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(Hypertension. 2000;35:673.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Department of Pharmacology (T.I., K.T., M.N., H.M., T.U.) and Department of Neurosurgery (T.I., N.S.), Gifu University School of Medicine, Gifu, Japan; and the Department of Biochemistry (H.I., K.K.), Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
Correspondence to Osamu Kozawa, MD, PhD, Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan.
| Abstract |
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Key Words: protein kinases vasopressins aorta muscle, smooth, vascular
| Introduction |
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Vascular SMC play a crucial role in the pathogenesis of hypertension and atherosclerosis.5 Arginine vasopressin (AVP) is a vasoactive agent that stimulates the proliferation of vascular SMC6 and induces vasoconstriction.7 As for intracellular signaling in these cells, it has been shown that AVP induces phosphoinositide hydrolysis by phospholipase C through its binding to the V1 receptor8 and activates phospholipase D,9 10 which hydrolyzes phosphatidylcholine. Both types of hydrolysis result in the formation of diacylglycerol, a physiological activator of protein kinase C (PKC).11 12 The activation of PKC by AVP plays an important role in inducing proliferation of vascular SMC.13 In a previous study14 we have shown that AVP stimulates the induction of HSP27 through activation of PKC in an aortic SMC line, A10 cells.
The mitogen-activated protein (MAP) kinase superfamily plays a central role in intracellular signal transduction pathways initiated by a variety of extracellular stimuli.15 16 The specificity of the cellular response is recognized to be determined by the activation of a particular MAP kinase pathway in response to a given stimulus. The 3 MAP kinases, p42/p44 MAP kinase, p38 MAP kinase, and SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) are known as central elements used by mammalian cells to transduce the diverse messages.16 As for vascular SMC, p42/p44 MAP kinase has been reported to be activated and involved in the pathway of platelet-derived growth factorstimulated proliferation.16 Both p42/p44 MAP kinase and p38 MAP kinase have been shown to be phosphorylated by angiotensin II.16 In addition, it has recently been reported that H2O2 activates p42/p44 MAP kinase and p38 MAP kinase.16 However, the exact roles of MAP kinase superfamily in vascular SMC have not yet been fully clarified.
In the present study, we studied the involvement of the MAP kinase superfamily in the AVP-stimulated HSP27 induction in aortic smooth muscle A10 cells. Our results show that p38 MAP kinase among MAP kinases takes part in the pathway of the AVP-stimulated induction of HSP27 and that p38 MAP kinase acts at a point downstream from PKC activation in the pathway.
| Methods |
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Cell Culture
Aortic smooth muscle A10 cells were obtained from the American
Type Culture Collection and maintained as previously
described.14 In brief, the cells were seeded into 35-mm
(1x105) or 90-mm (5x105) diameter dishes.
After 5 days, the medium was exchanged for serum-free DMEM. After 48
hours, the cells were stimulated by AVP or TPA in serum-free DMEM for
the indicated periods. When indicated, the cells were pretreated with
PD98059, SB203580, calphostin C, PD169316, ET-18-OCH3, or
bisindolylmaleimide for 60 minutes before the stimulation.
Immunoassay of HSP27
The cultured cells were stimulated by AVP or TPA, rinsed twice
with PBS, and then frozen at -80°C for a few days before
analysis. The frozen cells in each dish were collected and
suspended in 0.3 mL of PBS, and each suspension was sonicated and
centrifuged at 125 000g for 20 minutes at 4°C.
The supernatant was used for the immunoassay of HSP27. The
concentrations of HSP27 in soluble extracts of the cells were
determined by sandwich-type enzyme immunoassays, as described
previously.2 3 Protein concentrations in soluble
extracts were determined with the use of a protein assay kit (Bio-Rad)
with BSA as the standard protein. Rat HSP27, which was used as the
standard for the immunoassay, was purified from skeletal
muscle.2 3
Sucrose Density Gradient Centrifugation
Extract of A10 cells was layered over a 3.5-mL linear gradient
of sucrose (10% to 40%) in 50 mmol/L Tris/HCl, pH 7.0, that
contained 5 mmol/L EDTA and was centrifuged at 4°C at
130 000g for 16 hours in a swinging bucket rotor (RPS56T;
Hitachi). After centrifugation, each sample was
fractionated into 15 test tubes, each of which contained 0.25 mL of
0.1% BSA.
Western Blot Analysis of p42/p44 MAP Kinase, p38 MAP
Kinase, and SAPK/JNK
The cultured cells were stimulated by AVP for the indicated
periods. Western blot analysis was performed as described
previously2 3 with each of the MAP kinase antibodies and
peroxidase-labeled antibodies raised in goat against rabbit IgG as
second antibodies. Peroxidase activity on the nitrocellulose sheet was
visualized on x-ray film by use of the ECL Western blotting detection
system.
Isolation of RNA and Northern Blot Analysis of mRNA
for HSP27
The cultured cells were stimulated by AVP for 12 hours. The
cells were then rinsed twice with PBS and frozen at -80°C for a few
days before analysis. Total RNA was isolated with the use of a
QuickPrep Total RNA Extraction kit (Pharmacia Biotech). Then, 20 µg
of total RNA was subjected to electrophoresis on a 0.9% agarose2.2
mol/L formaldehyde gel and blotted onto a nitrocellulose membrane. For
Northern blotting, the membrane was allowed to hybridize with the cDNA
probe that had been labeled with a Multiprime DNA labeling system, as
described previously.17 A BamHI-Hind
III fragment of cDNA for mouse HSP2718 was kindly
provided by Dr Lyndon F. Cooper of the University of North
Carolina.
Statistical Analysis
The data were analyzed by a 1-way ANOVA, followed by the
Bonferroni method for multiple comparisons between pairs. A value of
P<0.05 was considered significant. All data are
presented as means±SD of triplicate determinations from 3
independent experiments.
| Results |
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Effect of PD98059 on AVP-Induced Accumulation of HSP27 in A10
Cells
To clarify the role of p42/p44 MAP kinase in the AVP-stimulated
induction of HSP27, we next examined the effect of PD98059, which is a
specific inhibitor of the upstream kinase that
activates p42/p44 MAP kinase.19 The concentration
of HSP27 in unstimulated A10 cells was 542±35 ng/mg protein. The
AVP-induced accumulation of HSP27 was not affected by PD98059 over the
range of 0.1 to 10 µmol/L (Figure 2). We found that PD98059 significantly
reduced the AVP-induced phosphorylation of p42/p44 MAP
kinase (Figure 3A).
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Effects of SB203580 or PD169316 on AVP-Induced Accumulation of
HSP27 in A10 Cells
To clarify the role of p38 MAP kinase in the AVP-stimulated
induction of HSP27, we examined the effect of SB203580, a specific
inhibitor of p38 MAP kinase,20 on the
accumulation of HSP27 by AVP. We found that SB203580 also inhibited the
AVP-induced phosphorylation of p38 MAP kinase without
affecting the phosphorylation of p42/p44 MAP kinase by
AVP (Figure 3, B and C). The AVP-induced accumulation of HSP27
was significantly reduced by SB203580, which alone did not affect on
the basal levels of HSP27 (Figure 4, A
and B). The viability of the cells treated with SB203580 was >90%, as
assessed by trypan blue staining. The inhibitory effect of
SB203580 was dose-dependent over the range of 0.1 to 10 µmol/L.
The maximum effect of SB203580 was observed at 10 µmol/L, a dose
that caused
90% reduction in the AVP effect. In addition, PD169316,
another inhibitor of p38 MAP kinase,21 reduced
the AVP-stimulated HSP27 accumulation (689±43 ng/mg protein for
0.1 µmol/L AVP; 396±35 ng/mg protein for 0.1 µmol/L AVP
with 10 µmol/L PD169316, as measured during the stimulation for
48 hours; values for unstimulated cells have been subtracted from each
data point).
|
Effects of PD98059 or SB203580 on TPA-Induced Accumulation of HSP27
in A10 Cells
We previously showed that AVP stimulates the induction of HSP27
through PKC activation in A10 cells.14 We confirmed that
the accumulation of HSP27 by AVP was reduced by bisindolylmaleimide, an
alternative inhibitor of PKC22 (675±40 ng/mg
protein for 0.1 µmol/L AVP; 359±30 ng/mg protein for 0.1
µmol/L AVP with 3 µmol/L bisindolylmaleimide, as measured
during the stimulation for 48 hours; values for unstimulated cells have
been subtracted from each data point). Thus we next examined the effect
of PD98059 or SB203580 on the accumulation of HSP27 by TPA, a
PKC-activating phorbol ester.12 The TPA-induced
accumulation of HSP27 was significantly reduced by 10 µmol/L
SB203580, although it was not affected by 10 µmol/L PD98059
(Table). SB20358 (10 µmol/L) caused
70% reduction in the TPA effect.
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Effects of Calphostin C or ET-18-OCH3 on AVP-Induced
Phosphorylation of p38 MAP Kinase in A10 Cells
To clarify the effect of PKC on the AVP-induced
phosphorylation of p38 MAP kinase, we examined the
effects of highly specific PKC inhibitors, calphostin
C23 and ET-18-OCH3,24 on
phosphorylation. These inhibitors markedly
reduced the AVP-induced phosphorylation of p38 MAP
kinase (Figure 5, A and B).
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Effects of SB203580 or PD169316 on mRNA Levels for HSP27 by AVP in
A10 Cells
We further examined the effects of SB203580 and PD169316 on the
expression levels of mRNA for HSP27 by AVP. The increase by AVP in the
mRNA levels for HSP27 was significantly reduced by SB203580 or PD169316
(Figure 6, A and B).
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Dissociation of Aggregated Form of HSP27 by AVP in A10
Cells
HSP27 exists in 2 forms, an aggregated form and a dissociated
form.25 It is known that the dissociation of HSP27 occurs
concomitantly with the phosphorylation of HSP27, as
previously described.25 Our specific immunoassay of HSP27
detects both an aggregated form and a dissociated form, as previously
described.25 We investigated the response to AVP of the
aggregated form of HSP27. Extracts of unstimulated A10 cells contained
both forms, an aggregated form and a dissociated form (Figure 7). On the other hand, HSP27 in
AVP-stimulated A10 cells mainly showed a dissociated form (Figure 7).
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| Discussion |
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We showed that the AVP-induced accumulation of HSP27 was not affected
by PD98059, which reduced the phosphorylation of
p42/p44 MAP kinase by AVP. Therefore it seems unlikely that p42/p44 MAP
kinase is involved in the AVP-stimulated HSP27 induction. On the other
hand, SB203580 and PD169316 reduced the AVP-induced accumulation of
HSP27. We found that SB203580 actually inhibited the p38 MAP kinase
phosphorylation by AVP without affecting the p42/p44
MAP kinase phosphorylation by AVP. Thus these results
suggest that p38 MAP kinase is involved in the HSP27 induction by AVP.
Furthermore, to elucidate the role of p38 MAP kinase in the
AVP-stimulated induction of HSP27, we examined whether or not
inhibitors of p38 MAP kinase affect the mRNA levels for
HSP27. The increase by AVP in the mRNA levels for HSP27 was
significantly reduced by SB203580 or PD169316. On the basis of our
findings, it is most likely that the activation of p38 MAP kinase is
required for the AVP-stimulated HSP27 induction in A10 cells. As far as
we know, this is one of the first studies to show that the p38 MAP
kinase pathway mediates induction of HSP27 in vascular SMC. The p38 MAP
kinase superfamily consists of at least 4 different homologous
proteins: p38
, p38ß, p38
, and p38
.16 Among
these isoforms, p38
and p38ß are ubiquitously expressed. The
expression of p38
is most prominent in muscle. Each of these
isoforms contains a dual phosphorylation site on
threonine and tyrosine. Many of the properties of these isoforms are
very similar.16 However, it has recently been reported
that p38
, unlike the other 3 p38 isoforms, shows a lack of
sensitivity to the inhibitory properties of pyridimyl
imidazoles such as SB203580.27 We showed herein that
SB203580 suppressed the HSP27 accumulation by AVP. Thus, taking these
results into account, it seems unlikely that p38
is involved in the
AVP-stimulated HSP27 induction in A10 cells.
The evidence of implication between p38 MAP kinase and HSP27 is accumulating in several cells, showing that the activation of p38 MAP kinase leads to the phosphorylation of HSP27.1 It is recognized that these phenomena are induced within 1 hour after stimulation. HSP27 exists in 2 forms, an aggregated form and a dissociated form. It is known that the dissociation of HSP27 occurs concomitantly with the phosphorylation of HSP27, as previously described.25 Thus we examined the possible existence of 2 forms of HSP27 in unstimulated or AVP-stimulated A10 cells. Extracts of unstimulated A10 cells contained both forms, an aggregated form and a dissociated form. On the contrary, HSP27 in AVP-stimulated A10 cells mainly showed a dissociated form. These findings suggest that AVP phosphorylates HSP27. We have reported that AVP significantly stimulates HSP27 induction after 36 hours from the stimulation.14 Taking our results into account, it is most likely that AVP not only phosphorylates HSP27 but also stimulates HSP27 induction in A10 cells.
In a previous study14 we have shown that AVP stimulates the induction of HSP27 by PKC activation in A10 cells. We demonstrated that the TPA-induced accumulation of HSP27 was reduced by SB203580. On the contrary, the HSP27 accumulation was not affected by PD98059. In addition, calphostin C and ET-18-OCH3, PKC inhibitors,23 24 suppressed the phosphorylation of p38 MAP kinase by AVP. Our results suggest that PKC acts at a point upstream from p38 MAP kinase in the AVP-stimulated induction of HSP27 in A10 cells.
Accumulating evidence indicates that HSP27 in vivo may act in the regulation of the structure and dynamics of actin filaments for an increased survival of cells recovering from stress.28 29 In addition, HSP27 has been reported to be associated with agonist-induced contraction of vascular SMC.30 On the other hand, it has been reported that the induction of HSP72 in vascular SMC before mechanical injury results in reduced proliferation without overt cytotoxicity.31 On the basis of these findings, it is probable that the induction of HSPs such as HSP27 by vasoactive agents might act protectively under hazardous conditions in vascular SMC. Investigations of HSPs in vascular SMC may contribute to vascular pathology such as hypertension, arteriosclerosis, and aging of vasculature. Further investigations would be required to clarify the details.
In conclusion, our results strongly suggest that p38 MAP kinase is required for AVP-stimulated HSP27 induction in vascular SMC, and the activation of p38 MAP kinase is dependent on PKC.
| Acknowledgments |
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Received February 12, 1999; first decision March 10, 1999; accepted September 29, 1999.
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