Apoptotic effect of sphingosine 1-phosphate and increased sphingosine 1-phosphate hydrolysis on mesangial cells cultured at low cell density

The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of the endothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 x 10(4) cells/cm(2)), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [(33)P]S1P, [(3)H]S1P, and [(3)H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [(3)H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novo ceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.     

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.     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H2O2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H2O2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H2O2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H2O2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H2O2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.     

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.     

Novel GPCR screening approach: indirect identification of S1P receptor agonists in antagonist screening using a calcium assay

To elucidate the physiological function of sphingosine 1-phosphate receptors 1-3 (S1P1-3) we aimed to identify selective ligands for these GPCRs. S1P2 and S1P3 are coupled to Gq, and are, therefore, linked to the phospholipase C/IP3/calcium pathway. S1P1 is solely coupled to Gi and was artificially linked to calcium signaling by coexpression of Galpha 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and "apparent" antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.     

Protein kinase Calpha and sphingosine 1-phosphate-dependent signaling in endothelial cell

Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCalpha and PKCepsilon activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P(3) stimulates PKCalpha and S1P1 leads to PKCepsilon activity. Infection of endothelial cells with dominant negative (DN)PKCalpha adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCalpha, as infection with DN PKCepsilon does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCalpha reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCalpha.     

Induction of antiproliferative connective tissue growth factor expression in Wilms' tumor cells by sphingosine-1-phosphate receptor 2

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration- and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P2, did not. Further, the specific S1P2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P2. This was confirmed by adenoviral transduction of S1P2 in WiT49 cells, which showed that overexpression of S1P2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH2-terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH2-terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P2 might act as a growth inhibitor in Wilms' tumor.     

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.     

Sphingosine 1-phosphate induces membrane ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and CrkII

Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of beta-adrenergic receptor kinase, indicating that the betagamma subunit of G(i) was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of G(i)-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.     

Activation of Src mediates PDGF-induced Smad1 phosphorylation and contributes to the progression of glomerulosclerosis in glomerulonephritis

Platelet-derived growth factor (PDGF) plays critical roles in mesangial cell (MC) proliferation in mesangial proliferative glomerulonephritis. We showed previously that Smad1 contributes to PDGF-dependent proliferation of MCs, but the mechanism by which Smad1 is activated by PDGF is not precisely known. Here we examined the role of c-Src tyrosine kinase in the proliferative change of MCs. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, MC proliferation and type IV collagen (Col4) expression peaked on day 6. Immunohistochemical staining for the expression of phospho-Src (pSrc), phospho-Smad1 (pSmad1), Col4, and smooth muscle α-actin (SMA) revealed that the activation of c-Src and Smad1 signals in glomeruli peaked on day 6, consistent with the peak of mesangial proliferation. When treated with PP2, a Src inhibitor, both mesangial proliferation and sclerosis were significantly reduced. PP2 administration also significantly reduced pSmad1, Col4, and SMA expression. PDGF induced Col4 synthesis in association with increased expression of pSrc and pSmad1 in cultured MCs. In addition, PP2 reduced Col4 synthesis along with decreased pSrc and pSmad1 protein expression in vitro. Moreover, the addition of siRNA against c-Src significantly reduced the phosphorylation of Smad1 and the overproduction of Col4. These results provide new evidence that the activation of Src/Smad1 signaling pathway plays a key role in the development of glomerulosclerosis in experimental glomerulonephritis.