The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a Gi-independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores partially blocked increases in [Ca2+]i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.

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Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G proteincoupled receptors, named S1P 1 to S1P 5 . In this study, we found that cross-communication between plateletderived growth factor receptor and S1P 2 serves as a negative damper of PDGF functions. Deletion of the S1P 2 receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P 1 and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P 2 deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P 2 -null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P 2 reduced DNA synthesis and expression of SphK1. Thus, S1P 2 serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed by activation of SphK by many stimuli, including platelet-derived growth factor (PDGF) (43, 48). As a specific ligand for a family of five G protein-coupled receptors (GPCRs), S1P 1 to S1P 5 (2, 48), S1P regulates a wide variety of important cellular processes, including cytoskeletal rearrangements and cell movement (17,25,45,49,57), angiogenesis and vascular maturation (14,16,26,32,57), and immunity and lymphocyte trafficking (33,34). Interestingly, all of the S1P receptors (S1PRs) have been shown to play critical roles in cytoskeletal reorganization and cell migration (13,26,57). Activation of S1P 1 or S1P 3 increases directional or chemotactic migration (14, 27, 57), and both mediate activation of Rac via G i (26,38). In contrast, ligation of S1P 2 decreases chemotaxis and membrane ruffling (49), due to suppression of Rac activation, probably by stimulation of a GTPase-activating protein for Rac (38). Interestingly, the repellant receptor S1P 2 and the attractant receptor S1P 3 similarly stimulate RhoA activity, likely via G 12/13 (21). Recent studies suggest that the balance of counteracting signals from the G i -and the G 12/13 -Rho pathways directs either positive or negative regulation of Rac and cell migration (49). Similar to its functions in lower organisms, including yeasts and plants, which do not have S1PRs, S1P may also have intracellular actions important for calcium homeostasis (36), cell growth (40, 56), and stress responses (9,11,37,42). S1P, like various other GPCR agonists, can activate growth factor tyrosine kinase receptors in the absence of added...

Various foods are associated with effects against metabolic diseases such as insulin resistance and type 2 diabetes; however, their mechanisms of action are mostly unclear. Fatty acids may contribute by acting as precursors of signalling molecules or by direct activity on receptors. The medium-and long-chain NEFA receptor FFA1 (free fatty acid receptor 1, previously known as GPR40) has been linked to enhancement of glucose-stimulated insulin secretion, whereas FFA4 (free fatty acid receptor 4, previously known as GPR120) has been associated with insulin-sensitising and anti-inflammatory effects, and both receptors are reported to protect pancreatic islets and promote secretion of appetite and glucose-regulating hormones. Hypothesising that FFA1 and FFA4 mediate therapeutic effects of dietary components, we screened a broad selection of NEFA on FFA1 and FFA4 and characterised active compounds in concentration -response curves. Of the screened compounds, pinolenic acid, a constituent of pine nut oil, was identified as a relatively potent and efficacious dual FFA1/FFA4 agonist, and its suitability for further studies was confirmed by additional in vitro characterisation. Pine nut oil and free and esterified pure pinolenic acid were tested in an acute glucose tolerance test in mice. Pine nut oil showed a moderately but significantly improved glucose tolerance compared with maize oil. Pure pinolenic acid or ethyl ester gave robust and highly significant improvements of glucose tolerance. In conclusion, the present results indicate that pinolenic acid is a comparatively potent and efficacious dual FFA1/FFA4 agonist that exerts antidiabetic effects in an acute mouse model. The compound thus deserves attention as a potential active dietary ingredient to prevent or counteract metabolic diseases.