Products by the sphingosine kinase/sphingosine 1-phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis

Sphingosine 1-phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P-degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild-type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over-expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P-accumulating cells (SPL-null cells and SPL-null cells over-expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL-dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.     

Aldose reductase regulates platelet-derived growth factor-induced proliferation through mediating cell cycle progression in rat mesangial cells

Aldose reductase (AR), the first and the rate-limiting enzyme of the polyol pathway, has been implicated in platelet-derived growth factor (PDGF)-induced proliferation of rat mesangial cells (MsCs). It is well known that AR plays an important role in various chronic diabetic complications, for example, diabetic nephropathy. Moreover, our previous studies have demonstrated that an AR inhibitor (ARI) significantly reduced the proliferation of rat MsCs induced by PDGF, however, the mechanism remains unclear. The aim of the present study was to elucidate the molecular mechanisms through which AR regulates PDGF-induced rat MsC proliferation. It was demonstrated that PDGF-induced MsC proliferation was significantly inhibited by pretreatment with ARI. Cell cycle analysis by flow cytometry revealed that ARI prevented the entry of cells from the G1 into the S?phase. Furthermore, the effect of the PI3K/Akt signaling pathway on the cell cycle was analyzed. The PI3K/Akt pathway was activated with PDGF treatment. However, ARI blocked Akt activation in response to PDGF. Moreover, PDGF increased the levels of p21Cip1 cyclin kinase inhibitor protein in MsC, which was markedly inhibited by pretreatment with ARI. Conversely, PDGF significantly reduced the levels of the p27Kip1 cyclin kinase inhibitor protein, which was also restored by pretreatment with ARI. In conclusion, AR is involved in PDGF-induced rat MsC proliferation, and may serve as a potential target for the inhibition of MsC proliferation in several types of glomerulonephritis.     

Rapamycin inhibits platelet-derived growth factor-induced collagen, but not fibronectin, synthesis in rat mesangial cells

Rapamycin, a macrocyclic lactone, is effective in reducing the incidence of acute rejection after renal transplantation. The inhibitory effects of rapamycin on lymphocyte proliferation and the molecular mechanisms that were involved have been described. However, its effects on glomerular mesangial cells have not been clearly understood, and here, we examined the effect of rapamycin on platelet-derived growth factor (PDGF)-induced extracellular matrix synthesis as well as cell proliferation in mesangial cells. Rat mesangial cells were isolated from the glomeruli of Sprague-Dawley rats and cultured with Dulbecco's modified Eagles medium containing 20% fetal bovine serum. Different concentrations of rapamycin were administered 1 hour before the addition of 10 ng/ml of PDGF into growth arrested and synchronized cells. Cell proliferation was assessed by [3H]thymidine incorporation, total collagen synthesis by [3H]proline incorporation, and fibronectin secretion into the medium by Western blot analysis. In the mesangial cells, PDGF increased cell proliferation by 4.6-fold, total collagen synthesis by 1.8-fold, and fibronectin secretion by 3.2-fold. Rapamycin above 10 nM significantly inhibited PDGF-induced proliferation and collagen synthesis, but the treatment of rapamycin up to 1 microM did not show any significant effects on PDGF-induced fibronectin secretion. These inhibitory effects of rapamycin on PDGF-induced mesangial cell proliferation and collagen synthesis reflect the potential value of rapamycin in the prevention and treatment of glomerulosclerosis in patients with chronic allograft nephropathy.     

Differential effect of FGF and PDGF on cell proliferation and migration in osteoblastic cells

It has been known that growth factors such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) can promote proliferation and migration in a variety of cell types including osteoblastic cells. However, the mechanism underlying their action has not been clearly defined. The present study was undertaken to examine the effect of FGF and PDGF on cell proliferation and migration and to determine the role of extracellular signal-regulated kinase (ERK) and Akt in action of FGF and PDGF in osteoblastic cells. FGF enhanced proliferation in a dose- and time-dependent manner, whereas it did not affect cell migration. FGF induced a transient activation of ERK, but not Akt, which was inhibited by an inhibitor of MEK, the upstream kinase of ERK, but not by inhibitors of PI3K/Akt (LY294002), epidermal growth factor receptor (EGFR, AG1478), and Src (PP2). FGF-induced proliferation was inhibited by inhibitors of MEK/ERK and Src pathways. Exposure of cells to FGF stimulated transition of cell cycle from the G1 phase to S phase and increased phosphorylation of Rb. FGF-induced phosphorylation of Rb was attenuated by inhibitors of MEK/ERK and Src pathways. Cell migration studies indicated that PDGF stimulated migration, but it had no effect on cell proliferation. PDGF induced activation of ERK and Akt. The ERK activatin was inhibited by the Src inhibitor and the Akt activation was inhibited by inhibitors of EGFR and Src. PDGF-induced migration was inhibited by inhibitors of MEK/ERK, PI3K/Akt, EGFR and Src pathways. Taken together, these findings suggest that the MEK/ERK and Src pathways play an important role in the FGF-induced proliferation and signaling pathways involving MEK/ERK, EGFR, Src and PI3K/Akt mediate the PDGF-induced migration. These data are of importance in understanding the roles of these growth factors in osteoblastic cell proliferation and migration.     

Sphingosine kinase 1 regulates differentiation of human and mouse lung fibroblasts mediated by TGF-beta1

Transforming growth factor beta (TGF-beta) contributes to the progression of pulmonary fibrosis through up-regulation of alpha-smooth muscle actin (alpha-SMA) as lung myofibroblast differentiation. Bioactive sphingosine 1-phosphate (S1P) has been shown to mimic TGF-beta signals; however, the function of S1P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK1 and alpha-SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of sphingosine kinase (SPHK) with TGF-beta1 stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK1 decreased alpha-SMA and fibronectin expression up-regulated by TGF-beta1. In the meantime, SPHK1 inhibition did not affect smad2 phosphorylation in response to TGF-beta1. Then we examined whether S1P receptors transactivation may affect TGF-beta signals. siRNA against S1P(2) and S1P(3), but not S1P(1), reduced alpha-SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-beta1 on the cell membrane where S1P(2) or S1P(3) was overexpressed. These data suggested that SPHK1 activation by TGF-beta1 leads to Rho-associated myofibroblasts differentiation mediated by transactivated S1P receptors in the lung fibrogenic process.     

Roles of extracellular and intracellular sphingosine 1-phosphate in cell migration

Sphingosine 1-phosphate (S1P) is an important factor for the regulation of cell motility acting both inside and outside the cells. The precise role of S1P in the control of cell motility, however, remains unclear. Here we describe the roles of S1P in the regulation of cell motility by dissecting them into intracellular and extracellular actions using a liposomal S1P transfer technique. In a Boyden chamber assay free S1P enhanced directional cell movement, whereas liposomal S1P induced nondirectional cell movement. Furthermore, inhibition of sphingosine kinase (SphK) 1 by several inhibitors or knockdown of the enzyme expression by siRNA caused reduced wound-faced cell polarity formation as assessed by wound-healing assay. Moreover, S1P-induced cell migration was strongly inhibited by SphK inhibitors. These results indicate that extracellular S1P acting through S1P receptors facilitates the formation of cell polarity, whereas S1P generated inside the cells functions as an intracellular mediator per se to enhance nondirectional cell movement, thus S1P enhances directional cell movement in a coordinated fashion.     

Oncogenic S1P signalling in EBV‐associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein–Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine‐1‐phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV‐encoded latent genes (EBNA1, LMP1, and LMP2A) can up‐regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV‐positive NPC patient‐derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P‐induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IL-10 induces mesangial cell proliferation via a PDGF-dependent mechanism

Interleukin-10 (IL-10) is a mesangial cell growth factor in vivo and in vitro. However, the mechanism by which IL-10 exerts its mitogenic activity is not known. The aim of this study was to determine whether IL-10 induces mesangial cell proliferation in a PDGF-dependent or independent fashion. A well--characterized rat mesangial cell line (1097) was used in a series of cell proliferation experiments in which cells were serum-starved and then incubated with recombinant IL-10 in the presence or absence of STI 571 (a specific inhibitor of signalling via the PDGF-alpha and beta receptors) or a neutralizing anti-PDGF-AB antibody. IL-10 induced significant mesangial cell proliferation at 24 and 48 h after cytokine addition. This response was inhibited totally by the addition of STI-571, demonstrating that IL-10 mitogenic activity has an absolute requirement for signalling through the PDGF receptor. In further studies, it was found that STI-571 could be added 24 h after IL-10 stimulation and still exert a profound inhibition of IL-10 mitogenic activity. The ability of a neutralizing anti-PDGF-AB antibody to inhibit completely IL-10-induced mesangial cell proliferation confirmed that IL-10 acts via induction of an autocrine PDGF response rather than the possibility that IL-10 may transactivate the PDGF receptor in a PDGF-independent fashion. In conclusion, this study has demonstrated that IL-10 induces mesangial cell proliferation via an autocrine PDGF-mediated mechanism. Thus, therapies which antagonize PDGF signalling will also inhibit any contribution of IL-10 to mesangial proliferation.     

Sphingosine 1-phosphate triggers apoptotic signal for B16 melanoma cells via ERK and caspase activation

The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), recently was reported to induce apoptosis of some cancer cells and neurons, although it generally known to exert mitogenic and antiapoptotic effects. In this study, we investigated the effects of S1P on the cell growth, melanogenesis, and apoptosis of cultured B16 mouse melanoma cells. In results, S1P was found to induce apoptosis in B16 melanoma cells in a dose- and time-dependent manner, but exerted minimal effects on melanogenesis. Although receptors of sphingosine 1-phosphate (endothelial differentiation gene 1 [Edg]/S1P(1), Edg5/S1P(2), Edg3/S1P(3)) were expressed in B16 melanoma cells, they were shown not to be associated with S1P-induced apoptosis. In addition, pertussis toxin did not block the apoptotic effects of S1P on B16 melanoma cells. S1P induced caspase-3 activation and the extracellular signal-regulated kinase (ERK) activation. Interestingly, the ERK pathway inhibitor, UO126, reversed the apoptotic effects of S1P on B16 melanoma cells. These results suggest that S1P induced apoptosis of B16 melanoma cells via an Edg receptor-independent, pertussis toxin-insensitive pathway, and appears to be associated with the ERK and caspase-3 activation.     

Lipoxins inhibit Akt/PKB activation and cell cycle progression in human mesangial cells

Lipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFbeta1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFbeta1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents.     

Regulation of platelet-derived growth factor-induced Ras signaling by poliovirus receptor Necl-5 and negative growth regulator Sprouty2

Necl-5, known as a poliovirus receptor and up-regulated in many cancer cells, enhances platelet-derived growth factor (PDGF)-induced activation of Ras-Raf-MEK-ERK signaling, but not PDGF-induced tyrosine phosphorylation of PDGF receptor, resulting in facilitation of cell proliferation. Here, we showed that Necl-5 interacted with Sprouty2, known to be a negative regulator of growth factor-induced signaling, and reduced the inhibitory effect of Sprouty2 on PDGF-induced Ras signaling. Necl-5 was reported to be down-regulated by its trans-interaction with nectin-3 upon cell-cell contact, initiating cooperative cell-cell adhesion with cadherin. This down-regulation of Necl-5 caused tyrosine phosphorylation of Sprouty2 by c-Src, which was activated by PDGF receptor in response to PDGF, and inhibited PDGF-induced Ras signaling. Thus, Necl-5 and Sprouty2 cooperatively regulate PDGF-induced Ras signaling. The roles of Necl-5 and Sprouty2 in contact inhibition for cell proliferation are also discussed.     

Sphingosine kinase mediates activation of extracellular signal-related kinase and Akt by respiratory syncytial virus

Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infected cells remain viable well into the infection. This prolonged survival results from RSV-induced activation of pro-survival pathways, including Akt and extracellular signal-related kinase (ERK). Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite with demonstrated links to cell survival. It is enzymatically generated by sequential activation of ceramidase (generation of sphingosine) and sphingosine kinase (generation of S1P). In these studies, we found that RSV stimulated neutral ceramidase and sphingosine kinase activities in lung epithelial cells. The combined effect of activation of these two enzymes would decrease proapoptotic ceramide and increase antiapoptotic S1P. S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection. RSV infection does eventually kill infected cells but activation of cell survival pathways significantly delays cell death. The studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection.     

Sphingosine-1-phosphate-induced intracellular Ca2+ mobilization in human endothelial cells.

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca2+ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca2+, S1P increased intracellular Ca2+ concentration ([Ca2+]i) and this increase was partially inhibited by La3+ (1 microM), indicating that S1P induces Ca2+ influx from extracellular pool and Ca2+ release from intracellular stores. S1P increased [Ca2+]i concentration dependently in Ca2+-free extracellular solution. The Hill coefficient (1.7) and EC50 (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca2+ store in the presence of ryanodine, S1P did not induce intracellular Ca2+ release. Furthermore, the Ca2+-induced Ca2+ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca2+ release. S1P-induced intracellular Ca2+ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca2+ release was not inhibited by the mitochondrial Ca2+ uptake inhibitor CCCP or the mitochondrial Ca2+ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca2+ from intracellular stores primarily via Ca2+-induced and IP3-induced Ca2+ release and this Ca2+ mobilization is independent of mitochondrial Ca2+ stores.     

Sphingosine 1-phosphate: a prototype of a new class of second messengers

Sphingosine 1-phosphate (SPP) is an important sphingolipid-derived second messenger in mammalian cells that acts to promote proliferation and to inhibit apoptosis. Various growth factors increase the intracellular concentration of SPP by activating sphingosine kinase, the molecular cloning of which has revealed that it defines a new type of lipid kinase. Cell fate is influenced by the balance between the intracellular concentration of SPP and that of ceramide, a pro-apoptotic sphingolipid metabolite. The observation that a similar "rheostat" is a determinant of cell survival in yeast cells exposed to heat shock indicates that it is an evolutionarily conserved mechanism of stress regulation. SPP also acts extracellularly to inhibit cell motility and to influence cell morphology, effects that appear to be mediated by the G protein-coupled receptor EDG1. These observations indicate that SPP is the prototype of a new class of lipid mediators that exert both intracellular and extracellular actions.     

Expression of sphingosine kinase 2 in synovial fibroblasts of rheumatoid arthritis contributing to apoptosis by a sphingosine analogue, FTY720

Gene expression profiles in synovial tissues from rheumatoid arthritis (RA) patients have yielded useful information on the pathogenetic process of the synovitis. In one group of them, sphingosine kinase 2 (SPHK2), a nuclear protein regulating cell proliferation, seemed to be highly expressed, undergoing a different pathogenetic process of synovitis. In the present study it was clarified that SPHK2 was expressed in the synovial fibroblasts of the synovial tissues obtained from the knee joints of the RA patients. In the cultured synovial fibroblasts from these patients, SPHK2 was more highly expressed than that in the human macrophage cell line, THP-1 and human dermal fibroblasts. SPHK2 was expressed in and around the nucleus and transferred to the cytoplasm and cell surface by the administration of epidermal growth factor, associated with the increased expression of sphingosine-1-phosphate. A sphingosine analogue, FTY720, which is activated by phosphorylation specifically by SPHK2, mediated apoptotic signaling of the cultured synovial fibroblasts. These findings suggest that SPHK2 may regulate the autonomous proliferation of synovial fibroblasts as one of the predisposing genes to RA and could be a target for a novel therapeutic strategy for RA.     

PI3K/Akt and ERK1/2 signalling pathways are involved in endometrial cell migration induced by 17beta-estradiol and growth factors

Cell motility and invasion are crucial events for endometrial cells, not only for the establishment of pathological states but also during the physiological tissue remodelling that occurs during the menstrual cycle and embryo implantation. We have characterized these phenomena in endometrial stromal cells evaluating cell migration-specific stimuli and the biochemical pathways involved. Ability of endometrial cells to migrate on collagen type IV substrate was evaluated by means of chemotaxis experiments. Modulation of this phenomenon by different growth factors and steroid hormones and their ability to activate extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/Akt signalling in this context were examined. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor and fibroblast growth factor-2 as chemoattractant agents stimulated basal migration of endometrial stromal cells through the rapid activation of both ERK1/2 and PI3K/Akt signalling pathways. Experiments using wortmannin and PD98059, specific inhibitors of the PI3K/Akt and ERK1/2 activity, respectively, showed that the activation of both pathways is required for growth-factor-induced cell motility responses. Similarly, 17beta-estradiol (10(-6)-10(-8) M) could enhance both constitutive and PDGF-induced migration of the cells and their rapid treatment with the hormone significantly increased phosphorylation of ERK1/2 and Akt. Conversely, progesterone did not interfere with the basal migration but inhibits the PDGF-induced motility of this cell type. Rapid activation of intracellular signalling cascades ERK1/2 and PI3K/Akt by growth factors and estrogens is involved in the migration of normal endometrial stromal cells.     

Inhibition of rac1 reduces PDGF-induced reactive oxygen species and proliferation in vascular smooth muscle cells.

In vascular smooth muscle cells, reactive oxygen species (ROS) were known to mediate platelet-derived growth factor (PDGF)-induced cell proliferation and NADH/NADPH oxidase is the major source of ROS. NADH/NADPH oxidase is controlled by rac1 in non-phagocytic cells. In this study, we examined whether the inhibition of rac1 by adenoviral-mediated gene transfer of a dominant negative rac1 gene product (Ad.N17rac1) could reduce the proliferation of rat aortic vascular smooth muscle cells (RASMC) stimulated by PDGF via decreasing intracellular ROS. RASMC were stimulated by PDGF (80 ng/mL) with or without N-acetylcysteine 1 mM or infected with 100 mutiplicity of infection of Ad.N17rac1. Intracellular ROS levels were measured at 12 hr using carboxyl-2', 7'-dichlorodihydrofluorescein diacetate confocal microscopy. At 72 hr, cellular proliferation was evaluated by cell number counting and XTT assay. Compared with control, ROS levels were increased by 2-folds by PDGF. NAC and Ad.N17rac1 inhibited PDGF-induced increase of ROS by 77% and 65%, respectively. Cell number was increased by PDGF by 1.6-folds compared with control. NAC and Ad.N17rac1 inhibited PDGF-induced cellular growth by 45% and 87%, respectively. XTT assay also showed similar results. We concluded that inhibition of rac1 in RASMCs could reduce intracellular ROS levels and cellular proliferation induced by PDGF.     

ZNF580在1-磷酸鞘氨醇诱导内皮细胞迁移和增殖中的作用

 目的：研究人锌指蛋白ZNF580在1-磷酸鞘氨醇(sphingosine 1-phosphate, S1P)诱导内皮细胞迁移和增殖中的作用,为探讨ZNF580功能提供科学依据。方法：RT-PCR检测S1P受体在EA.hy926细胞的表达情况;不同浓度(0~10 μmol/L) S1P刺激EA.hy926细胞不同时间(0~12 h)后,RT-PCR和Western blotting检测S1P对ZNF580表达的影响;利用p38 MAPK信号通路特异性抑制剂SB203580研究S1P是否通过此信号通路影响ZNF580的表达;脂质体转染法获得瞬时过表达和瞬时低表达ZNF580的EA.hy926细胞;Transwell实验及MTT比色法分析ZNF580对内皮细胞迁移和增殖活性的影响。结果：EA.hy926细胞表达S1P1、S1P3和S1P5三种受体,其cDNA的特异性扩增产物分别为352 bp、701 bp和236 bp;S1P刺激EA.hy926细胞后,ZNF580的表达呈现剂量和时间依赖性增高;SB203580能够抑制S1P诱导的ZNF580的上调作用;ZNF580过表达（低表达）后内皮细胞迁移和增殖活性明显增强（减弱）。结论：S1P通过p38 MAPK信号通路影响ZNF580的表达;ZNF580在内皮细胞迁移和增殖过程中起着重要的调控作用。     

Signalling of sphingosine-1-phosphate in Müller glial cells via the S1P/EDG-family of G-protein-coupled receptors

Signalling of sphingosine-1-phosphate (S1P) via G-protein-coupled receptors of the Endothelial Differentiation Gene family differentially regulates cellular processes such as migration, proliferation and morphogenesis in a variety of cell types. Proliferation and migration of retinal Müller glial cells are involved in pathological events such as proliferative vitreoretinopathy and proliferative diabetic retinopathy. Investigation of possible functional roles of S1P receptors might thus open new insights into Müller cell pathophysiology. Here we show that cultured Müller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC₅₀ 11 nM). This calcium increase consists of two components; an initial fast peak and a slow plateau component. The initial transient is caused by a release of calcium from intracellular stores and is suppressed by U-73122, a selective phospholipase C inhibitor. The slow plateau component is caused by a calcium influx. These results suggest that the S1P-induced calcium response in Müller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Müller cell migration but no proliferation. Thus, the data indicate that Müller cells might be involved in S1P signalling in the retina.