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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 I (cGK I
), 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 I
induction of MKP-1 and
consequent inactivation of MAPKs.
nitric oxide; guanosine 3',5'-cyclic monophosphate; cGMP-dependent
protein kinase I; platelet-derived growth factor; hypertension
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-1. 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 I (cGK I
), the downstream
effector of the NO/cGMP signaling pathway, enhances the inhibitory
effects of insulin on PDGF-directed VSMC migration.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
PDGF-BB, cell culture reagents, fetal bovine serum, and antibiotics
were purchased from Life Technologies (Grand Island, NY). [-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 -actin was used to assess the purity (>99%) of the
smooth muscle cell cultures. Unless otherwise indicated, primary
cultures of VSMCs were maintained in
-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
-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
-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
-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 I and MKP-1.
The adenoviral vector for expressing cGK I
was constructed by first
cloning the cDNA of human cGK I
into the multiple cloning site of
the adenoviral transfer plasmid pCMVI/
E1sp1A, itself generated by
cloning the expression cassette of the pCI expression vector (Promega,
Madison, WI) into the plasmid p
E1sp1A containing NH2-terminal E1-deleted sequences of Ad5 (Microbix
Biosystems, Toronto, Canada). The resulting plasmid pCMVI/cGK I
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 I
) was generated via homologous recombination between pCMVI/cGK I
and pJM17, screened for
by using the polymerase chain reaction, and then recovered and
plaque-purified as described previously for Ad5-cGK I
(33). MKP-1- and
-galactosidase-expressing adenoviruses
were generated by cotransfection of plasmid pACCMVpLpA-MKP-1 or
pACCMVpLpA-
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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).
|
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 8 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 5 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).
|
|
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 -galactosidase (Ad.
-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).
|
|
Ad.cGK I 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 I
, 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 I
infection of
VSMCs was accompanied by an increase in cGK I
protein expression
10-fold higher than that of uninfected cells. Furthermore, cGK I
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 I
(Fig. 7C)
compared with that in noninfected VSMCs (Fig. 7C, lane
2). Most importantly, insulin and 8-BrcGMP, which activate cGK
I
(6), decreased PDGF-induced MAPK phosphorylation by
80 and 90%, respectively, in cGK I
-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 I
expression enhances
insulin-mediated inhibition of MAPK phosphorylation in PDGF-stimulated
cells via MKP-1 expression.
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 I, 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 I
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 I
directly, was more potent than
insulin in inhibiting VSMC migration in cGK I
/MKP-1-expressing
VSMCs. This observation suggests that, in addition to cGK I
, cGMP
generation is a major determinant of insulin-mediated inhibition of
VSMC migration via MKP-1 expression. It should be noted that cGK I
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 I
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- 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 I
, 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 I
(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 I 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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, H,
Yamada N,
Kamata K,
Kuwaki T,
Shimada M,
Osuga J,
Shionoiri F,
Yahagi N,
Kadowaki T,
Tamemoto H,
Ishibashi S,
Yazaki Y,
and
Makuuchi M.
Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1.
J Clin Invest
101:
1784-1788,
1998
2.
Abedi, H,
and
Zachary I.
Signalling mechanisms in the regulation of vascular cell migration.
Cardiovasc Res
30:
544-556,
1995[ISI][Medline].
3.
Begum, N,
Duddy N,
Sandu O,
Rienzie J,
and
Ragolia L.
Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase dependent signaling pathways.
Mol Endocrinol
14:
1365-1376,
2000
4.
Begum, N,
and
Ragolia L.
High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation.
Am J Physiol Cell Physiol
278:
C81-C91,
2000
5.
Begum, N,
Ragolia L,
Rienzie J,
McCarthy M,
and
Duddy N.
Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension.
J Biol Chem
273:
25164-25170,
1998
6.
Begum, N,
Sandu OA,
Ito M,
Lohmann SM,
and
Smolenski A.
Active Rho kinase (ROK-a) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.
J Biol Chem
277:
6214-6222,
2002
7.
Begum, N,
Song Y,
Rienzie J,
and
Ragolia L.
Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension.
Am J Physiol Cell Physiol
275:
C42-C49,
1998
8.
Bornfeld, KE,
Raines EW,
Nakano T,
Graves LM,
Krebs EG,
and
Ross R.
IGF-1 and PDGF-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation.
J Clin Invest
93:
1266-1274,
1994[ISI][Medline].
9.
Brizzi, MF,
Formato L,
Dentelli P,
Rosso A,
Pavan M,
Garbarino G,
Pegoraro M,
Camussi G,
and
Pegoraro L.
Interleukin-3 stimulates migration and proliferation of vascular smooth muscle cells: a potential role in atherogenesis.
Circulation
103:
549-554,
2001
10.
Cospedal, R,
Abedi H,
and
Zachary I.
Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases.
Cardiovasc Res
41:
708-721,
1999[ISI][Medline].
11.
Dubey, RK,
Jackson EK,
and
Luscher TF.
Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. Role of cyclic-nucleotides and angiotensin1 receptors.
J Clin Invest
96:
141-149,
1995[ISI][Medline].
12.
Gockerman, A,
Prevette T,
Jones JI,
and
Clemmons DR.
Insulin like growth factor-1 binding protein inhibits the smooth muscle cell migration responses to IGF-1 and IGF II.
Endocrinology
136:
4168-4173,
1995[Abstract].
13.
Goetze, S,
Xi XP,
Kawano H,
Gotlibowski T,
Fleck E,
Hsueh W,
and
Law RE.
PPAR gamma-ligands inhibit migration mediated by multiple chemoattractants in vascular smooth muscle cells.
J Cardiovasc Pharmacol
33:
798-806,
1999[ISI][Medline].
14.
Goetze, S,
Xi XP,
Kawano Y,
Kawano H,
Fleck E,
Hsueh W,
and
Law RE.
TNF--induced migration of vascular smooth muscle cells is MAPK dependent.
Hypertension
33:
183-189,
1999
15.
Gomez-Foix, AM,
Coats WS,
Baque S,
Alam T,
Gerard RD,
and
Newgard CW.
Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism.
J Biol Chem
267:
25129-25134,
1992
16.
Grotendorst, GR,
Chang T,
Seppa HEJ,
Kleinman HK,
and
Martin GR.
Platelet derived growth factor is a chemoattractant for vascular smooth muscle.
J Cell Physiol
113:
261-266,
1983[ISI].
17.
Ishigami, M,
Swertfeger DK,
Granholm NA,
and
Hui DY.
Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase.
J Biol Chem
273:
20156-20161,
1998
18.
Kahn, AM,
Allen JC,
Seidel CL,
and
Zang S.
Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase.
Hypertension
35:
303-306,
2000
19.
Lundberg, MS,
Curto KA,
Bilato C,
Monticone RE,
and
Crow MT.
Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways.
J Mol Cell Cardiol
30:
2377-2389,
1998[ISI][Medline].
20.
Mansfield, PJ,
Shayman JA,
and
Boxer LA.
Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase.
Blood
95:
2407-2412,
2000
21.
Montagnani, M,
and
Quon MJ.
Insulin action in vascular endothelium: potential mechanisms linking insulin resistance with hypertension.
Diabetes Obes Metab
2:
285-292,
2000[ISI][Medline].
22.
Nakao, J,
Ito H,
Kanayasu T,
and
Murota S.
Stimulatory effect of insulin on smooth muscle cell migration induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and its modulation by elevated extracellular glucose levels.
Diabetes
34:
185-191,
1985[Abstract].
23.
Nelson, PR,
Yamamura S,
Mureebe L,
Itoh H,
and
Kent C.
Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase.
J Vasc Surg
27:
117-125,
1998[ISI][Medline].
24.
Pauly, RR,
Passaniti A,
Bilato C,
Monticone R,
Cheng L,
Papadopoulos N,
Gluzband YA,
Smith L,
Weinstein C,
Lakatta EG,
and
Crow MT.
Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation.
Circ Res
75:
41-54,
1994[Abstract].
25.
Ross, R.
The pathogenesis of atherosclerosis: a perspective for the 1990s.
Nature
362:
801-809,
1993[ISI][Medline].
26.
Scherrer, U,
Randin D,
Vollenweider P,
Vollenweider L,
and
Nicod P.
Nitric oxide release accounts for insulin's vascular effects in humans.
J Clin Invest
94:
2511-2515,
1994[ISI][Medline].
27.
Shoemaker, JK,
and
Bonen A.
Vascular actions of insulin in health and disease.
Can J Appl Physiol
20:
127-154,
1995[ISI][Medline].
28.
Steinberg, HO,
Brechtel G,
Johnson A,
Fineberg N,
and
Baron AD.
Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release.
J Clin Invest
94:
1172-1179,
1994[ISI][Medline].
29.
Steinberg, HO,
Chaker H,
Leaming R,
Johnson A,
Brechtel G,
and
Baron AD.
Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance.
J Clin Invest
97:
2601-2610,
1996
30.
Smith, PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Gartner FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
and
Klenk DC.
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:
76-85,
1985[ISI][Medline].
31.
Sugimoto, T,
Haneda M,
Togawa M,
Isono M,
Shikano T,
Araki S,
Nakagawa T,
Kashiwagi A,
Guan KL,
and
Kikkawa R.
Atrial natriuretic peptide induces the expression of MKP-1, a mitogen-activated protein kinase phosphatase, in glomerular mesangial cells.
J Biol Chem
271:
544-547,
1996
32.
Takehara, N,
Kawabe J,
Aizawa Y,
Hasebe N,
and
Kikuchi K.
High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells.
Biochim Biophys Acta
1497:
244-252,
2000[ISI][Medline].
33.
Vaandrager, AB,
Tilly BC,
Smolenski A,
Schneider-Rasp S,
Bot AG,
Edixhoven M,
Scholte BJ,
Jarchau T,
Walter U,
Lohmann SM,
Poller WC,
and
de Jonge HR.
cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl channels co-expressed with cGMP-dependent protein kinase type II but not type I
.
J Biol Chem
272:
4195-4200,
1997
34.
Yamboliev, IA,
Chen J,
and
Gerthoffer WT.
PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs.
Am J Physiol Cell Physiol
281:
C709-C718,
2001
35.
Yasunari, K,
Kohno M,
Kano H,
Yokokawa K,
Minami M,
and
Yoshikawa Y.
Mechanisms of action of troglitazone in the prevention of high glucose-induced migration and proliferation of cultured coronary smooth muscle cells.
Circ Res
81:
953-962,
1997