied by a fourfold increase in tyrosine phosphorylation in SHR. Our findings are in agreement with a recent report demonstrating transient MAPK activation in rat aortic tissues in response to an acute elevation in blood pressure induced by either restraint or administration of hypertensive agents (i.e., phenylephrine and ANG II) (33). The increase in insulin-mediated MAPK activation observed in SHR in the present study may be due to a combination of increased signaling via p21ras/MAPK pathway (5) and defective insulin-mediated induction of MKP-1, a transcriptionally regulated dual-specificity phosphatase, responsible for inactivation of MAPK family members both in vivo as well as in vitro (30). In the present study, we observed that low levels of MKP-1 mRNA are present in the basal state in WKY and insulin as well as IGF-I caused a rapid induction of MKP-1 mRNA (>2-fold over basal levels) in VSMCs isolated from WKY. In contrast, VSMCs from SHR exhibit marked reductions in basal levels of MKP-1 mRNA and insulin and IGF-I failed to induce MKP-1 mRNA expression adequately. To our knowledge, this is the first study to demonstrate insulin-mediated induction of MKP-1 mRNA in VSMCs and its abnormal regulation in hypertension. Other studies have demonstrated induction of MKP-1 expression by FBS, ANG II, and platelet-derived growth factor in VSMCs (10, 11). A recent study by Lai et al. (17) reported that balloon injury of rat carotid artery was accompanied by decreased expression of MKP-1 mRNA, whereas p44 MAPK activity was increased. The results presented in this study add a new dimension to the observations that sustained MAPK activation seen in hypertension may be due to inherent reductions in MKP-1 mRNA expression resulting from defective regulation of MKP-1 gene expression, especially in response to insulin and IGF-I. This resistance to insulin of MKP-1 transcription may largely be responsible for the observed acceleration of VSMC growth seen during hypertension and atherosclerosis. Regarding the mechanism of MKP-1 expression, studies in NIH/3T3 fibroblasts indicate that MKP-1 induction is mediated by the SAPK signaling pathway and that the ERK pathway is not involved in MKP-1 induction (32). Our preliminary studies with anisomycin, a potent stimulator of JNK/SAPK, indicate that MKP-1 expression can be induced by anisomycin in VSMCs with a resultant inhibition of mitogenesis. Moreover, inhibition of ERKs and p38 MAPK signaling with PD-98059 and SB-203580, respectively, blocked insulin-mediated induction of MKP-1 expression. The above findings suggest the existence of a complex cross talk between the ERKs/SAPK and p38 MAPK signaling cascades. It remains to be seen whether insulin causes activation of SAPK in VSMCs. In this connection, it is interesting to note that studies by Moxham et al. (20) reported activation of JNK by insulin in rat skeletal muscle. We found that inhibition of PI3-kinase signaling pathway by treatment with wortmannin abrogates insulin's effects on MAPK activation, mitogenesis, and insulin-induced MKP-1 mRNA expression.

In summary, the results of this study indicate that insulin differentially stimulates mitogenesis in primary cultures of VSMCs isolated from SHR by activating MAPK cascade. An increased activation of MAPK due to defective induction of MKP-1 in hyperinsulinemic/hypertensive states may lead to VSMC proliferation and hypertrophy.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical support given by Priya Kumar and Deborah Nucatola in analyzing the effects of IGF-I receptor antibody on insulin-stimulated mitogenesis.

    FOOTNOTES

This work was supported in part by a "grant-in-aid" from the American Heart Association, New York State Affiliate, and the medical education funds from Winthrop University Hospital.

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. §1734 solely to indicate this fact.

Address for reprint requests: N. Begum, The Diabetes Research Laboratory, Winthrop Univ. Hospital, 259 First St., Mineola, NY 11501.

Received 18 February 1998; accepted in final form 20 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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