Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.     

CREG inhibits migration of human vascular smooth muscle cells by mediating IGF-II endocytosis

We previously determined that the cellular repressor of E1A-stimulated genes, (CREG) plays a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). This study aimed to identify the role of CREG in modulating the migration of SMCs. Recombinant virus-mediated CREG expression inhibited the cellular migration of cultured SMCs associated with down-regulated activity of matrix metalloproteinase-9 (MMP-9). In contrast, CREG knockdown via the retroviral transfer of short hairpin RNAs promoted cellular migration. Enzyme-linked immunosorbent assay and endocytosis analysis revealed that CREG knockdown attenuated the internalization and increased secretion of insulin-like growth factor (IGF)-II. Western blot analysis demonstrated that both phosphoinositide 3-kinase (PI3K) and phosphatase Akt were enhanced in CREG knockdown SMCs. Furthermore, the effect of CREG knockdown on SMC migration was abrogated in a dose-dependent manner by the addition of either IGF-II neutralizing antibody or the PI3K inhibitor, LY294002. These results indicate that the CREG knockdown-mediated increase in IGF-II secretion promoted cellular migration in SMCs via the PI3K/Akt signal pathway. Additionally, blockage of IGF-II binding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF2R) by IGF2R antibody or recombinant IGF2R fragment attenuated the endocytosis of IGF-II in cells overexpressing CREG. This indicates that M6P/IGF2R is involved in the regulation of CREG-mediated IGF-II endocytosis. In summary, these data demonstrate for the first time that CREG plays a critical role in the inhibition of SMC migration, as well as maintaining SMCs in a mature phenotype. These results may provide a new therapeutic target for vascular disease associated with neointimal hyperplasia.     

Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells

Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to the long-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism for fumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cells are resistant to fumonisin B1 toxicity; 25 microM fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, while it did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study, DL-threo-dihydrosphingosine, the sphingosine kinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to fumonisin B1. Treatment of these cells with 25 microM fumonisin B1 and 2.5 microM SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell cultures induced apoptosis by themselves and their effects were potentiated by SKI or fumonisin B1. Addition of physiological amounts of sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and fumonisin B1. Results indicated that HEK-293 cells are resistant to fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival properties. Taken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical event in rendering the HEK-293 cells relatively resistant to fumonisin B1-induced apoptosis.     

Jagged-1/Notch3 signaling transduction pathway is involved in apelin-13-induced vascular smooth muscle cells proliferation

The apelin/apelin receptor （APJ, apelin-angiotensin receptor-like 1） system is a newly deorphanized G protein- coupled receptor system. Both apelin and APJ that are important regulatory factors are expressed in the cardio- vascular system. Our previous studies demonstrated that apelin-13 significantly stimulated vascular smooth muscle cell （VSMC） proliferation. In this paper, our data sug- gested that the Jagged-l/Notch3 signaling transduction pathway is involved in apelin-13-induced VSMC prolifer- ation by promoting the expression of Cyclin D1. Results indicated that apelin-13 stimulates the proliferation of VSMC and the expression of Jagged-1 and Notch3 in con- centration- and time-dependent manners. The increased expression of Jagged-1 and Notch3 induced by apelin-13 could be abolished by extracellular signal-regulated protein kinase （ERK） blockade. PD98059 （ERK inhibitor） can inhibit the activation of Jagged-I/Notch3 induced by apelin- 13. Down-regulation of Notch3 using small interfering RNA inhibits the expression of Cyclin DI and prevents apelin- 13-induced VSMC proliferation. In conclusion, Jagged-I/ Notch3 signaling transduction pathway is involved in VSMC proliferation induced by apelin-13.     

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca(2+)-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.     

Sphingosine 1-phosphate induces EGFR expression via Akt/NF-kappaB and ERK/AP-1 pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3-kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P-induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P-induced EGFR upregulation was blocked by a selective NF-kappaB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P-induced activation of NF-kappaB was blocked by wortmannin, but not by U0126, suggesting that activation of NF-kappaB was mediated through PI3K/Akt. Moreover, S1P-induced EGFR expression was inhibited by an AP-1 inhibitor curcumin and tanshinone IIA. S1P-stimulated AP-1 subunits (c-Jun and c-Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP-1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF-stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF-kappaB and ERK/AP-1 pathways independently regulated S1P-induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P-induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

LncRNA MALAT1 promotes proliferation and migration of airway smooth muscle cells in asthma by downregulating microRNA-216a

Asthma is a difficult chronic airway inflammation, if it cannot be treated and relieved in time, it will seriously affect the health and quality of life of patients. Airway remodeling is relevant to asthma, but there is currently no effective treatment for airway remodeling. Regulating the biological function of airway smooth muscle cells (AMSCs) may be an important method to inhibit airway remodeling. LncRNA MALAT1 and microRNA-216a are involved in the regulation of AMSCs respectively, but there is no research to prove that they can regulate airway remodeling of asthma through mutual combination. Hence, the aim of the present study was performed to investigate the function of lncRNA MALAT1 and microRNA-216a on AMSCs in asthma. The relationship between lncRNA MALAT1, microRNA-216a and AMSCs was studied by MTT, qPCR, Western blot, Transwell and flow cytometry. The results revealed that lncRNA MALAT1 was up-regulated and microRNA-216a was down-regulated in asthma. lncRNA MALAT1 inhibited microRNA-216a targetedly. Whether downregulating lncRNA MALAT1 or upregulating microRNA-216a, cell proliferation, migration and invasion were reduced and apoptosis increased. Therefore, it is believed that lncRNA MALAT1 promotes proliferation and migration of asthma AMSCs by downregulating microRNA-216a. Since lncRNA MALAT1 and microRNA-216a take part in asthma by jointly regulating the proliferation of airway smooth muscle cells and other biological functions, it would be interesting to study if they become biomarkers of asthma, and relationship between the two in asthma diagnosis and poor prognosis.     

Involvement of released sphingosine 1-phosphate/sphingosine 1-phosphate receptor axis in skeletal muscle atrophy

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known.Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts.Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.     

MicroRNA-638 inhibits human airway smooth muscle cell proliferation and migration through targeting cyclin D1 and NOR1

Abnormal airway smooth muscle cell (ASMC) proliferation and migration contribute significantly to increased ASM mass associated with asthma. MicroRNA (miR)-638 is a primate-specific miRNA that plays important roles in development, DNA damage repair, hematopoiesis, and tumorigenesis. Although it is highly expressed in ASMCs, its function in ASM remodeling remains unknown. In the current study, we found that in response to various mitogenic stimuli, including platelet-derived growth factor-two B chains (PDGF-BB), transforming growth factor β1, and fetal bovine serum, the expression of miR-638, as determined by quantitative real-time polymerase chain reaction (qRT-PCR), was significantly downregulated in the proliferative human ASMCs. Both gain- and loss-of-function studies were performed to study the role of miR-638 in ASMC proliferation and migration. We found that adenovirus-mediated miR-638 overexpression markedly inhibits ASMC proliferation and migration, while ablation of miR-638 by anti-miR-638 markedly increases cell proliferation and migration, as determined by WST-8 proliferation and scratch wound assays. Dual-luciferase reporter assay, qRT-PCR, and immunoblot analysis were used to investigate the effects of miR-638 on the expression of the downstream target genes in ASMCs. Our results demonstrated that miR-638 overexpression significantly reduced the expression of downstream target cyclin D1 and NOR1, both of which have been shown to be essential for cell proliferation and migration. Together, our study provides the first in vitro evidence highlighting the antiproliferative and antimigratory roles of miR-638 in human ASMC remodeling and suggests that targeted overexpression of miR-638 in ASMCs may provide a novel therapeutic strategy for preventing ASM hyperplasia associated with asthma.     

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Intracellular Ca²⁺ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca²⁺ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca²⁺ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [³H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca²⁺ and cAMP signaling pathways. Histamine stimulated Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors in hBASMCs. β₂-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca²⁺ signals. Responses to other Ca²⁺-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E₂ (PGE₂), through EP₂ and EP₄ receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca²⁺ signals. There was no consistent relationship between the inhibition of Ca²⁺ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP₂ and EP₄ receptors, through cAMP and PKA, selectively inhibit Ca²⁺ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H₁ receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.     

Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.     

H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal KATP channel

Here we explored the impact of hydrogen sulfide (H₂S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H₂S in the range of 10–275 μM, and H₂S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24 h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H₂S synthesizing enzyme) and ATP-sensitive potassium (K_ATP) channels. The K_ATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the K_ATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H₂S causes the relaxation of human ASM and implicate as well the role for sarcolemmal K_ATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H₂S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.     

Phytosphingosine 1-phosphate: a high affinity ligand for the S1P(4)/Edg-6 receptor

It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors [1] and [2]. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P₄ receptor has generated interest due to its lymphoid tissue distribution. While the S1P₄ receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4d-hydroxysphinganine 1-phosphate, more commonly known as phytosphingosine 1-phosphate (PhS1P), binds to S1P₄ with higher affinity. Using radiolabeled S1P (S1³³P), the affinity of PhS1P for the S1P₄ receptor is 1.6 nM, while that of S1P is nearly 50-fold lower (119±20 nM). Radiolabeled PhS1P proved to be superior to S1³³P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS1³³P, for in vitro studies of the S1P₄ receptor pharmacology.     

TLR2/1 and sphingosine 1-phosphate modulate inflammation,myofibroblast differentiation and cell migration in fibroblasts

Dermal fibroblasts are important regulators of inflammatory and immune responses in the skin. The aim of the present study was to elucidate the interaction between two key players in inflammation, Toll-like receptors (TLRs) and sphingosine 1-phosphate (S1P), in normal human fibroblasts in the context of inflammation, fibrosis and cell migration. We demonstrate that TLR2 ligation strongly enhances the production of the pro-inflammatory cytokines IL-6 and IL-8. S1P significantly induces pro-inflammatory cytokines time- and concentration-dependently via S1P receptor (S1PR)2 and S1PR3. The TLR2/1 agonist Pam₃CSK₄ and S1P (> 1 μM) or TGF-β markedly upregulate IL-6 and IL-8 secretion. Pam₃CSK₄ and S1P alone promote myofibroblast differentiation as assessed by significant increases of α-smooth muscle actin and collagen I expression. Importantly, costimulation with S1P (> 1 μM) induces differentiation into myofibroblasts. In contrast, Pam₃CSK₄ and low S1P concentrations (< 1 μM) accelerate cell migration. These results suggest that TLR2/1 signaling and S1P cooperate in pro-inflammatory cytokine production and myofibroblast differentiation and promote cell migration of skin fibroblasts in a S1P-concentration dependent manner. Our findings provide significant insights into how infectious stimuli or danger signals and sphingolipids contribute to dermal inflammation which may be relevant for skin tissue repair after injury or disease.     

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca²⁺ signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.