p38 MAP Kinase Is Required for Vasopressin-Stimulated HSP27 Induction in Aortic Smooth Muscle Cells
Abstract—We previously showed that arginine vasopressin (AVP) stimulates heat shock protein 27 (HSP27) induction through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in the AVP-stimulated HSP27 induction in A10 cells. AVP stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. On the contrary, AVP had little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) phosphorylation. The HSP27 accumulation by AVP was not affected by PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, suppressed the AVP-induced accumulation of HSP27. 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C, induced accumulation of HSP27 and was not inhibited by PD98059 but was inhibited by SB203580. Calphostin C and ET-18-OCH3, inhibitors of protein kinase C, reduced the phosphorylation of p38 MAP kinase by AVP. SB203580 and PD169316 suppressed the AVP-increased levels in mRNA for HSP27. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by AVP. These results strongly suggest that p38 MAP kinase takes part in the pathway of the AVP-stimulated induction of HSP27 in vascular smooth muscle cells.
When cells or organs are exposed to stress such as heat, they produce a set of proteins known as heat shock proteins (HSPs).1 HSPs are divided into 2 groups, low- and high-molecular-weight HSPs such as HSP90 and HSP70. HSP27 is a member of the family of low-molecular-weight HSPs with molecular masses from 15 to 30 kDa. HSP27 is present in various tissues and cells, especially in skeletal muscle cells and smooth muscle cells (SMC), as we showed previously.2 3 The functions of HSP27 are less understood than those of high-molecular-weight HSPs. As one of the functions, it is recognized that HSP27 may act as chaperones,1 like high-molecular-weight HSPs. In addition, evidence is accumulating that HSP27 participates in physiological processes such as modulation of actin filament and stability, growth, and secretion in several types of cells.1 These observations suggest that HSP27 may have important roles in diverse cellular functions. In vascular SMC, it has been reported that restraint stress induces HSP27 mRNA.4 However, neither the exact mechanism of HSP27 induction nor the roles have been fully clarified.
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 factor–stimulated 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.
AVP was purchased from Peptide Institute, Inc. PD98059, SB203580, PD169316, ET-18-OCH3, and bisindolylmaleimide were from Calbiochem-Novabiochem. 12-O-tetradecanoylphorbol 13-acetate (TPA) was from Sigma Chemical Co. Calphostin C was from Funakoshi Pharmaceutical Co. Phosphospecific p42/p44 MAP kinase antibodies, p42/p44 MAP kinase antibodies, phosphospecific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phosphospecific SAPK/JNK antibodies, and SAPK/JNK antibodies were from New England BioLabs, Inc. The ECL Western blotting detection system was from Amersham. Other materials and chemicals were obtained from commercial sources. PD98059, SB203580, calphostin C, TPA, PD169316, ET-18-OCH3, and bisindolylmaleimide were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the measurement made in the immunoassay of HSP27, Western blot analysis, nor Northern blot analysis.
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 (1×105) or 90-mm (5×105) 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% agarose–2.2 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.
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.
Effect of AVP on Phosphorylation of p42/p44 MAP Kinase, p38 MAP Kinase, and SAPK/JNK in A10 Cells
We first examined the effect of AVP on the phosphorylation of p42/p44 MAP kinase, p38 MAP kinase, and SAPK/JNK. Stimulation by vasopressin significantly induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase (Figure 1⇓). The time courses in the phosphorylation of the 2 MAP kinases by AVP appeared to be quite different from each other. The phosphorylation of p42/p44 MAP kinase by AVP reached the peak at 5 minutes. On the other hand, the phosphorylation of p38 MAP kinase reached the peak at 20 minutes. However, AVP had little effect on the phosphorylation of SAPK/JNK (Figure 1⇓).
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⇓).
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.
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).
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).
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⇓).
We previously showed that AVP stimulates the induction of HSP27 in aortic smooth muscle A10 cells and that the activation of PKC acts as a positive regulator in the induction of HSP27.14 In the present study, we examined whether or not the MAP kinase superfamily is involved in the AVP-stimulated HSP27 induction in A10 cells. AVP stimulated the significant phosphorylation of p42/p44 MAP kinase and p38 MAP kinase while having little effect on the phosphorylation of SAPK/JNK. It is well recognized that MAP kinase is activated by dual phosphorylation on threonine and tyrosine by specific MAP kinase kinase.26 Thus our findings suggest that AVP activates p42/p44 MAP kinase and p38 MAP kinase in A10 cells.
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: p38α, p38β, p38γ, and p38δ.16 Among these isoforms, p38α and p38β are ubiquitously expressed. The expression of p38γ 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 p38δ, 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 p38δ 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.
We are grateful to Kouseki Hirade, Daijiro Hatakeyama, and Masaichi Miwa for their skillful technical assistance.
- Received February 12, 1999.
- Revision received March 10, 1999.
- Accepted September 29, 1999.
Benjamin IJ, Mcmillan DR. Stress (heat shock) proteins: molecular chaperons in cardiovascular biology and disease. Circ Res. 1998;83:117–132.
Inaguma Y, Goto S, Shinohara H, Hasegasa K, Ohshima K, Kato K. Physiological and pathological changes in levels of 2 small stress proteins, HSP27 and αB crystallin, in rat hind limb muscles. J Biochem. 1993;114:378–384.
Udelsman R, Blake MJ, Stagg CA, Li DG, Putney DJ, Holbrook NJ. Vascular heat shock protein expression in response to stress. J Clin Invest. 1993;91:465–473.
Scott-Burden T, Hahn AWA, Buhler FR, Resink TL. Vasoactive peptides and growth factors in the pathophysiology of hypertension. J Cardiovasc Pharmacol. 1992;20:S55–S64.
Grillone LR, Clark MA, Godfrey RW, Stassen F, Crooke ST. Vasopressin induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholine-specific phospholipase C and stimulates the release of arachidonic acid by at least 2 pathways in the smooth muscle cell line, A-10. J Biol Chem. 1988;263:2658–2663.
Billah MM, Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J. 1990;269:281–291.
Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986;233:305–312.
Cramelo C, Okada K, Tsai P, Schrier RW. Phorbol esters and arginine vasopressin in vascular smooth muscle cell activation. Am J Physiol. 1989;256:F875–F881.
Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev. 1999;79:143–180.
Kato K, Goto S, Hasegawa K, Inaguma Y. Coinduction of 2 low-molecular-weight stress proteins, αB crystallin and HSP28, by heat or arsenite stress in human glioma cells. J Biochem. 1993;114:640–647.
Cooper LF, Uoshima K. Differential estrogenic regulation of small Mr heart shock protein expression in osteoblasts. J Biol Chem. 1994;269:7869–7873.
Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.
Kummer JL, Rao PK, Heidenreich KA. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem. 1997;282:20490–20494.
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F. The bisindolylmaleimide GF109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991;266:15771–15781.
Kato K, Hasegawa K, Goto S, Inaguma Y. Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol Chem. 1994;269:11274–11278.
Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem. 1995;270:7420–7426.
Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Padova FD, Ulevitvh RJ, Han J. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinase, p38δ. J Biol Chem. 1997;282:30122–30128.
Lavoie JN, Hickey E, Weber LA, Landry J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem. 1993;268:24210–24214.