epidermal-growth-factor and safingol

Our previous studies show that epidermal growth factor (EGF) stimulates phospholipase D (PLD)-induced phosphatidic acid (PA) formation in rat calvarial osteoblastic cells. This study investigated the effects of PA on cytosolic calcium ([Ca2+]i) and proliferation, and the possible involvement of the PLD pathway in EGF effects on [Ca2+]i and proliferation in rat calvarial osteoblastic cells. PA markedly increased [Ca2+]i. This response was unaffected by thapsigargin, which depletes [Ca2+]i pools, blocked by verapamil, a calcium channel blocker, and enhanced by propanolol, an inhibitor of PA-phosphohydrolase. PA also reduced the EGF dependent-[Ca2+]i increase by 60%, while a PLD inhibitor blocked these effects. Furthermore, PA significantly increased cell proliferation (P < 0.05) which was inhibited by verapamil and enhanced by H-7 (PKC inhibitor). The PLD inhibitor significantly (P < 0.05) reduced the EGF-induced increase in proliferation. In summary, PA stimulates rat calvarial osteoblastic cell proliferation and mobilization of [Ca2+]i using extracellular pools, and EGF's mitogenic effect on these cells requires activation of PLD.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Division; Cells, Cultured; Cytoplasm; Epidermal Growth Factor; Osteoblasts; Phosphatidic Acids; Phospholipase D; Propranolol; Protein Kinase C; Rats; Rats, Sprague-Dawley; Skull; Sphingosine; Thapsigargin; Verapamil

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Kinetics; Lysophospholipids; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Receptors, Platelet-Derived Growth Factor; Second Messenger Systems; Signal Transduction; Sphingosine; Transcription Factor AP-1

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.

Topics: 3T3 Cells; Animals; Antibodies; Binding, Competitive; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Division; Ceramides; Cholera Toxin; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; G(M1) Ganglioside; Lysophospholipids; Mice; Mitogens; Peptide Fragments; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Sphingolipids; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

The inhibitory effect of fumonisin B1 (FB1) on epidermal growth factor (EGF)-induced DNA synthesis in primary rat hepatocytes was investigated by monitoring the incorporation of [3H]thymidine in the DNA. A pulse-labelling technique was adapted to determine the incorporation of the radioactivity in the DNA (S-phase) quantitatively. FB1 inhibits the EGF-induced DNA synthesis up to 90% when incorporated at concentrations of 150 to 300 microM for a period of 44 h. A continued presence of FB1 is required to exhibit this inhibition as (i) the subsequent removal of FB1 resulted in a reversal of the effect, (ii) a higher stimulatory response in EGF-treated hepatocytes was found when the exposure period of hepatocytes in FB1 was reduced, and (iii) pretreatment of hepatocytes with FB1 only slightly reduced (not significantly) DNA synthesis induced by EGF. Whilst the growth inhibitory effect of FB1 was not associated with a cytotoxic effect, binding studies using [125I]EGF indicated that the growth factor-receptor interaction was not altered. No relationship was found between the disruption of the sphingolipid biosynthesis by FB1 and (i) the mitoinhibitory effect on the EGF response and (ii) the cytotoxicity of FB1 in primary hepatocytes.

Topics: Animals; Cell Division; Cells, Cultured; DNA Replication; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fumonisins; Liver; Male; Mycotoxins; Rats; Rats, Inbred F344; Sphingosine; Time Factors

Growth signalling networks that use glycerophospholipid metabolites as second messengers have been well characterized, but less is known of the second messengers derived from sphingolipids, another major class of membrane lipids. A tantalizing link between sphingolipids and cellular proliferation has emerged from the discovery that the sphingolipid metabolites sphingosine and sphingosine-1-phosphate stimulate growth of quiescent Swiss 3T3 fibroblasts by a pathway that is independent of protein kinase C. Sphingosine-1-phosphate is rapidly produced from sphingosine and may mediate its biological effects. Furthermore, sphingosine-1-phosphate triggers the dual signal transduction pathways of calcium mobilization and activation of phospholipase D, prominent events in the control of cellular proliferation. Here we report that activation of sphingosine kinase and the formation of sphingosine-1-phosphate are important in the signal transduction pathways activated by the potent mitogens platelet-derived growth factor (PDGF) and fetal calf serum (FCS).

Topics: 3T3 Cells; Animals; Cattle; Cell Division; DNA; Enzyme Activation; Epidermal Growth Factor; Fetal Blood; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Second Messenger Systems; Sphingosine