Osamu Kozawa

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.

Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01)

Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.

Insulin-like growth factor-I stimulates45Ca-accumulation in cultures of osteoblast-like cells

SummaryThe effect of insulin-like growth factor-I (IGF-I) on calcification in cloned osteoblast-like MC3T3-E1 cells was studied by measuring accumulation of45Ca into sodium dodecyl sulfate-insoluble, EDTA-extractable structure that appeared after 2 weeks of culture. The accumulation of45Ca was markedly enhanced by incubating cells with IGF-I after 6 weeks of culture. The enhancement was dose-dependent in a range between 0.1 nM and 100 nM. IGF-I stimulated alkaline phosphatase activity in the cells cultured for 7 weeks dose-dependently between 0.1 nM and 100 nM. DNA synthesis examined in the cells cultured for 7 weeks was not influenced by 100 nM IGF-I. These results suggest that IGF-I stimulates Ca-accumulation in osteoblast-like cells without stimulating their proliferation.