Minodronate suppresses prostaglandin F2alpha-induced vascular endothelial growth factor synthesis in osteoblasts.

In our previous study, we showed that prostaglandin F2alpha (PGF2alpha) stimulates vascular endothelial growth factor (VEGF) synthesis via activation of p44/p42 mitogen-activated protein (MAP) kinase via protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In addition, we demonstrated that incadronate amplified, and tiludronate suppressed PGF2alpha-induced VEGF synthesis among bisphosphonates, while alendronate or etidronate had no effect. In the present study, we investigated the effects of minodronate, a newly developed bisphosphonate, on PGF (2alpha)-induced VEGF synthesis in MC3T3-E1 cells. Minodronate significantly reduced VEGF synthesis induced by PGF2alpha dose-dependently at levels between 3 and 100 microM. PGF2alpha-stimulated phosphorylation of Raf-1, MEK1/2 and p44/p42 MAP kinase were suppressed by minodronate. 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator VEGF synthesis induced by PKC, was inhibited by minodronate. Minodronate inhibited Raf-1, MEK1/2 and p44/p42 MAP kinase phosphorylation induced by TPA. Mevalonate failed to affect the suppressive effect of minodronate on PGF2alpha-induced VEGF synthesis. Taken together, these results indicate that minodronate suppresses PGF2alpha-stimulated VEGF synthesis at the point between PKC and Raf-1 in osteoblasts.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Platelet-derived growth factor-BB amplifies PGF2alpha-stimulated VEGF synthesis in osteoblasts: function of phosphatidylinositol 3-kinase

We have reported that prostaglandin F2alpha (PGF2alpha) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that phosphatidylinositol 3 (PI3)-kinase activated by platelet-derived growth factor-BB (PDGF-BB) negatively regulates the interleukin-6 synthesis in these cells. In the present study, we investigated the effect of PDGF-BB on the PGF2alpha-induced VEGF synthesis in MC3T3-E1 cells. PDGF-BB, which alone did not affect the levels of VEGF, significantly enhanced the PGF2alpha-stimulated VEGF synthesis. The amplifying effect of PDGF-BB was dose dependent in the range between 10 and 70 ng/ml. LY294002 or wortmannin, specific inhibitors of PI3-kinase, which by itself failed to affect the PGF2alpha-stimulated VEGF synthesis, significantly suppressed the amplification by PDGF-BB. PD98059, a specific inhibitor of MEK1/2, suppressed the amplification by PDGF-BB of the PGF2alpha-stimulated VEGF synthesis similar to the levels of PGF2alpha with PD98059. PDGF-BB itself induced the phosphorylation of p44/p42 MAP kinase in these cells, and the effects of PDGF-BB and PGF2alpha on the phosphorylation of p44/p42 MAP kinase were additive. Moreover, LY294002 had little effect on the phosphorylation of p44/p42 MAP kinase induced by PGF2alpha with PDGF-BB. These results strongly suggest that PGF2alpha-stimulated VEGF synthesis is amplified by PI3-kinase-mediating PDGF-BB signaling in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Sphingosine 1-phosphate amplifies phosphoinositide hydrolysis stimulated by prostaglandin f2 alpha in osteoblasts: involvement of p38MAP kinase

We previously showed that sphingosine 1-phosphate phosphorylates p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine 1-phosphate on phospholipase C-catalyzing phosphoinositide hydrolysis induced by prostaglandin F2alpha (PGF2 alpha) in these cells. Sphingosine 1-phosphate significantly amplified the inositol phosphates formation by PGF2 alpha. Sphingosine 1-phosphate did not enhance the formation induced by NaF, a direct activator of heterotrimeric GTP-binding proteins. PD98059, an inhibitor of the kinase that activates p42/p44 MAP kinase, had little effect on the amplification by sphingosine 1-phosphate. SB203580, an inhibitor of p38 MAP kinase, reduced the effect of sphingosine 1-phosphate on the formation of inositol phosphates by PGF2 alpha. The phosphorylation of p42/p44 MAP kinase by PGF alpha was attenuated by PD98059. SB203580 suppressed the phosphorylation of p38 MAP kinase by PGF2 alpha. Tumor necrosis factor-alpha enhanced the PGF2 alpha-stimulated formation of inositol phosphates. These results strongly suggest that sphingosine 1-phosphate amplifies PGF2 alpha-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in osteoblasts.     

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.     

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f(2alpha)-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) activates both phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells and then induces the activation of protein kinase C (PKC). In this study, we investigated the effect of PGF(2alpha) on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein, in these cells. PGF(2alpha) significantly induced the accumulation of HSP27 dose-dependently within the range of 10 nM to 10 microM. PGF(2alpha) stimulated the increase in the levels of mRNA for HSP27. A total of 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, induced the accumulation of HSP27. The stimulative effect of PGF(2alpha) was reduced in the PKC down-regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF(2alpha)-induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF(2alpha) was reduced by U-73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF(2alpha) and TPA stimulated p42/p44 mitogen-activated protein (MAP) kinase. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, suppressed the induction of HSP27 stimulated by PGF(2alpha) or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF(2alpha). These results indicate that PGF(2alpha) stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts.     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.     

(--)-epigallocatechin gallate enhances prostaglandin F2alpha-induced VEGF synthesis via upregulating SAPK/JNK activation in osteoblasts

Catechin, one of the major flavonoids presented in plants such as tea, reportedly suppresses bone resorption. We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. To clarify the mechanism of catechin effect on osteoblasts, we investigated the effect of (--)-epigallocatechin gallate (EGCG), one of the major green tea flavonoids, on the VEGF synthesis by PGF(2alpha) in MC3T3-E1 cells. The PGF(2alpha)-induced VEGF synthesis was significantly enhanced by EGCG. The amplifying effect of EGCG was dose dependent between 10 and 100 microM. EGCG did not affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, and SP600125, a specific inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), reduced the PGF(2alpha)-induced VEGF synthesis. EGCG markedly enhanced the phosphorylation of SAPK/JNK induced by PGF(2alpha) without affecting the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. SP600125 markedly reduced the amplification by EGCG of the SAPK/JNK phosphorylation. In addition, the PGF(2alpha)-induced phosphorylation of c-Jun was amplified by EGCG. These results strongly suggest that EGCG upregulate PGF(2alpha)-stimulated VEGF synthesis resulting from amplifying activation of SAPK/JNK in osteoblasts.     

Involvement of p38 and p42/44 MAP kinases and protein kinase C in the interferon-gamma and interleukin-1alpha-induced phosphorylation of 85-kDa cytosolic phospholipase A(2) in primary human bronchial epithelial cells

Interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) play an important role in the modulation of acute and chronic airway inflammation. Both IFN-gamma and IL-1 are known to increase the release of arachidonic acid (AA) from airway epithelial cells, suggesting that AA metabolites may mediate the cytokine-induced inflammation. This study was designed to examine the direct effect of IFN-gamma and IL-alpha on the phosphorylation of 85-kDa cytosolic phospholipase A(2) (cPLA(2)) and AA release in primary normal human bronchial epithelial (NHBE) cells. Treatment with IFN-gamma and IL-1alpha for 15 min induced a rapid increase of AA release from NHBE cells, which was blocked by the cPLA(2) inhibitor MAFP (p<0.05) but not by the sPLA(2) inhibitor LY311727 or iPLA(2) inhibitor HELSS. Immunoprecipitation and Western blot analysis showed that both IFN-gamma and IL-1alpha induced a rapid phosphorylation of cPLA(2). The IFN-gamma and IL-1alpha-induced cPLA(2) phosphorylation and AA release in the NHBE cells were inhibited by the p38 MAP kinase (MAPK) inhibitor SB203580, p42/44 MAPK inhibitor PD98059 and protein kinase C (PKC) inhibitor bisindolylmaleimide I. These results demonstrate the involvement of p38 and p42/44 MAPKs as well as PKC in the IFN-gamma and IL-1alpha-induced cPLA(2) phosphorylation and AA release in human airway epithelial cells.     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

Involvement of MAP kinases in TGF-beta-stimulated vascular endothelial growth factor synthesis in osteoblasts

Transforming growth factor-beta (TGF-beta) reportedly induces vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. We have recently shown that TGF-beta activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in these cells. In the present study, we investigated the exact mechanism of TGF-beta behind the synthesis of VEGF in MC3T3-E1 cells. PD98059 and U-0126, specific inhibitors of MEK, suppressed the VEGF synthesis induced by TGF-beta. U-0126 inhibited the TGF-beta-induced p44/p42 MAP kinase phosphorylation. SB203580 and PD169316, inhibitors of p38 MAP kinase, reduced the TGF-beta-stimulated VEGF synthesis. SB202474, a negative control for p38 MAP kinase inhibitor, did not affect the VEGF synthesis. A combination with PD98059 and SB203580 almost completely suppressed the TGF-beta-induced VEGF synthesis. Retinoic acid, which alone failed to affect VEGF synthesis, markedly enhanced the VEGF synthesis stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-increased levels of VEGF mRNA. The amplifications by retinoic acid of TGF-beta-increased VEGF synthesis and levels of VEGF mRNA were reduced by PD98059 or SB203580. The combination of PD98059 and SB203580 almost completely suppressed the enhancement by retinoic acid of VEGF synthesis induced by TGF-beta. Taken together, our results strongly suggest that both p44/p42 MAP kinase and p38 MAP kinase take part in TGF-beta-stimulated VEGF synthesis in osteoblasts, and that retinoic acid upregulates the VEGF synthesis.     

Thrombin Mediates Migration of Rat Brain Astrocytes via PLC, Ca2+, CaMKII, PKCα, and AP-1-Dependent Matrix Metalloproteinase-9 Expression

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca²⁺-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca²⁺ concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca²⁺/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.     

(-)-Epigallocatechin gallate suppresses endothelin-1-induced interleukin-6 synthesis in osteoblasts: inhibition of p44/p42 MAP kinase activation

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.     

Role of MAPK in chronic cerebral vasospasm

This study was undertaken to investigate the role of p44/42 MAPK in a dog double hemorrhage model of subarachnoid hemorrhage (SAH), and whether MEK inhibitors can alter the degree of SAH-induced vasoconstriction. The diameter of the basilar artery, which was compared with day 0 angiogram, decreased gradually in a time-dependent manner from day 3 (80%), day 5 (68%) through day 7 (53.5%). The level of MAPK (p44/42) immunoprecipitation peaked on day 3 and remained enhanced through day 7 (P < 0.05). MEK inhibitor PD98059 significantly reduced p44/42 MAPK immunoprecipitation and significantly reversed vasospasm and increased residual diameter to 79.0% on day 7. These results demonstrated that p44/42 MAPK kinase is involved in the pathogenesis of cerebral vasospasm. The MEK inhibitor PD98059 might be useful in the treatment of vasospasm.     

C(2)-ceramide-induced circular smooth muscle cell contraction involves PKC-epsilon and p44/p42 MAPK activation in cat oesophagus. Mitogen-activated protein kinase

We investigated the mechanism of C(2)-ceramide (C(2))-induced circular smooth muscle cell contraction in cat oesophagus. C(2) produced contraction of smooth muscle cells isolated by enzymatic digestion, peaked at 30 s and was sustained at a plateau at 5 min. The response to C(2) was concentration-dependent. H-7 or chelerythrine inhibited C(2)-induced contraction, while the diacylglycerol (DAG) kinase inhibitor, R59949, had no effect, suggesting that the contraction is protein kinase C (PKC) pathway-dependent. To test if PKC-mediated contraction may be isozyme-specific, we examined the effects of PKC isozymes antibodies on contraction. PKC-epsilon antibody inhibited the contraction by C(2) but not by PKC-betaII or -gamma, suggesting that PKC-epsilon mediates the contraction by C(2). To characterize the specific PKC isozymes that mediate contraction of the smooth muscle cells, we used, as an inhibitor, N-myristoylated peptides (myr-PKC) derived from the pseudosubstrate sequences of PKC-(alpha)(beta)(gamma), -alpha, -delta, or -epsilon. myr-PKC-epsilon only inhibited the contraction, which was concentration-dependent, suggesting that PKC-epsilon isozyme is involved in the contraction. To examine which mitogen-activated protein kinases (MAPKs) are involved in C(2)-induced contraction, specific MAPK inhibitors (MEK inhibitor, PD98059, and p38 MAPK inhibitor, SB202190) are used. Preincubation of PD98059 blocked the contraction induced by C(2) in a concentration-dependent manner. However, SB202190 had no effects on contraction. C(2) increased the intensity of the bands identified by phosphospecific p44/p42 MAPK antibody and preincubation of PD98059 decreased the intensity of bands as compared with C(2)-stimulated cells. In conclusion, C(2) produced the contraction of smooth muscle cells of cat oesophagus. The contraction is mediated by PKC-epsilon, resulting in the activation of p44/p42 MAPK.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Activation of mitogen-activated protein kinases is required for alpha1-adrenergic agonist-induced cell scattering in transfected HepG2 cells

Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.