<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> lyase (S<em>1</em>PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S<em>1</em>PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(<em>1</em>(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, <em>1</em>) led to the discovery of 2 (LX293<em>1</em>) and 4 (LX2932). The immunological phenotypes observed in S<em>1</em>PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S<em>1</em>PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to <em>1</em>80 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends on the weak proteolytic activity of its own zymogen as well as its cognate inhibitor, hepatocyte growth factor activator inhibitor <em>1</em> (HAI-<em>1</em>). Oligomerization of matriptase zymogens and HAI-<em>1</em>, and probably its interaction with other proteins, has been proposed to occur during matriptase activation. In the present study, we examined the cellular events associated with matriptase activation triggered either by the physiological inducer <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) or by a chemical inducer, the polyanionic compound suramin. S<em>1</em>P-induced matriptase translocation to cell-cell contacts, where it is activated, is an F-actin polymerization-dependent process. Conversely, suramin-induced matriptase accumulation and activation at vesicle-like structures is an F-actin polymerization-independent process. While matriptase activation can occur at different subcellular locations, both S<em>1</em>P- and suramin-induced matriptase accumulation form unique subcellular structures, termed activation foci, where oligomerization of matriptase zymogens and HAI-<em>1</em> may occur, promoting matriptase activation. Furthermore, matriptase activation may be regulated by intracellular signaling, because Ro 3<em>1</em>-8220, a bisindolylmaleimide protein kinase C inhibitor, inhibited both S<em>1</em>P- and suramin-induced activation. The requirement of HAI-<em>1</em> for matriptase activation and the coincidence of HAI-<em>1</em> and matriptase in activation foci apparently provide rapid access of HAI-<em>1</em> for the inhibition of matriptase immediately after its activation. Indeed, all activated matriptase was detected in complexes with HAI-<em>1</em> only 5 min after suramin stimulation. The close temporospatial coupling of matriptase activation with its inhibition suggests that the proteolytic activity of this enzyme must be well controlled and that the proteolysis of matriptase substrates may be tightly regulated by this mechanism.

Dietary L-arginine (Arg) supplementation reduces white-fat gain in diet-induced obese rats but the underlying mechanisms are unknown. This study tested the hypothesis that Arg treatment affects expression of genes related to lipid metabolism in adipose tissue. Four-week-old male Sprague-Dawley rats were fed a low-fat (LF) or high-fat (HF) diet for <em>1</em>5 weeks. Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing <em>1</em>.5<em>1</em>% Arg-HCl or 2.55% L: -alanine (isonitrogenous control). After <em>1</em>2 weeks of Arg supplementation, rats were euthanized to obtain retroperitoneal adipose tissue for analyzing global changes in gene expression by microarray. The results were confirmed by RT-PCR analysis. HF feeding decreased mRNA levels for lipogenic enzymes, AMP-activated protein kinase, glucose transporters, heme oxygenase 3, glutathione synthetase, superoxide dismutase 3, peroxiredoxin 5, glutathione peroxidase 3, and stress-induced protein, while increasing expression of carboxypeptidase-A, peroxisome proliferator activated receptor (PPAR)-alpha, caspase 2, caveolin 3, and diacylglycerol kinase. In contrast, Arg supplementation reduced mRNA levels for fatty acid binding protein <em>1</em>, glycogenin, protein <em>phosphates</em> <em>1</em>B, caspases <em>1</em> and 2, and hepatic lipase, but increased expression of PPARgamma, heme oxygenase 3, glutathione synthetase, insulin-like growth factor II, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor, and stress-induced protein. Biochemical analysis revealed oxidative stress in white adipose tissue of HF-fed rats, which was prevented by Arg supplementation. Collectively, these results indicate that HF diet and Arg supplementation differentially regulate gene expression to affect energy-substrate oxidation, redox state, fat accretion, and adipocyte differentiation in adipose tissue. Our findings provide a molecular mechanism to explain a beneficial effect of Arg on ameliorating diet-induced obesity in mammals.

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S<em>1</em>PRs). Here, we studied the role of S<em>1</em>PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S<em>1</em>PR2-specific antagonist (JTE0<em>1</em>3), we found that S<em>1</em>PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S<em>1</em>pr2(+/+) → S<em>1</em>pr2(+/+), S<em>1</em>pr2(+/+) → S<em>1</em>pr2(-/-), and S<em>1</em>pr2(-/-) → S<em>1</em>pr2(+/+)) indicate the critical role of S<em>1</em>PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE0<em>1</em>3 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S<em>1</em>PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S<em>1</em>PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S<em>1</em>PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S<em>1</em>PR2 as a novel therapeutic target for vascular disorders.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) in animal models of ALI with reductions in lung edema. As S<em>1</em>P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S<em>1</em>P lyase (S<em>1</em>PL), the enzyme that irreversibly degrades S<em>1</em>P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S<em>1</em>PL expression, decreased S<em>1</em>P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[<em>1</em>(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S<em>1</em>PL or S<em>1</em>PL(+/-) mice resulted in increased S<em>1</em>P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S<em>1</em>PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S<em>1</em>P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac<em>1</em> activation. These results identify a novel role for intracellularly generated S<em>1</em>P in protection against ALI and suggest S<em>1</em>PL as a potential therapeutic target.

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin <em>1</em> (IL-<em>1</em>) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK<em>1</em> is a lipid kinase that produces the pro-growth, anti-apoptotic <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-<em>1</em> correlates with the expression of SphK<em>1</em> in glioblastoma cells, and neutralizing anti-IL-<em>1</em> antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-<em>1</em> up-regulates SphK<em>1</em> mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-<em>1</em>-induced SphK<em>1</em> up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK<em>1</em> expression by IL-<em>1</em> occurs on the level of transcription and is mediated via a novel AP-<em>1</em> element located within the first intron of the sphk<em>1</em> gene. In summary, our results suggest that SphK<em>1</em> expression is transcriptionally regulated by IL-<em>1</em> in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.

Sphingolipids are biologically active lipids that play important roles in various cellular processes and the sphingomyelin metabolites ceramide, <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> can act as signalling molecules in most cell types. With the recent development of the immunosuppressant drug FTY720 (Fingolimod) which after phosphorylation in vivo acts as a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist, research on the role of sphingolipids in the immune and other organ systems was triggered enormously. Since it was reported that FTY720 induced a modest, but significant transient decrease in heart rate in animals and humans, the question was raised which pharmacological properties of drugs targeting sphingolipid signalling will affect cardiovascular function in vivo. The answer to this question will most likely also indicate what type of drug could be used to treat cardiovascular disease. The latter is becoming increasingly important because of the increasing population carrying characteristics of the metabolic syndrome. This syndrome is, amongst others, characterized by obesity, hypertension, atherosclerosis and diabetes. As such, individuals with this syndrome are at increased risk of heart disease. Now numerous studies have investigated sphingolipid effects in the cardiovascular system, can we speculate whether certain sphingolipids under specific conditions are good, bad or maybe both? In this review we will give a brief overview of the pathophysiological role of sphingolipids in cardiovascular disease. In addition, we will try to answer how drugs that target sphingolipid signalling will potentially influence cardiovascular function and whether these drugs would be useful to treat cardiovascular disease.

STAT3 plays a crucial role in promoting progression of human cancers, including several types of B-cell lymphoma. However, as a transcription factor lacking its own enzymatic activity, STAT3 remains difficult to target with small-molecule drugs in the clinic. Here we demonstrate that persistent activated STAT3 colocalizes with elevated expression of S<em>1</em>PR<em>1</em>, a G-protein-coupled receptor for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), in the tumor cells of the activated B cell-like subtype of diffuse large B-cell lymphoma patient specimens. Inhibition of S<em>1</em>PR<em>1</em> expression by shRNA in the lymphoma cells validates that blocking S<em>1</em>PR<em>1</em> affects expression of STAT3 downstream genes critically involved in tumor cell survival, proliferation, tumor invasion, and/or immunosuppression. Using S<em>1</em>PR<em>1</em> shRNA, or FTY720, an antagonist of S<em>1</em>P that is in the clinic for other indications, we show that inhibiting S<em>1</em>PR<em>1</em> expression down-regulates STAT3 activity and causes growth inhibition of the lymphoma tumor cells in vitro and in vivo. Our results suggest that targeting S<em>1</em>P/S<em>1</em>PR<em>1</em> using a clinically relevant and available drug or other approaches is potentially an effective new therapeutic modality for treating the activated B cell-like subtype of diffuse large B-cell lymphoma, a subset of lymphoma that is less responsive to current available therapies.

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-<em>1</em> (Ang-<em>1</em>) or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-<em>1</em> and S<em>1</em>P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.

Interleukin <em>1</em>beta (IL-<em>1</em>beta) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Cer has been proposed to play a role in the down-regulation of cytochrome P450 2C<em>1</em><em>1</em> (CYP2C<em>1</em><em>1</em>) and induction of alpha<em>1</em>-acid glycoprotein (AGP) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (<em>1</em>995) J. Biol. Chem. 270, 25233-25238). Nonetheless, some of the features of the down-regulation of CYP2C<em>1</em><em>1</em> do not fit a simple model of Cer as a second messenger as follows: N-acetylsphinganine (C2-DHCer) is as potent as N-acetyl<em>sphingosine</em> (C2-Cer) in suppression of CYP2C<em>1</em><em>1</em>; the IL-<em>1</em>beta concentration dependence for SM turnover is different from that for the increase in Cer; and the increase in Cer mass is not equivalent to the amount of SM hydrolyzed nor the time course of SM hydrolysis. In this article, we report that these discrepancies are due to activation of ceramidase by the low concentrations of IL-<em>1</em>beta ( approximately 2.5 ng/ml) that maximally down-regulate CYP2C<em>1</em><em>1</em> expression, whereas higher IL-<em>1</em>beta concentrations (that induce AGP) do not activate ceramidase and allow Cer accumulation. This bimodal concentration dependence is demonstrated both by in vitro ceramidase assays and in intact hepatocytes using a fluorescence Cer analog, 6-((N-(7-nitrobenz-2-oxa-<em>1</em>, 3-diazol-4-yl)amino)-Cer (NBD-Cer), and following release of the NBD-fatty acid. IL-<em>1</em>beta increases both acid and neutral ceramidase activities, which appear to be regulated by tyrosine phosphorylation because pretreatment of hepatocytes with sodium vanadate increases (and 25 microM genistein reduces) the basal and IL-<em>1</em>beta-stimulated ceramidase activities. Since these findings suggested that <em>sphingosine</em> (and, possibly, subsequent metabolites) is the primary mediator of the down-regulation of CYP2C<em>1</em><em>1</em> by IL-<em>1</em>beta, the effects of exogenous <em>sphingosine</em> and C2-Cer on expression of this gene were compared. <em>Sphingosine</em> was more potent than C2-Cer in down-regulation of CYP2C<em>1</em><em>1</em> when added alone or with fumonisin B<em>1</em> to block acylation of the exogenous <em>sphingosine</em>. Furthermore, the suppression of CYP2C<em>1</em><em>1</em> by C2-Cer (and C2-DHCer) is probably mediated by free sphingoid bases, rather than the short chain Cer directly, because both are hydrolyzed by hepatocytes and increase cellular levels of <em>sphingosine</em> and sphinganine. From these observations we conclude that <em>sphingosine</em>, possibly via <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, is a mediator of the regulation of CYP2C<em>1</em><em>1</em> by IL-<em>1</em>beta in rat hepatocytes and that ceramidase activation provides a "switch" that determines which sphingolipids are elevated by this cytokine to produce multiple intracellular responses.

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with <em>sphingosine</em> kinase, metabolizes ceramide to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.

Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor <em>1</em> (OGR<em>1</em>) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR<em>1</em> (5<em>1</em>%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF<em>1</em>0A cells. These effects are insensitive to treatment with BN5202<em>1</em>, WEB-2<em>1</em>70, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = <em>1</em>59 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), PAF, psychosine (Psy), glucosyl-beta<em>1</em>'<em>1</em>-<em>sphingosine</em> (Glu-Sph), galactosyl-beta<em>1</em>'<em>1</em>-ceramide (Gal-Cer), or lactosyl-beta<em>1</em>'<em>1</em>-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a receptor with high affinity to SPC and low affinity to LPC, and that multiple cellular functions can be transduced via this receptor.

The bioactive lipid molecule <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) binds to specific cell surface receptors and regulates several cellular processes. S<em>1</em>P is abundant in plasma, and physiologically its most important target cells are lymphocytes and vascular endothelial cells. S<em>1</em>P plays a pivotal role in the immune system by regulating lymphocyte egress from the thymus and secondary lymphoid organs. The immunomodulator FTY720 impairs this egress, causing lymphopenia. Platelets had long been considered to be the major source of plasma S<em>1</em>P, however recent studies revealed the importance of erythrocytes as a major supply. The <em>sphingosine</em> analog FTY720 is a prodrug, and FTY720 <em>phosphate</em> (FTY720-P) its functional form. Although both erythrocytes and platelets can produce S<em>1</em>P, only platelets synthesize and release FTY720-P. This review will focus on the recent advances in our understanding of the metabolism and release of S<em>1</em>P and FTY720-P, especially in platelets and erythrocytes.

ATP is a physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of P(2) purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier enhancement evoked by ATP has been described. ATP effects on EC barrier function and vascular permeability, however, remain poorly characterized. Although the mechanisms involved are unclear, we previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier augmentation induced by simvastatin and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and therefore examined the role of these molecules in ATP-induced EC barrier enhancement. ATP induced rapid, dose-dependent barrier enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25 min (39% increase, <em>1</em>0 microM) and persisting at 2 h. These effects were associated with rearrangement of the EC actin cytoskeleton, early myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (<em>1</em>0 microM)-treated EC demonstrated a significant increase in Rac activation relative to controls, with a maximal effect (approximately 4-fold increase) at <em>1</em>0 min. Finally, ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to controls. Our results suggest for the first time that ATP-mediated barrier protection is associated with cytoskeletal activation and is dependent on both Rac activation and cortactin.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S<em>1</em>P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-<em>1</em><em>1</em>79, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S<em>1</em>P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S<em>1</em>P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S<em>1</em>P-induced eNOS enzyme activation, as well as S<em>1</em>P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-<em>1</em><em>1</em>79, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S<em>1</em>P-induced endothelial cell migration was maximum at <em>1</em> microM (approximately 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (<em>1</em>0 ng/ml) (approximately 2.5-fold increase) or hepatocyte growth factor (HGF) (approximately 2.5-fold increase). Migration induced by S<em>1</em>P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-<em>1</em> and Edg-3 (endothelial differentiation gene) S<em>1</em>P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a betagamma subunit inhibitory peptide of the beta-adrenergic receptor kinase, or an <em>1</em><em>1</em>-amino-acid peptide that inhibits G(<em>1</em>alpha2) signaling. Various strategies to interrupt Rho family signaling, including C(3) exotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S<em>1</em>P- but not VEGF-induced migration. Conversely, pharmacologic inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase reduced basal endothelial cell migration and abolished VEGF-induced endothelial cell migration but did not inhibit the increase in S<em>1</em>P-induced migration. Whereas VEGF and S<em>1</em>P increased both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein (MAP) kinase activities, only p38 MAP kinase inhibition significantly reduced VEGF- and S<em>1</em>P-stimulated migration. These data confirm S<em>1</em>P as a potent endothelial cell chemoattractant through G(<em>1</em>alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 MAP kinase activation. The divergence in signaling pathways evoked by S<em>1</em>P and VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.

Platelet-derived growth factor (PDGF) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) act via PDGF beta receptor-S<em>1</em>P(<em>1</em>) receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S<em>1</em>P(<em>1</em>) antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF beta receptor-S<em>1</em>P(<em>1</em>) receptor complex. Third, treatment of cells with antisense S<em>1</em>P(<em>1</em>) receptor plasmid construct reduced the PDGF- and S<em>1</em>P-dependent activation of p42/p44 MAPK. Fourth, S<em>1</em>P and/or PDGF induced the formation of endocytic vesicles containing both PDGF beta receptors and S<em>1</em>P(<em>1</em>) receptors, which was required for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S<em>1</em>P, suggesting the absence of a sequential mechanism. However, <em>sphingosine</em> kinase <em>1</em> is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous <em>sphingosine</em>. Thus, the presentation of <em>sphingosine</em> from other cell types and its conversion to S<em>1</em>P by the kinase exported from airway smooth muscle cells might enable S<em>1</em>P to act with PDGF on the PDGF beta receptor-S<em>1</em>P(<em>1</em>) receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-protein-coupled receptor agonists and/or sequential release and action of S<em>1</em>P in response to PDGF.

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S<em>1</em>P<em>1</em> and S<em>1</em>P4 receptors and demonstrate a reduced chemotactic response to S<em>1</em>P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) catalyzes the phosphorylation of <em>sphingosine</em> to produce the potent lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK<em>1</em>-interacting protein. Heregulin stimulated SphK<em>1</em> activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK<em>1</em> was required for heregulin-induced migration, lamellipodia formation, activation of PAK<em>1</em>, and subsequent FLNa phosphorylation. S<em>1</em>P directly stimulated PAK<em>1</em> kinase, suggesting that it may be a target of intracellularly generated S<em>1</em>P. Heregulin also induced colocalization of S<em>1</em>P(<em>1</em>) (promotility S<em>1</em>P receptor) but not S<em>1</em>P(2), with SphK<em>1</em> and FLNa at membrane ruffles. Moreover, an S<em>1</em>P(<em>1</em>) antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK<em>1</em> and S<em>1</em>P(<em>1</em>) to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK<em>1</em>, FLNa, and PAK<em>1</em>, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.

It is generally recognized that CpG islands are not methylated in normal tissues. SPHK<em>1</em> is a key enzyme catalyzing the production of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a novel signaling molecule for the proliferation and differentiation of various cells, including neural cells. Sequencing of genomic DNA and cDNA reveals that rat Sphk<em>1</em>a consists of six exons encoding 383 amino acids. Furthermore, we identified six alternative first exons for mRNA subtypes (Sphk<em>1</em>a, -b, -c, -d, -e, and -f) within a 3.7-kb CpG island. The CpG island contains a tissue-dependent, differentially methylated region (T-DMR; approximately 200 bp), which is located - 800 bp upstream of the first exon of Sphk<em>1</em>a. T-DMR is hypomethylated in the adult brain where Sphk<em>1</em>a is expressed, whereas it is hypermethylated in the adult heart where the gene is not expressed. In fetal tissues, hypomethylation of T-DMR is not associated with expression of Sphk<em>1</em>a, which suggests that differential availability of transcription factors is also likely to be involved in the mechanism of its expression. Here, we identify rat Sphk<em>1</em>, using multiple alternative first exons for the subtypes, and demonstrate that there is a CpG island bearing T-DMR.

BACKGROUND

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase <em>1</em> (MT<em>1</em>-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S<em>1</em>P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT<em>1</em>-MMP in this process.

RESULTS

LPA stimulated while S<em>1</em>P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT<em>1</em>080 fibrosarcoma cells migrated in response to both lipids. HT<em>1</em>080 cells also markedly invaded 3D collagen matrices (approximatly 700 microm over 48 hours) in response to either lipid. siRNA targeting of LPA<em>1</em> and Rac<em>1</em>, or S<em>1</em>P<em>1</em>, Rac<em>1</em>, and Cdc42 specifically inhibited LPA- or S<em>1</em>P-induced HT<em>1</em>080 invasion, respectively. Analysis of LPA-induced HT<em>1</em>080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (approximately <em>1</em>25 microm vs. approximately 45 microm) and velocity of invasion (approximately 0.09 microm/min vs. approximately 0.03 microm/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-<em>1</em>, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MT<em>1</em>-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT<em>1</em>-MMP nor invade in the presence of LPA, were transfected with MT<em>1</em>-MMP cDNA, and subsequently invaded in response to LPA. When HT<em>1</em>080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT<em>1</em>-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT<em>1</em>-MMP in this process.

CONCLUSIONS

LPA is a fundamental regulator of MT<em>1</em>-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S<em>1</em>P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT<em>1</em>-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/S<em>1</em>P receptors and Rho GTPases to migrate, and additionally, require MT<em>1</em>-MMP in order to invade collagen matrices during neoplastic progression.

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S<em>1</em>P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bis<em>phosphate</em>-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac<em>1</em> from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac<em>1</em> colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bis<em>phosphate</em>, moesin, actin, and rac<em>1</em> act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.

<em>Sphingosine</em> kinase (SphK) phosphorylates <em>sphingosine</em> into <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). S<em>1</em>P plays a critical role in angiogenesis, inflammation, and various pathologic conditions. To date, two mammalian isoenzymes, SphK<em>1</em> and SphK2, have been identified. Although both SphK<em>1</em> and SphK2 share overall homology and produce the common product, S<em>1</em>P, it has been proposed they display different unique and separate functions. In this study, we examined the role of SphK<em>1</em> and SphK2 in a murine collagen-induced arthritis model by down-regulating each isoenzyme via specific small interfering RNA (siRNA). Prophylactic i.p. administration of SphK<em>1</em> siRNA significantly reduced the incidence, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK<em>1</em> siRNA also down-regulated serum levels of S<em>1</em>P, IL-6, TNF-alpha, IFN-gamma, and IgG2a anti-collagen Ab. Ex vivo analysis demonstrated significant suppression of collagen-specific proinflammatory/Th<em>1</em> cytokine (IL-6, TNF-alpha, IFN-gamma) release in SphK siRNA-treated mice. Interestingly, mice received with SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-alpha, and IFN-gamma; and proinflammatory cytokine production to collagen in vitro when compared with control siRNA recipients. Together, these results demonstrate the distinct immunomodulatory roles of SphK<em>1</em> and SphK2 in the development of inflammatory arthritis by regulating the release of proinflammatory cytokines and T cell responses. These findings raise the possibility that drugs which specifically target SphK<em>1</em> activity may play a beneficial role in the treatment of inflammatory arthritis.

Acute lung injury (ALI) is a severe inflammatory disease for which no specific treatment exists. As glucocorticoids have potent immunosuppressive effects, their application in ALI is currently being tested in clinical trials. However, the benefits of this type of regimen remain unclear. Here we identify a mechanism of glucocorticoid action that challenges the long-standing dogma of cytokine repression by the glucocorticoid receptor. Contrarily, synergistic gene induction of <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) by glucocorticoids and pro-inflammatory stimuli via the glucocorticoid receptor in macrophages increases circulating <em>sphingosine</em> <em>1</em>-<em>phosphate</em> levels, which proves essential for the inhibition of inflammation. Chemical or genetic inhibition of SphK<em>1</em> abrogates the therapeutic effects of glucocorticoids. Inflammatory p38 MAPK- and mitogen- and stress-activated protein kinase <em>1</em> (MSK<em>1</em>)-dependent pathways cooperate with glucocorticoids to upregulate SphK<em>1</em> expression. Our findings support a critical role for SphK<em>1</em> induction in the suppression of lung inflammation by glucocorticoids, and therefore provide rationales for effective anti-inflammatory therapies.