Masaichi Miwa

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.

Involvement of pertussis toxin-sensitive GTP-binding protein in prostaglandin F2α-induced phosphoinositide hydrolysis in osteoblast-like cells

Prostaglandin F2 alpha (PGF2 alpha) stimulated the formation of inositol phosphates in a dose-dependent manner in cloned osteoblast-like MC3T3-E1 cells. This reaction was markedly inhibited dose-dependently by pertussis toxin. In the cell membranes, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGF2 alpha. These results suggest that pertussis toxin-sensitive GTP-binding protein is involved in the coupling of PGF2 alpha receptor to phospholipase C in these cells.

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 α–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 1μM and 30 μM. 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.

Effects of hypergravity on proliferation and differentiation of osteoblast-like cells

We investigated the effects of hypergravity on DNA synthesis and alkaline phosphatase (ALP) activity in cloned osteoblast-like cells, MC3T3-E1. Hypergravity (5 x g) stimulated DNA synthesis in these cells in a time-dependent manner and increased it approximately up to 150% of that of the control (1 x g). 12-O-Tetra-decanoylphorbol-13-acetate (TPA), a protein kinase C activator, and insulin-like growth factor I (IGF-I) enhanced DNA synthesis additively with hypergravity (5 x g). An increase in ALP activity induced by 10% fetal calf serum (FCS) was suppressed by hypergravity (2 x g, 5 x g). Five x g completely suppressed the increase in ALP activity. TPA and hypergravity (2 x g) suppressed the increase in ALP activity induced by FCS additively. Hypergravity (5 x g) showed no significant effect on cAMP nor cGMP production in these cells, but increased prostaglandin E2 (PGE2) production. Exogenous PGE2 stimulated DNA synthesis in these cells but had little effect on 10% FCS-induced ALP activity. These results suggest that hypergravity stimulates proliferation but suppresses differentiation of osteoblast-like cells through a pathway independent of the activation of protein kinase C and the production of cyclic nucleotides, and that hypergravity and IGF-I stimulate proliferation of these cells through an independent signal transduction pathway. Moreover, our data strongly suggest that PGE2 mediates the signalling of hypergravity on the proliferation of osteoblast-like cells.

MITOGEN-ACTIVATED PROTEIN (MAP) KINASES ARE INVOLVED IN INTERLEUKIN-1 (IL-1)-INDUCED IL-6 SYNTHESIS IN OSTEOBLASTS : MODULATION NOT OF P38 MAP KINASE, BUT OF P42/P44 MAP KINASE BY IL-1-ACTIVATED PROTEIN KINASE C

We previously reported that interleukin-1α (IL-1α)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1α itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1α-induced IL-6 synthesis in MC3T3-E1 cells. IL-1α time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1α-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1α. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1α-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1...

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.

Regulation of proliferation by vasopressin in aortic smooth muscle cells : function of protein kinase C

Aim: To investigate the effect of arginine vasopressin‐stimulated prostaglandin synthesis and the activation of protein kinase C on DNA synthesis in rat aortic smooth muscle cells. Methods: The effects of arginine vasopressin on the release of arachidonic acid and the synthesis of prostaglandin (PG) E2 and prostacyclin (PGI2) were determined. The effects of 12‐o‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C‐activating phorbol ester, and of 1‐oleoyl‐2‐acetylglycerol, a specific activator of protein kinase C, were evaluated in cultured rat aortic smooth muscle cells. The effects of arginine vasopressin and prostaglandins on the progression from the late G1 to the S phase of the cell cycle were evaluated by measuring the DNA synthesis, and the effects of TPA on them were evaluated. Results: Arginine vasopressin dose‐dependently stimulated arachidonic acid release. TPA and 1‐oleoyl‐2‐acetylglycerol dose‐dependently increased the vasopressin‐induced arachidonic acid release. Vasopressin stimulated the synthesis of both PGE2 and PGI2. TPA increased the vasopressin‐stimulated prostaglandin synthesis as well as the arachidonic acid release. Vasopressin, added at the G0/G1 phase of the cell cycle, stimulated DNA synthesis of aortic smooth muscle cells. Exogenous PGE2 and PGI2 inhibited the DNA synthesis and showed maximum inhibition when added at the late G1 phase. TPA alone, added at the late G1 phase, reduced the DNA synthesis stimulated by vasopressin at the G0/G1 phase to about 45%, but vasopressin alone, added at the late G1 phase, had little effect. However, with TPA pretreatment, vasopressin significantly suppressed the DNA synthesis by about 70%. Staurosporine, a protein kinase C inhibitor, reduced the suppression by TPA alone or by vasopressin with TPA pretreatment almost to the control level. Indomethacin, a cyclo‐oxygenase inhibitor, reduced the suppression by vasopressin with TPA pretreatment almost to the level of TPA alone. Conclusions: These results suggest that arginine vasopressin has a suppressive effect on DNA synthesis in rat aortic smooth muscle cells by inhibiting progression from the late G1 into the S phase of the cell cycle through the synthesis of PGE2 and PGI2, and that protein kinase C acts as an amplifier of this mechanism.

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f2?-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F2α (PGF2α) 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 PGF2α on the induction of heat shock protein 27 (HSP27), a low‐molecular‐weight heat shock protein, in these cells. PGF2α significantly induced the accumulation of HSP27 dose‐dependently within the range of 10 nM to 10 μM. PGF2α 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 PGF2α was reduced in the PKC down‐regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF2α‐induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF2α was reduced by U‐73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF2α 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 PGF2α or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF2α. These results indicate that PGF2α stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts. J. Cell. Biochem. 75:610–619, 1999.

Mechanism of prostaglandin E2-induced arachidonic acid release in osteoblast-like cells: independence from phosphoinositide hydrolysis.

We previously reported that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in the coupling of prostaglandin E2 (PGE2) receptor to phospholipase C in osteoblast-like MC3T3-E1 cells (1). In the present study, we analyzed the mechanism of PGE2-induced arachidonic acid (AA) release in MC3T3-E1 cells. PGE2 stimulated the release of AA and the formation of inositol trisphosphate (IP3) dose dependently in the range between 1 nM and 10 microM. The effect of PGE2 on AA release (ED50 was 80 nM) was more potent than that on IP3 formation (ED50 was 0.8 microM). Quinacrine, a phospholipase A2 inhibitor, suppressed the PGE2-induced AA release but had little effect on the IP3 formation. NaF, a GTP-binding protein activator, mimicked PGE2 by stimulating the AA release. The AA release stimulated by a combination of PGE2 and NaF was not additive. PTX had little effect on the PGE2-induced AA release. These results strongly suggest that the AA release and the phosphoinositide hydrolysis are separately stimulated by PGE2 in osteoblast-like cells, and the PGE2-induced AA release is mediated by PTX-insensitive GTP-binding protein.

Activation of protein kinase C inhibits 45Ca-accumulation in cultures of osteoblast-like cells: possible involvement of insulin-like growth factor-I

In a previous report, we have demonstrated that exogenous insulin-like growth factor-I (IGF-I) stimulates 45Ca-accumulation into extracellular matrix in long-term cultures of osteoblast-like MC3T3-E1 cells and that 45Ca-accumulation occurs even in the cultures without exogenous IGF-I. In this study, effects of protein kinase C (PKC) on IGF-I secretion and 45Ca-accumulation into extracellular matrix were examined in 6-week cultured MC3T3-E1 cells. The MC3T3-E1 cells secreted IGF-I spontaneously. The PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed IGF-I secretion in a dose-dependent manner. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), which is inactive for PKC, had little effect on the secretion. 1-Oleoyl-2-acetylglycerol, a specific activator for PKC, also suppressed the IGF-I secretion dose dependently. H-7, a PKC inhibitor, recovered the inhibitory effect of TPA. On the other hand, TPA inhibited the 45Ca-accumulation into extracellular matrix in cultures of these cells dose dependently, whereas 4 alpha-PDD was ineffective in this capacity. The TPA-induced inhibition of 45Ca-accumulation was recovered almost to the control level by H-7. Exogenous IGF-I recovered the inhibitory effect of TPA on 45Ca-accumulation. In spite of the inhibitory effects of TPA as above, TPA had little effect on DNA synthesis in these cells. These results suggest that the activation of PKC inhibits calcification via suppression of IGF-I secretion in osteoblast-like cells.