Haruhiko Tokuda

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.

Interleukin-6 Synthesis Induced by Prostaglandin E2: Cross-Talk Regulation by Protein Kinase C

We previously showed that prostaglandin E2 (PGE2) stimulates multiple intracellular signaling pathways as follows: by activation of adenylate cyclase; phosphoinositide (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D; and by induction of Ca2+ influx in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of PGE2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGE2 significantly stimulated IL-6 secretion in a dose-dependent manner in the range between 1 nmol/L and 10 micromol/L. A23187, a calcium ionophore, or dibutyryl-cAMP significantly induced IL-6 secretion. The effect of a combination of A23187 and dibutyryl-cAMP on IL-6 secretion was additive. The depletion of extracellular Ca2+ by EGTA reduced the PGE2-induced IL-6 secretion. EP1 receptor antagonist inhibited the PGE2-induced IL-6 secretion. H-89, an inhibitor of cAMP-dependent protein kinase, decreased the PGE2-induced IL-6 secretion. EP2 receptor agonist alone stimulated IL-6 secretion. However, EP4 receptor antagonist had little effect on IL-6 secretion. Calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the secretion of IL-6 induced by PGE2. The stimulative effect of PGE2 on IL-6 secretion was significantly enhanced in PKC downregulated MC3T3-E1 cells. Pertussis toxin enhanced PGE2-induced IL-6 secretion. These results strongly suggest that PGE2 stimulates IL-6 synthesis through both Ca2+ mobilization from extracellular space via EP1 receptor and cAMP production via EP2 receptor in osteoblast-like cells, and that the PKC activation by PGE2 itself regulates oversynthesis of IL-6.

Tumor Necrosis Factor-α Autoregulates Interleukin-6 Synthesis via Activation of Protein Kinase C FUNCTION OF SPHINGOSINE 1-PHOSPHATE AND PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE C

We investigated the mechanism of interleukin-6 (IL-6) synthesis induced by tumor necrosis factor-α (TNF) in osteoblast-like MC3T3-E1 cells. TNF stimulated the synthesis of IL-6 dose dependently in the range between 1 and 30 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the TNF-induced synthesis of IL-6. 1-Oleoyl-2-acetylglycerol, a specific activator of PKC, inhibited the TNF-induced IL-6 synthesis. The stimulative effect of TNF was markedly increased in the PKC down-regulated cells. TNF produced diacylglycerol. TNF had little effect on the formation of inositol phosphates and choline. On the contrary, TNF significantly stimulated the formation of phosphocholine dose dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the TNF-induced diacylglycerol production. The TNF-induced IL-6 synthesis was significantly enhanced by D-609. TNF induced sphingomyelin hydrolysis. Neither C2-ceramide nor sphingosine but sphingosine 1-phosphate significantly stimulated the synthesis of IL-6. PKC down-regulation amplified the IL-6 synthesis by sphingosine 1-phosphate. These results strongly suggest that sphingosine 1-phosphate may act as a second messenger for TNF-induced IL-6 synthesis and that TNF autoregulates IL-6 synthesis due to PKC activation via phosphatidylcholine-specific phospholipase C 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.

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.

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.

Tiludronate inhibits interleukin‐6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3‐E1 cells

In previous studies, we have reported that PGF2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP‐binding protein in osteoblast‐like MC3T3‐E1 cells, and that PGF2α and PGE1 induce interleukin‐6 (IL‐6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF2α‐ and PGE1‐induced IL‐6 synthesis in these cells. Tiludronate significantly suppressed the PGF2α‐induced IL‐6 secretion in a dose‐dependent manner in the range between 0.1 and 30 μM. However, the IL‐6 secretion induced by PGE1 or (Bu)2cAMP was hardly affected by tiludronate. The choline formation induced by PGF2α was reduced by tiludronate dose‐dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF2α‐induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP‐binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF‐induced IL‐6 secretion in human osteoblastic osteosarcoma Saos‐2 cells. These results strongly suggest that tiludronate inhibits PGF2α‐induced IL‐6 synthesis via suppression of phosphatidylcholine‐hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP‐binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998.

Proliferative effect of PGD2 on osteoblast-like cells; Independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2α

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.

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.

Effect of vitamin D3 on interleukin-6 synthesis induced by prostaglandins in osteoblasts

In previous studies, we have shown that prostaglandin F2alpha (PGF2alpha) stimulates interleukin-6 (IL-6) synthesis via activation of protein kinase C in osteoblast-like MC3T3-E1 cells, and that prostaglandin E1 (PGE1) induces the synthesis of IL-6 through protein kinase A activation. In the present study, we investigated the effect of vitamin D3 on IL-6 synthesis in MC3T3-E1 cells. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, inhibited the IL-6 synthesis induced by PGF2alpha or PGE1. On the contrary, 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, had no effect. 1,25-(OH)2D3 did not affect the IL-6 synthesis stimulated by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of protein kinase C. The IL-6 synthesis induced by cholera toxin or forskolin was significantly inhibited by 1,25-(OH)2D3. However, 1,25-(OH)2D3 had little effect on the IL-6 synthesis induced by dibutyryl cAMP. These results strongly suggest that 1,25-(OH)2D3, an active form of vitamin D3, inhibits IL-6 synthesis at both the protein kinase C pathway and the protein kinase A pathway in osteoblasts.