Raloxifene enhances spontaneous microaggregation of platelets through upregulation of p44/p42 MAP kinase: a case report.

UNLABELLED: A 60-year-old postmenopausal woman diagnosed as primary osteoporosis began to take raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated after the treatment, concomitant with the significant upregulation of p44/p42 mitogen-activated protein (MAP) kinase induced by adenosine diphosphate (ADP). After the cessation of raloxifene, the spontaneous microaggregates of platelets and the acceleration of ADP-induced p44/p42 MAP kinase phosphorylation was diminished. We concluded that raloxifene caused platelet hyperaggregability to shear stress and p44/p42 MAP kinase was involved in the pathological state. INTRODUCTION: A 60-year-old postmenopausal woman suffering from severe lumbago was diagnosed as primary osteoporosis with combined vertebral fractures. After the acute phase, she began to take 60 mg daily of oral raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated significantly after 8 weeks from the beginning of raloxifene treatment and observed at 12 weeks. RESULTS: The platelet aggregation induced by ADP was little changed; however, low doses (0.3 and 1 microM) of ADP significantly induced the phosphorylation of p44/p42 MAP kinase in the platelets obtained at 12 weeks. Although there were few subjective complaints except for paroxysmal headache, the medication was stopped with her consent to avoid any adverse effects. The spontaneous microaggregates of platelets gradually decreased after the cessation of medication. At 12 weeks after the cessation, the phosphorylation of p44/p42 MAP kinase induced by low doses of ADP was no more observed. CONCLUSION: These results strongly suggest that raloxifene caused platelet hyperaggregability to shear stress and subclinical thrombus formation in this case and that p44/p42 MAP kinase was involved in the pathological state.

Overexpression of protein kinase C-delta plays a crucial role in interleukin-6-producing pheochromocytoma presenting with acute inflammatory syndrome: a case report.

Pheochromocytomas are tumors that may produce a variety of substances in addition to catecholamines. To date, among several cases of systemic inflammatory syndrome associated with interleukin-6 (IL-6) secretion, IL-6-producing pheochromocytomas, have been reported. However, the mechanism underlying IL-6 oversecretion in these cases has not yet been clarified. This report describes a patient with pheochromocytoma who exhibited pyrexia and marked inflammatory signs including C-reactive protein elevation. The inflammatory symptoms were easily controlled by the administration of loxoprofen, a nonsteroidal anti-inflammatory drug. The plasma concentration of IL-6 and 11-d-TXB(2), a stable metabolite of thromboxane A(2) (TXA(2)), were significantly elevated in parallel with an elevation of norepinephrine in the samples obtained by selective venous sampling. A left adrenalectomy was performed, and the acute inflammatory symptoms naturally diminished without loxoprofen. Cultured tumor cells obtained from the resected specimen spontaneously released IL-6, and indomethacin inhibited the IL-6 release. According to a cDNA microarray analysis, mRNA of protein kinase C-delta (PKC-delta), prostaglandin D synthase, and arachidonate release-relating enzymes were significantly overexpressed in the tumor tissue in comparison to the adjacent nontumor tissue. The constitutive phosphorylation of PKC-delta was observed in the tumor tissue. These results strongly suggest that the systemic inflammatory syndrome in IL-6-producing pheochromocytoma, at least in part, is caused by the overexpression of PKC-delta, resulting in an excess of arachidonate derivatives such as prostaglandins.

Phosphatidylinositol 3-kinase/Akt plays a part in tumor necrosis factor-alpha-induced interleukin-6 synthesis in osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl-CHIRO-inositol 2-( R)-2- O-methyl-3-O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.

Inhibition of chemotactic peptide-induced phosphoinositide hydrolysis by phorbol esters through the activation of protein kinase C in differentiated human leukemia (HL-60) cells.

Phorbol-12,13-dibutyrate (PDBU), a tumor-promoting and protein kinase C-activating phorbol ester, inhibited formylmethionylleucyl-phenylalanine-induced generation of inositol mono-, bis-, and tris-phosphates from the hydrolysis of phosphoinositides in human leukemia (HL-60) cells, which had been differentiated to polymorphonuclear leukocyte-like cells by pretreatment with dibutyryl cyclic adenosine 3':5'-monophosphate. PDBU did not alter the binding of formylmethionylleucyl-phenylalanine to the cells. Other protein kinase C-activating substances such as 12-O-tetradecanoylphorbol-13-acetate and 1-oleoyl-2-acetyl-glycerol could substitute for PDBU, but 4 alpha-phorbol-12,13-didecanoate, which is inactive for both tumor promotion and protein kinase C activation, was ineffective in this capacity. Prolonged treatment of the cells with PDBU resulted in the down-regulation and decrease of protein kinase C activity to the level of 30-40% of that in the control cells. In the down-regulated cells, formylmethionylleucylphenylalanine still induced generation of the phosphorylated inositols to the same extent as that in the control cells, but the inhibition of this reaction by PDBU was reduced to 30-50% as compared with that in the control cells. These results strongly suggest that tumor-promoting phorbol esters inhibit the agonist-induced phosphoinositide hydrolysis through the activation of protein kinase C in the differentiated HL-60 cells.

Basic fibroblast growth factor induces interleukin-6 synthesis in osteoblasts: autoregulation by protein kinase C.

We previously reported that basic fibroblast growth factor (bFGF) stimulates both phospholipases C and D via independent pathways in osteoblastlike MC3T3-E1 cells. In this study, we investigated the effect of bFGF on interleukin-6 (IL-6) synthesis in these cells. bFGF stimulated the IL-6 synthesis dose-dependently in the range between 1 and 30 ng/ml. The depletion of extracellular Ca2+ by EGTA suppressed the bFGF-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, also inhibited the IL-6 synthesis by bFGF. bFGF stimulated the Ca2+ influx from extracellular space. Genistein, a tyrosine kinase inhibitor, suppressed the bFGF-induced Ca2+ influx. Staurosporine, an inhibitor for protein kinases, enhanced the bFGF-induced IL-6 synthesis. Calphostin C, a highly potent and specific inhibitor for protein kinase C (PKC), also enhanced the IL-6 synthesis by bFGF. The bFGF-induced IL-6 synthesis was amplified in PKC down-regulated cells. U-73122, a phospholipase C inhibitor, enhanced the bFGF-induced IL-6 synthesis. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, also enhanced the IL-6 synthesis by bFGF. These results strongly suggest that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilization in osteoblastlike cells, and that the IL-6 synthesis by bFGF is autoregulated due to PKC activation.

Autoregulation of prostaglandin E2-induced Ca2+ influx in osteoblast-like cells: inhibition by self-induced activation of protein kinase C.

In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.

Effect of ceramide on interleukin-6 synthesis in osteoblast-like cells.

We previously showed that prostaglandin (PG) E1 stimulates the synthesis of interleukin-6 (IL-6) through activation of protein kinase (PK) A in osteoblast-like MC3T3-E1 cells and that PGF2alpha induces IL-6 synthesis through PKC activation. In other studies, we demonstrated that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilisation in these cells and that tumour necrosis factor-alpha (TNF) induces IL-6 synthesis through sphingosine 1-phosphate, a product of sphingomyelin turnover. In the present study, among sphingomyelin metabolites, we examined the effect of ceramide on the IL-6 synthesis induced by various agonists in MC3T3-E1 cells. C2-ceramide, a cell-permeable ceramide analogue, suppressed the PGE1-induced IL-6 synthesis. C2-ceramide inhibited the IL-6 synthesis induced by PGF2alpha or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. C2-ceramide reduced the IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP. C2-ceramide inhibited the IL-6 synthesis induced by thrombin. The IL-6 synthesis stimulated by thapsigargin, which is known to stimulate Ca2+ mobilisation from intracellular Ca2+ stores, or A23187, a Ca-ionophore, was also inhibited by C2-ceramide. C2-ceramide did not affect the IL-6 synthesis induced by interleukin-1. On the contrary, C2-ceramide enhanced the TNF-induced IL-6 synthesis. D,L-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, inhibited the enhancement by C2-ceramide as well as the TNF-effect. These results strongly suggest that ceramide modulates the IL-6 synthesis stimulated by various agonists 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.

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis.

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.

Effects of retinoic acid on signalling by prostaglandin E2 in osteoblast-like cells.

We investigated the effects of retinoic acid (RA) on the signalling pathways by prostaglandin E2 (PGE2) in osteoblast-like MC3T3-E1 cells. The pretreatment with RA significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner in the range between 0.1 nM and 0.1 microM, without affecting protein contents in the cultured cells. This effect of RA was dependent on the time of pretreatment up to 8 h. However, RA had little effect on the formation of inositol phosphates induced by NaF, a GTP-binding protein activator. On the other hand, RA significantly inhibited PGE2-induced cAMP accumulation in a dose-dependent manner between 0.1 nM and 0.1 microM. This effect of RA was dependent on the time of pretreatment up to 8 h. RA also inhibited the cAMP accumulation induced by NaF or forskolin which directly activates adenylate cyclase. These results strongly suggest that RA modulates the signalling by PGE2 in osteoblast-like cells as follows: the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between PGE2 receptor and GTP-binding protein, and the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase.

Genistein inhibits potentiation by wortmannin of protein kinase C-activated phospholipase D in osteoblast-like cells.

We previously showed that protein kinase C (PKC) induces phosphatidylcholine-hydrolysing phospholipase D activation in osteoblast-like MC3T3-E1 cells and that tyrosine kinase is involved in this activation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, markedly enhanced the formation of choline induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC in MC3T3-E1 cells. The effect of wortmannin was dose-dependent between 0.1 microM and 10 microM. ML-7, an inhibitor of myosin light chain kinase, had little effect on the TPA-induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, significantly suppressed the potentiation by wortmannin. These results strongly suggest that phosphatidylinositol 3-kinase is involved in the regulation of phospholipase D activation by PKC in osteoblast-like cells.

Pituitary adenylate cyclase-activating polypeptide induces cAMP production independently from vasoactive intestinal polypeptide in osteoblast-like cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) isolated from ovine hypothalamic tissue is a novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures. In osteoblasts, the detail of intracellular signalling systems of PACAP has not yet been clarified. In this study, we investigated the effects of PACAP on cAMP accumulation, phosphoinositide hydrolysis and Ca2+ influx in osteoblast-like MC3T3-E1 cells, compared with those of vasoactive intestinal polypeptide (VIP), which shows a considerable homology with PACAP in the N-terminal sequence. PACAP stimulated cAMP accumulation in a dose-dependent manner in the range between 0.1 nM and 0.1 microM in these cells. VIP also stimulated cAMP accumulation dose-dependently between 1 nM and 0.1 microM. The effect of PACAP on cAMP accumulation (EC50 = 3 nM) was more potent than that of VIP (EC50 = 30 nM). The cAMP accumulation stimulated by a combination of PACAP (3 nM) and VIP (30 nM) was additive. [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP, and antagonist for the VIP receptor which markedly inhibited the VIP-induced cAMP accumulation, had little effect on the PACAP-induced cAMP accumulation. Either PACAP or VIP had little effect on the formation of inositol phosphates and Ca2+ influx in these cells. These results strongly suggest that PACAP stimulates cAMP production via an independent binding site from VIP in osteoblast-like MC3T3-E1 cells and that PACAP has no effect on the activation of protein kinase C nor the intracellular Ca2+ mobilization in these cells.

Prostaglandin E1 stimulates interleukin-6 secretion via protein kinase A in osteoblast-like cells.

We investigated the effect of prostaglandin E1 (PGE1) on the secretion of interleukin-6 (IL-6) in osteoblast-like MC3T3-E1 cells. PGE1, which induced cAMP accumulation, stimulated IL-6 secretion time-dependently up to 48 h. The stimulative effect of PGE1 was dose-dependent in the range between 10 nM and 10 microM. Cholera toxin, an activator of Gs, stimulated IL-6 secretion in MC3T3-E1 cells. Forskolin, which directly activates adenylate cyclase, significantly induced IL-6 secretion in a dose-dependent manner in the range between 1 and 50 microM. Dibutyryl cAMP (Bt2-cAMP) stimulated IL-6 secretion time-dependently up to 48 h. The effect of Bt2-cAMP on IL-6 secretion was dose-dependent in the range between 0.1 and 3 mM. N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline-sulfonamide (H-89), a potent and selective inhibitor of protein kinase A, which suppressed the IL-6 secretion induced by forskolin or Bt2-cAMP, significantly inhibited the IL-6 secretion induced by PGE1. These results indicate that PGE1 stimulates IL-6 secretion via the activation of protein kinase A in osteoblast-like cells.

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase.

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.

Mechanism of phospholipase D activation induced by prostaglandin D2 in osteoblast-like cells: function of Ca2+/calmodulin.

Prostaglandin D2 (PGD2) stimulated the formation of choline in a dose-dependent manner in the range between 10 nM and 10 microM. The effect of PGD2 on the formation of inositol phosphates (EC50 was 20 nM) was more potent than that on the formation of choline (EC50 was 0.5 microM). The formation of choline stimulated by a combination of PGD2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, was additive. Staurosporine, an inhibitor for protein kinases, enhanced the PGD2-induced formation of choline, but H-7, another inhibitor for protein kinases, had little effect. PGD2 stimulated Ca2+ influx from extracellular space dose-dependently. The depletion of extracellular Ca2+ by EGTA reduced the PGD2-induced formation of choline. W-7 and trifluoperazine dihydrochloride, antagonists of calmodulin, dose-dependently inhibited the PGD2-induced choline formation. These results strongly suggest that PGD2 activates phospholipase D in a Ca2+/calmodulin dependent manner in osteoblast-like cells, and that protein kinase C is not essential for the PGD2-induced activation of phospholipase D.

Involvement of protein kinase C activation in endothelin-1-induced secretion of interleukin-6 in osteoblast-like cells.

We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.

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.

Basic fibroblast growth factor stimulates vascular endothelial growth factor release in osteoblasts: divergent regulation by p42/p44 mitogen-activated protein kinase and p38 mitogen-activated protein kinase.

We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.

Effect of prostaglandin E2 on phospholipase D activity in osteoblast-like MC3T3-E1 cells.

Recent evidence indicates that phosphatidylcholine breakdown by phospholipase D (PLD) is an important cellular control mechanism. We investigated the signaling pathway participating in prostaglandin E2 (PGE2)-induced PLD activation in osteoblast-like MC3T3-E1 cells. PGE2 stimulated PLD activity, as measured by choline generated from phosphatidylcholine, just after the stimulation. The reaction reached a plateau 15 minutes later. PGE2 stimulated PLD activity in a dose-related manner and also increased inositol phosphate (IP) formation. However, the EC50 value for PGE2-induced IP formation is lower than that for PLD activation. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, stimulated PLD activity, and a combination of PGE2 and TPA potentiated it in an additive manner. Although NaF, a heterotrimeric GTP-binding protein activator, significantly stimulated PLD activity, this effect was not augmented by combination with PGE2. PGE2-induced PLD activity was markedly suppressed by either chelating extracellular Ca2+ by EGTA or pertussis toxin. These findings suggest that osteoblasts might have at least two PLD activation mechanisms which involve PKC-dependent or -independent pathways. However, present results indicate that PKC is unlikely to be essential to PGE2-induced PLD activation. On the contrary, pertussis toxin-sensitive GTP-binding protein and extracellular Ca2+ might play important roles in the pathway of PGE2-induced PLD activation.

Sphingosine 1-phosphate induces heat shock protein 27 via p38 mitogen-activated protein kinase activation in osteoblasts.

We previously showed that sphingosine 1-phosphate acts as a second messenger for tumor necrosis factor alpha-induced interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells and that the synthesis by sphingosine 1-phosphate is dependent on p42/p44 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27) in MC3T3-E1 cells. Not C2-ceramide, but sphingosine and sphingosine 1-phosphate significantly induced HSP27 accumulation dose dependently in the range between 1microM and 30 microM. DL-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, markedly inhibited the sphingosine-induced HSP27 accumulation. Sphingosine 1-phosphate induced increase in the levels of the mRNA for HSP27. Sphingosine 1-phosphate stimulated the phosphorylation of p38 MAP kinase. The sphingosine 1-phosphate-induced HSP27 accumulation was dose dependently suppressed by SB203580, an inhibitor of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 reduced the sphingosine 1-phosphate-induced increase of mRNA for HSP27. These results strongly suggest that sphingosine 1-phosphate-stimulated HSP27 induction is mediated via p38 MAP kinase activation in osteoblasts.