Jun Kotoyori

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.

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E2 (PGE2). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca2+ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1 microM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE2 synthesis and DNA synthesis in these cells. The inhibitory effect by 50 microM indomethacin on the DNA synthesis was reversed by adding 10 microM PGE2. These results strongly suggest that extracellular ATP stimulates Ca2+ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE2 synthesis.

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

Abstract 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 in 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 Ca 2+ 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. PACOA stimulated cAMP accumulation in a dose-dependent manner in the range between 0.1 nM and 0.1 μM in these cells. VIP also stimulated cAMP accumulation dose-dependently between 1 nM and 0.1 μM. The effect of PACAP on cAMP accumulation ( ec 50 = 3 nM ) was more potent than that of VIP ( ec 50 = 30 nM ). The cAMP accumulation stimulated by a combinatiion of PACAP (3 nM) and VIP (30 nM) was additive. [Lys 1 , Pro 2,5 , Arg 3,4 , Tyr 6 ]-VIP, an 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 Ca 2+ influx in these cells. These results strongly suggest that PACAP stimulates cAMP production via an independent binding site from VIP osteoblast-like MC3AT3-E1 cells and that PACAp has no effect on the activation of protein kinase C nor the intracellular CA 2+ mobilization in these 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.

Glucocorticoid inhibits cAMP production induced by vasoactive agents in aortic smooth muscle cells

It is well-known that atherosclerotic change and hypertension are common manifestations in patients with glucocorticoid excess. We previously reported that pituitary adenylate cyclase activating polypeptide (PACAP), prostaglandin E2 (PGE2) and carbacyclin, a stable analog of prostacyclin, have suppressive effects on vasopressin-induced DNA synthesis of rat aortic smooth muscle cells through cAMP production (Murase et al., J. Hypertens., 10 (1992) 1505; Oiso et al., Biochem. Cell. Biol., 71 (1993) 156). In the present study, we investigated the effect of glucocorticoid on cAMP production induced by PACAP, PGE2 and carbacyclin in aortic smooth muscle cells. The pretreatment with dexamethasone significantly inhibited cAMP accumulation induced by these vasoactive agents in a dose dependent manner in the range between 10 pM and 10 nM. These inhibitory effects of dexamethasone were dependent on the time of pretreatment up to 8 h. Dexamethasone inhibited cAMP accumulation induced by NaF, a GTP-binding protein activator, and forskolin which directly activates adenylate cyclase. Moreover, forskolin-induced adenylate cyclase activity was significantly reduced in membranes prepared from the cells treated with dexamethasone. These results strongly suggest that glucocorticoid inhibits cAMP production induced by vasoactive agents in primary cultured rat aortic smooth muscle cells and the inhibitory effect is exerted at the level of adenylate cyclase.

Modulation of prostaglandin E2-induced Ca2+ influx by steroid hormones in osteoblast-like cells

In osteoblast-like MC3T3-E1 cells, we previously reported that prostaglandin E2 (PGE2), a potent bone resorbing agent, stimulates Ca2+ influx (H. Tokuda, M. Miwa, Y. Oiso and O. Kozawa, Cell Signal 1992; 4: 261-266). In this study, we examined the effects of various hormones belonging to the steroid hormone superfamily on PGE2-induced Ca2+ influx in MC3T-E1 cells. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], an active form of vitamin D3, dexamethasone and retinoic acid significantly inhibited the PGE2-induced Ca2+ influx in a dose-dependent manner in these cells. The effects of these hormones were dependent on the time of pretreatment and submaximum inhibitions were observed at 6 h. In contrast, 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], an inactive form of vitamin D3, 17 beta-estradiol, progesterone, testosterone and triiodothyronine had little effect on the PGE2-induced Ca2+ influx in these cells. These results suggest that, in the steroid hormone superfamily, 1,25-dihydroxyvitamin D3, glucocorticoid and retinoic acid modulate bone metabolism through the inhibition of Ca2+ influx induced by PGE2 in osteoblast-like cells.

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.

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.

Effect of vitamin D3 on prostaglandin E2 synthesis in osteoblast-like cells

We previously showed that prostaglandin (PG) F2 alpha stimulates PGE2 synthesis in osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) amplifies the effect of PGF2 alpha through the potentiation of phospholipase A2 activity (H. Tokuda, Y. Oiso and O. Kozawa, J Cell Biochem. 48: 262-268, 1992). In the present study, we investigated the effects of vitamin D3 on PGF2 alpha-induced PGE2 synthesis in MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], an active form of vitamin D3, significantly inhibited the PGF2 alpha-induced PGE2 synthesis in a dose-dependent manner in the range between 1 pM and 1 nM. This inhibitory effect of 1,25-(OH)2D3 was dependent on the time of pretreatment up to 8 h. On the contrary, the pretreatment with 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, had little effect on the PGF2 alpha-induced PGE2 synthesis in these cells. However, the pretreatment with 1,25-(OH)2D3 no longer affected the amplification of PGF2 alpha-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, a PKC activator. These results strongly suggest that 1,25-(OH)2D3 inhibits PGF2 alpha-induced PGE2 synthesis in osteoblast-like cells, however, the activation of PKC reverses this inhibitory effect of 1,25-(OH)2D3.