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Abstract
We have previously reported that extracellular ATP stimulates Ca2+ influx from extracellular space, resulting in the production of prostaglandin E2 which mediates, at least in part, its proliferative effect on osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) stimulates phospholipase D in these cells. In the present study, we examined the effect of extracellular ATP on phosphatidylcholine-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stimulated the formation of both choline and inositol phosphates dose-dependently in the range between 0·1 and 1 mm. The formation of choline by a combination of ATP and NaF, an activator of GTP-binding protein, was synergistic, whereas that of inositol phosphates was not. A combination of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Staurosporine, an inhibitor of PKC, had little effect on ATP-stimulated formation of choline. Choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA, while being inhibited by W-7, an antagonist of calmodulin. These results suggest that extracellular ATP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in osteoblast-like cells, and that neither PKC activation nor GTP-binding protein is involved in this mechanism.
Journal of Endocrinology (1995) 145, 81–86
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We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.
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We previously reported that basic fibroblast growth factor (FGF-2) activates p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in FGF-2-induced VEGF release in these cells. FGF-2 markedly induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, markedly reduced the FGF-2-induced VEGF release. SP600125 suppressed the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase induced by FGF-2. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase, failed to affect the FGF-2-induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the FGF-2-stimulated VEGF release in an additive manner. These results strongly suggest that FGF-2 activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in FGF-2-induced VEGF release.
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Department of Pharmacology, Department of Clinical Laboratory, Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
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Department of Pharmacology, Department of Clinical Laboratory, Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
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Department of Pharmacology, Department of Clinical Laboratory, Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
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We have previously reported that protein kinase C negatively regulates basic fibroblast growth factor (FGF-2)-stimulated synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. To further clarify the mechanism underlying the synthesis of IL-6 in osteoblasts, we investigated whether p70 S6 kinase is involved in the FGF-2-stimulated IL-6 synthesis in these cells. Rapamycin, an inhibitor of p70 S6 kinase, significantly enhanced the FGF-2-stimulated IL-6 synthesis in a dose-dependent manner. Downregulation of p70 S6 kinase by siRNA markedly amplified the FGF-2-stimulated IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C, induced the phosphorylation of p70 S6 kinase. Go6976 and bisindolylmaleimide I, inhibitors of protein kinase C, suppressed the TPA-stimulated phosphorylation of p70 S6 kinase. Additionally, protein kinase C inhibitors markedly reduced the phosphorylation of p70 S6 kinase induced by FGF-2. These results strongly suggest that p70 S6 kinase functions at a point downstream of protein kinase C and limits the FGF-2-stimulated IL-6 synthesis in osteoblasts.
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We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.
