epidermal-growth-factor and sphingosine-kinase

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Epidermal Growth Factor; Female; Humans; Lysophospholipids; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; RNA, Small Interfering; Signal Transduction; Sphingosine; Triple Negative Breast Neoplasms

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Lysophospholipids; Mice; Muscle, Skeletal; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Satellite Cells, Skeletal Muscle; Signal Transduction; Sphingosine

Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.

Topics: Cell Movement; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; HeLa Cells; Humans; Immunoblotting; Lysophospholipids; Microscopy, Confocal; Mutation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; RNA Interference; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

We have previously shown that high expression levels of the lipid kinase sphingosine kinase-1 (SphK1) correlate with poor survival of glioblastoma (GBM) patients. In this study we examined the regulation of SphK1 expression by epidermal growth factor receptor (EGFR) signaling in GBM cells. As the EGFR gene is often overexpressed and mutated in GBM, and EGFR has been shown to regulate SphK1 in some cell types, we examined the effect of EGF signaling and the constitutively active EGFRvIII mutant on SphK1 in GBM cells. Treatment of glioma cell lines with EGF led to increased expression and activity of SphK1. Expression of EGFRvIII in glioma cells also activated and induced SphK1. In addition, siRNA to SphK1 partially inhibited EGFRvIII-induced growth and survival of glioma cells as well as ERK MAP kinase activation. To further evaluate the connection between EGFR and SphK1 in GBM we examined primary neurosphere cells isolated from fresh human GBM tissue. The GBM-derived neurosphere cell line GBM9, which forms GBM-like tumors intracranially in nude mice, maintained expression of EGFRvIII in culture and had high levels of SphK1 activity. EGFR inhibitors modestly decreased SphK1 activity and proliferation of GBM9 cells. More extensive blockage of SphK1 activity by a SphK inhibitor, potently blocked cell proliferation and induced apoptotic cell death of GBM9 cells. Thus, SphK1 activity is necessary for survival of GBM-derived neurosphere cells, and EGFRvIII partially utilizes SphK1 to further enhance cell proliferation.

Topics: Animals; Annexin A5; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Mice, Nude; Mutation; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Signal Transduction; Time Factors

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK1 and production of S1P, only E(2) stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E(2)-induced S1P and dihydro-S1P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK1/2, indicating that E(2) activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E(2)-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogens; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Electrospray Ionization; Sphingosine

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER(+)) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P(3) is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P(3) induces a migratory phenotype via an SK1-dependent mechanism in ER(+) MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER(+) breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p21-Activated Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Receptors, Estrogen; RNA, Small Interfering; Signal Transduction; Sphingosine

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation; Glioblastoma; Humans; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Plasminogen Activator Inhibitor 1; Protein Kinase C-alpha; Protein Kinase C-delta; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; STAT Transcription Factors; Transcription Factor AP-1

Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.

Topics: Amino Acid Sequence; Cell Line, Tumor; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Mitogen-Activated Protein Kinase 3; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Sequence Homology, Amino Acid; Threonine

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Estradiol; Humans; Phosphotransferases (Alcohol Group Acceptor); Prolactin; Promoter Regions, Genetic; RNA, Small Interfering; Up-Regulation

Sphingosine 1-phosphate (S1P), produced by two sphingosine kinase isoenzymes, denoted SphK1 and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK1, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethylsphingosine, a competitive inhibitor of both SphKs, implicating S1P generation in this process. Down-regulating expression of SphK1 in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.

Topics: Amino Acid Sequence; Cell Movement; Cells, Cultured; Chemotaxis; Enzyme Activation; Epidermal Growth Factor; Humans; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor)

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [(3)H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; ras Proteins

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.

Topics: Calcium Signaling; Cell Line; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Inositol 1,4,5-Trisphosphate; Kidney; Lysophospholipids; MAP Kinase Signaling System; Phosphatidylinositol Diacylglycerol-Lyase; Phosphotransferases (Alcohol Group Acceptor); Receptors, Muscarinic; Sphingosine; Type C Phospholipases; Virulence Factors, Bordetella

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

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine-1-phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin-like growth factor-1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4-fold increase in Vmax. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.

Topics: Animals; Carbazoles; Carrier Proteins; Cytosol; Enzyme Activation; Epidermal Growth Factor; Growth Substances; Indole Alkaloids; Membrane Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; PC12 Cells; Pheochromocytoma; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptor, trkA; Time Factors

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

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