Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.     

Possible involvement of phosphatidylinositol 3-kinase/Akt signal pathway in vasopressin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells

We previously reported that p38 mitogen-activated protein (MAP) kinase takes a part in arginine vasopressin (AVP)-induced heat shock protein 27 (HSP27) phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the phosphorylation of HSP27 in these cells. AVP time-dependently induced the phosphorylation of PI3K and Akt. Akt inhibitor, 1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, partially suppressed the phosphorylation of HSP27. The AVP-induced HSP27 phosphorylation was attenuated by LY294002, a PI3K inhibitor. The combination of Akt inhibitor and SB203580, a p38 MAP kinase inhibitor, completely suppressed the AVP-induced phosphorylation of HSP27. Furthermore, LY294002 or Akt inhibitor did not affect the AVP-induced phosphorylation of p38 MAP kinase and SB203580 did not affect the phosphorylation of PI3K or Akt. These results suggest that PI3K/Akt plays a part in the AVP-induced phosphorylation of HSP27, maybe independently of p38 MAP kinase, in aortic smooth muscle A10 cells.     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.     

Thrombin stimulates dissociation and induction of HSP27 via p38 MAPK in vascular smooth muscle cells

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells.     

Midazolam suppresses thrombin-induced heat shock protein 27 phosphorylation through inhibition of p38 mitogen-activated protein kinase in cardiac myocytes

It has been shown that anesthetics have effects of cardiac preconditioning. Heat shock proteins (HSPs) function as molecular chaperone. Among them, HSP27, a low-molecular-weight HSP, abundantly exist in heart. However, the relationship between anesthetics and HSP27 in heart is not yet clarified. We investigated whether thrombin induces or phosphorylates HSP27 in primary cultured mouse myocytes and the effect of midazolam on the thrombin-stimulated HSP27 phosphorylation and the mechanism behind it. Thrombin time dependently phosphorylated HSP27 at Ser-15 and Ser-85 while having no effect on the levels of HSP27. Midazolam markedly suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. Thrombin induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase without affecting stress-activated protein kinase/c-Jun N-terminal kinase. In addition, midazolam attenuated the phosphorylation of thrombin-induced p38 MAP kinase but not that of p44/p42 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. These results strongly suggest that thrombin induces the HSP27 phosphorylation at least through the p38 MAP kinase activation in cardiac myocytes and that midazolam inhibits the thrombin-induced HSP27 phosphorylation via suppression of p38 MAP kinase activation.     

Phosphatidylinositol 3-kinase/Akt plays a role in sphingosine 1-phosphate-stimulated HSP27 induction in osteoblasts

We previously reported that p38 mitogen-activated protein (MAP) kinase plays a part in sphingosine 1-phosphate-stimulated heat shock protein 27 (HSP27) induction in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the induction of HSP27 in these cells. Sphingosine 1-phosphate time dependently induced the phosphorylation of Akt. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduced the HSP27 induction stimulated by sphingosine 1-phosphate. The sphingosine 1-phosphate-induced phosphorylation of GSK-3beta was suppressed by Akt inhibitor. The sphingosine 1-phosphate-induced HSP27 levels were attenuated by LY294002 or wortmannin, PI3K inhibitors. Furthermore, LY294002 or Akt inhibitor did not affect the sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase and SB203580, a p38 MAP kinase inhibitor, had little effect on the phosphorylation of Akt. These results suggest that PI3K/Akt plays a part in the sphingosine 1-phosphate-stimulated induction of HSP27, maybe independently of p38 MAP kinase, in osteoblasts.     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.     

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.     

Modulation by the steroid/thyroid hormone superfamily of TGF-beta-stimulated VEGF release from vascular smooth muscle cells

We previously reported that transforming growth factor-beta (TGF-beta) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen-activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF-beta-stimulated VEGF release from A10 cells. Retinoic acid or 1,25-dihydroxyvitamin D3 enhanced TGF-beta-induced VEGF release in a concentration-dependent manner, whereas dexamethasone or corticosterone suppressed TGF-beta-induced VEGF release. 1,25-Dihydroxyvitamin D3 and TGF-beta stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF-beta or 1,25-dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25-dihydroxyvitamin D3, and glucocorticoids affect TGF-beta-stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25-dihydroxyvitamin D3 occurs, in part, via modification of TGF-beta-induced activation of p38 MAP kinase.     

Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases

We investigated whether transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-beta increased the level of HSP27 but had no effect on the HSP70 level. TGF-beta stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-beta induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-beta was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-beta-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-induced increase of mRNA for HSP27. The amplification of TGF-beta-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-beta-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-beta-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.     

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.     

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.     

Alpha2 adrenoreceptor agonist regulates protein kinase C-induced heat shock protein 27 phosphorylation in C6 glioma cells

Dexmedetomidine (Dexmd), a potent and highly specific α₂ adrenoreceptor agonist, is an efficient therapeutic agent for sedation. Dexmd has been recently reported to have a neuroprotective effect. Heat shock protein (HSP) 27, a low-molecular weight HSP has been shown to be expressed following cerebral ischemia in astrocytes but not in neurons. HSP27 expression is involved in ischemic tolerance of the brain. This study investigated the effect of Dexmd on HSP27 in rat C6 glioma cells. 12- O -tetradecanoylphorbol-13-actate (TPA), a direct activator of protein kinase C (PKC), stimulated the phosphorylation of HSP27 at Ser82, but not Ser15 in a time-dependent manner. Prostaglandin (PG) E₁ or PGE₂ which activates the adenylyl cyclase-cAMP system as well as forskolin and dibutyryl-cAMP, suppressed the TPA-induced phosphorylation of HSP27. Dexmd reversed the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system. Therefore, these results strongly suggest that Dexmd reverses the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system activation through the inhibition of its system in C6 cells. α₂ Adrenoreceptor agonists may therefore show a neuroprotective effect through the modification of HSP27 phosphorylation induced by PKC activation.     

Platelet-derived growth factor-BB phosphorylates heat shock protein 27 in cardiac myocytes

It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet-derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF-BB induced the phosphorylation of HSP27 at Ser-15 and -85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF-BB time dependently phosphorylated HSP27 at Ser-15 and -85. PDGF-BB stimulated the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF-BB-stimulated phosphorylation of HSP27 at both Ser-15 and -85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF-BB phosphorylates HSP27 at Ser-15 and -85 via p38 MAP kinase in cardiac myocytes.     

A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.

Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1beta, and transforming growth factor-beta. Cellular migration was blocked by SB203580, an inhibitor of p38(MAPK). Time course experiments demonstrated increased phosphorylation of p38(MAPK). Activation of p38(MAPK) resulted in the phosphorylation of HSP27 (heat shock protein 27), which may modulate F-actin polymerization. Inhibition of p38(MAPK) activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38(MAPK), increased cell migration, whereas overexpression of p38alpha MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38(MAPK) pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.     

Mechanism of simvastatin on induction of heat shock protein in osteoblasts

It has recently been reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) stimulate bone formation. However, the mechanism of stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether simvastatin induces heat shock protein (HSP) 27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Simvastatin increased the levels of HSP27 while having little effect on the levels of HSP70 or HSP90. The effect of simvastatin on HSP27 accumulation was dose dependent. Cycloheximide reduced the accumulation. Simvastatin induced an increase in the levels of mRNA for HSP27. Actinomycin D suppressed the mRNA levels. Simvastatin induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase among the MAP kinase superfamily. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by simvastatin while SB202474, a negative control of p38 MAP kinase inhibitor, had no effect. SB203580 reduced the simvastatin-increased mRNA levels for HSP27. Lovastatin, another statin, also induced the HSP27 accumulation and SB203580 suppressed the HSP27 accumulation. These results strongly suggest that statins such as simvastatin do not stimulate the induction of HSP70 and HSP90, but do stimulate the induction of HSP27 in osteoblasts and that p38 MAP kinase plays a role in this induction.     

Enhancement by sphingosine 1‐phosphate in vasopressin‐induced phosphoinositide hydrolysis in aortic smooth‐muscle cells: Involvement of p38 MAP kinase

We previously reported that sphingosine 1‐phosphate (S‐1‐P), a sphingomyelin metabolite, activates p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in aortic smooth‐muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C‐catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C₂‐ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S‐1‐P, which alone slightly stimulated the IPs formation, dose‐dependently amplified the AVP‐induced formation of IPs. Tumor necrosis factor‐α enhanced the AVP‐induced formation of IPs. However, S‐1‐P did not enhance the formation of IPs by NaF, a heterotrimeric GTP‐binding protein activator. Pertussis toxin inhibited the effect of S‐1‐P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S‐1‐P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S‐1‐P on the formation of IPs by AVP. SB203580 inhibited the AVP‐induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S‐1‐P. These results indicate that S‐1‐P amplifies AVP‐induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth‐muscle cells. J. Cell. Biochem. 80:46–52, 2000. © 2000 Wiley‐Liss, Inc.     

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells

Although it is known that transforming growth factor (TGF)-beta induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF-beta stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The VEGF synthesis induced by TGF-beta was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF-beta in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF-beta-stimulated synthesis of VEGF (each in a dose-dependent manner). PD169316 or SB203580 attenuated the TGF-beta-induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF-beta stimulates the synthesis of VEGF in aortic smooth muscle cells.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.