OBJECTIVE

Plasma high-density lipoprotein (HDL) level is inversely correlated with the risk of atherosclerosis. However, the cellular mechanism by which HDL exerts antiatherogenic actions is not well understood. In this study, we focus on the lipid components of HDL as mediators of the lipoprotein-induced antiatherogenic actions.

RESULTS

HDL and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulated the migration and survival of human umbilical vein endothelial cells. These responses to HDL and S<em>1</em>P were almost completely inhibited by pertussis toxin and other specific inhibitors for intracellular signaling pathways, although the inhibition profiles of migration and survival were different. The HDL-stimulated migration and survival of the cells were markedly inhibited by antisense oligonucleotides against the S<em>1</em>P receptors EDG-<em>1</em>/S<em>1</em>P<em>1</em> and EDG-3/S<em>1</em>P3. Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S<em>1</em>P<em>1</em>. The S<em>1</em>P-rich fraction and chromatographically purified S<em>1</em>P from HDL stimulated cell migration, but the rest of the fraction did not, as was the case of the cell survival.

CONCLUSIONS

HDL-induced endothelial cell migration and survival may be mediated by the lipoprotein component S<em>1</em>P and the lipid receptors S<em>1</em>P<em>1</em> and S<em>1</em>P3.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-<em>1</em> and TNF. S<em>1</em>P administration via the airways but not via the vasculature induces lung leakage. Using receptor-null mice, we show that S<em>1</em>P, acting on S<em>1</em>P3 receptor expressed on both type I and type II alveolar epithelial cells but not vascular endothelium, induces pulmonary edema by acute tight junction opening. WT but not S<em>1</em>P3-null mice showed disruption of pulmonary epithelial tight junctions and the appearance of paracellular gaps between epithelial cells by electron microscopy within <em>1</em> h of airways exposure to S<em>1</em>P. We further show by fluorescence microscopy that S<em>1</em>P induced rapid loss of ZO-<em>1</em> reactivity, an essential component of the cytoplasmic plaque associated with tight junctions, as well as of the tetraspannin Claudin-<em>1</em>8, an integral membrane organizer of tight junctions. S<em>1</em>P shows synergistic activity with the proinflammatory cytokine TNF, showing both pulmonary edema and mortality at subthreshold S<em>1</em>P doses. Specifically, preexposure of mice to subthreshold doses of TNF, which alone induced no lung edema, exacerbated S<em>1</em>P-induced edema and impaired survival. S<em>1</em>P, acting through S<em>1</em>P3, regulates epithelial integrity and acts additively with TNF in compromising respiratory barrier function. Because S<em>1</em>P3-null mice are resistant to S<em>1</em>P-induced pulmonary leakage, either alone or in the presence of TNF, S<em>1</em>P3 antagonism may be useful in protecting epithelial integrity in pulmonary disease.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) phosphorylates <em>sphingosine</em> to generate <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK<em>1</em> is of considerable interest. We report that SK<em>1</em>, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK<em>1</em> with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK<em>1</em>-PA interaction maps to the C terminus of SK<em>1</em> and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK<em>1</em> constitutes a novel, physiologically relevant PA effector.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N<em>1</em>E-<em>1</em><em>1</em>5 neuronal cells, S<em>1</em>P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S<em>1</em>P is approximately <em>1</em>00-fold more potent than LPA in evoking these shape changes, with an EC50 as low as <em>1</em>.5 nM. Microinjection of S<em>1</em>P has no effect, neither has addition of <em>sphingosine</em> or ceramide. As with LPA, S<em>1</em>P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S<em>1</em>P and LPA do not show cross-desensitization. We conclude that S<em>1</em>P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S<em>1</em>P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor, S<em>1</em>P(<em>1</em>), and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S<em>1</em>P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S<em>1</em>P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S<em>1</em>P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.

The novel immunomodulator FTY720 is remarkably effective in models of transplantation and autoimmunity. Recent data show that phosphorylated FTY720 is an agonist at four <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors. Stimulation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors leads to sequestration of lymphocytes in secondary lymphatic tissues and thus away from inflammatory lesions and graft sites.

<em>Sphingosine</em> kinase (SPHK) catalyzes the formation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em><em>1</em>). S<em>1</em>P plays an important role in regulation of a variety of biological processes through intracellular and extracellular actions. S<em>1</em>P has recently been shown to be the ligand for the EDG-<em>1</em> family of G-protein-coupled receptors. To date, seven cloned SPHKs have been reported with confirmed SPHK activity, including human, mouse, yeast, and plant. A computer search of various databases suggests that a new SPHK family is emerging. The cloning and manipulation of SPHK genes will no doubt provide us with important information about the functions of S<em>1</em>P in a wide range of organisms.

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme <em>sphingosine</em> <em>1</em>-<em>phosphate</em> lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.

The sphingolipid metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a serum-borne lipid that regulates many vital cellular processes. S<em>1</em>P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S<em>1</em>P. It has been suggested that S<em>1</em>P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S<em>1</em>P has been challenging, but there is ample evidence to suggest that the balance between S<em>1</em>P and ceramide and/or <em>sphingosine</em> levels in cells is an important determinant of cell fate.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S<em>1</em>P(<em>1</em>) to S<em>1</em>P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S<em>1</em>P(2) serves as a negative damper of PDGF functions. Deletion of the S<em>1</em>P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S<em>1</em>P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S<em>1</em>P(<em>1</em>) and <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), the enzyme that produces S<em>1</em>P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S<em>1</em>P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase <em>1</em>/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S<em>1</em>P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK<em>1</em> expression and activity. Conversely, reintroduction of S<em>1</em>P(2) reduced DNA synthesis and expression of SphK<em>1</em>. Thus, S<em>1</em>P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK<em>1</em> expression. Our results suggest that a complex interplay between PDGFR and S<em>1</em>P receptors determines their functions.

FTY720 (fingolimod) is an oral <em>sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP-9 and increased its counterregulator--tissue inhibitor of metalloproteinase, TIMP-<em>1</em>--resulting in a proteolytic balance that favors preservation of blood-brain-barrier (BBB) integrity. Furthermore, FTY720 reduced S<em>1</em>P lyase that increases the S<em>1</em>P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S<em>1</em>P receptors on resident brain cells. This is accompanied by an increase in S<em>1</em>P(<em>1</em>) and S<em>1</em>P(5) in contrast with the attenuation of S<em>1</em>P(3) and S<em>1</em>P(4). Late-stage rescue therapy with FTY720, even up to <em>1</em> month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis, acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S<em>1</em>P(<em>1</em>-5)). <em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>), the enzyme responsible for S<em>1</em>P production, is overexpressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of "hypoxic regions," characterized by cells expressing high levels of hypoxia-inducible factors HIF-<em>1</em>alpha and HIF-2alpha, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far, nothing is known about the role and the regulation of SK<em>1</em> during tumor-induced hypoxia or about SK<em>1</em> regulation and HIFs. Here we investigated the role of HIF-<em>1</em>alpha and HIF-2alpha in the regulation of SK<em>1</em> during hypoxic stress in glioma-derived U87MG cells. We report that hypoxia increases SK<em>1</em> mRNA levels, protein expression, and enzyme activity, followed by intracellular S<em>1</em>P production and S<em>1</em>P release. Interestingly, knockdown of HIF-2alpha by small interfering RNA abolished the induction of SK<em>1</em> and the production of extracellular S<em>1</em>P after CoCl(2) treatment, whereas HIF-<em>1</em>alpha small interfering RNA resulted in an increase of HIF-2alpha and of SK<em>1</em> protein levels. Moreover, using chromatin immunoprecipitation analysis, we demonstrate that HIF-2alpha binds the SK<em>1</em> promoter. Functionally, we demonstrate that conditioned medium from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S<em>1</em>P receptor-dependent manner. These studies provide evidence of a link between S<em>1</em>P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors.

<em>Sphingosine</em> kinase catalyzes the formation of the bioactive sphingolipid metabolite <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney <em>sphingosine</em> kinase 6 x <em>1</em>0(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified <em>sphingosine</em> kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify <em>sphingosine</em> kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified <em>sphingosine</em> kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of <em>1</em> M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified <em>sphingosine</em> kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. <em>Sphingosine</em> kinase shows substrate specificity for D-erythro-<em>sphingosine</em> and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydro<em>sphingosine</em>, or N,N-dimethyl<em>sphingosine</em>. However, the latter two sphingolipids were potent competitive inhibitors. With <em>sphingosine</em> as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for <em>sphingosine</em> and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

Radiotherapy is widely used as a radical treatment for prostate cancer, but curative treatments are elusive for poorly differentiated tumors where survival is just <em>1</em>5% at <em>1</em>5 years. Dose escalation improves local response rates but is limited by tolerance in normal tissues. A <em>sphingosine</em> analogue, FTY720 (fingolimod), a drug currently in phase III studies for treatment of multiple sclerosis, has been found to be a potent apoptosis inducer in prostate cancer cells. Using in vitro and in vivo approaches, we analyzed the impact of FTY720 on sphingolipid metabolism in hormone-refractory metastatic prostate cancer cells and evaluated its potential as a radiosensitizer on cell lines and prostate tumor xenografts. In prostate cancer cell lines, FTY720 acted as a <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) inhibitor that induced prostate cancer cell apoptosis in a manner independent of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors. In contrast, γ irradiation did not affect SphK<em>1</em> activity in prostate cancer cells yet synergized with FTY720 to inhibit SphK<em>1</em>. In mice bearing orthotopic or s.c. prostate cancer tumors, we show that FTY720 dramatically increased radiotherapeutic sensitivity, reducing tumor growth and metastasis without toxic side effects. Our findings suggest that low, well-tolerated doses of FTY720 could offer significant improvement to the clinical treatment of prostate cancer.

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S<em>1</em>P is produced by two <em>sphingosine</em> kinases, SphK<em>1</em> and SphK2. Expression of SphK<em>1</em> is elevated in many cancers. Here, we report that LPA markedly enhanced SphK<em>1</em> mRNA and protein in gastric cancer MKN<em>1</em> cells but had no effect on SphK2. LPA also up-regulated SphK<em>1</em> expression in other human cancer cells that endogenously express the LPA(<em>1</em>) receptor, such as DLD<em>1</em> colon cancer cells and MDA-MB-23<em>1</em> breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(<em>1</em>) receptor. An LPA(<em>1</em>) receptor antagonist or down-regulation of its expression prevented SphK<em>1</em> and S<em>1</em>P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG<em>1</em>478 attenuated the increased SphK<em>1</em> and S<em>1</em>P(3) expression induced by LPA. Moreover, down-regulation of SphK<em>1</em> attenuated LPA-stimulated migration and invasion of MNK<em>1</em> cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S<em>1</em>P(3), but not S<em>1</em>P(<em>1</em>), also reduced LPA-stimulated migration and invasion of MKN<em>1</em> cells. Collectively, our results suggest that SphK<em>1</em> is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S<em>1</em>P, which have all been implicated in regulation of motility and invasiveness of cancer cells.

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor signaling system has biological and medical importance and is the first lipid G protein-coupled receptor (GPCR) structure to be solved to 2.8-Å resolution. S<em>1</em>P binds to five high-affinity GPCRs generating multiple downstream signals that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and routine oral treatment of multiple sclerosis. Genetics, chemistry, and now structural biology have advanced this integrated biochemical system. The S<em>1</em>P receptors have a novel N-terminal fold that occludes access to the binding pocket from the extracellular environment as well as orthosteric and bitopic ligands with very different physicochemical properties. S<em>1</em>P receptors and metabolizing enzymes have been deleted, inducibly deleted, and knocked in as tagged or altered receptors in mice. An array of genetic models allows analysis of integrated receptor function in vivo. We can now directly understand causal relationships among protein expression, signal, and control points in physiology and pathology.

Lymphocytes egress from lymphoid organs in response to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P); minutes later they migrate from blood into tissue against the S<em>1</em>P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here, we show that heterotrimeric guanine nucleotide-binding protein-coupled receptor kinase-2 (GRK2) functions in down-regulation of S<em>1</em>P receptor-<em>1</em> (S<em>1</em>PR<em>1</em>) on blood-exposed lymphocytes. T and B cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S<em>1</em>P-deficient mice. In the spleen, B cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2 deficiency and by mutation of an S<em>1</em>PR<em>1</em> desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S<em>1</em>PR<em>1</em> desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein-coupled receptors on the cell surface (S<em>1</em>P<em>1</em>-5), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S<em>1</em>P action in human glioblastoma cells are not well defined. S<em>1</em>P receptors (<em>1</em>-5) and S<em>1</em>P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S<em>1</em>P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S<em>1</em>P showed a significant increase in migration, whereas U<em>1</em><em>1</em>8 and U<em>1</em>38 cell lines were strongly inhibited. S<em>1</em>P-mediated inhibition correlated with S<em>1</em>P2 receptor expression. FTY720-P, an S<em>1</em>P analogue that binds all S<em>1</em>P receptors except S<em>1</em>P2, did not inhibit glioblastoma cell migration. Overexpression of S<em>1</em>P2 further suppressed migration, and blockage of S<em>1</em>P2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S<em>1</em>P2 receptor stimulation, both Rac<em>1</em> and RhoA GTPases were activated by S<em>1</em>P treatment in native cells and cells overexpressing S<em>1</em>P2. Treatment of U<em>1</em><em>1</em>8 cells with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S<em>1</em>P stimulated U<em>1</em><em>1</em>8 cells overexpressing beta-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S<em>1</em>P2 overexpression, partially blocked by S<em>1</em>P<em>1</em>, or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S<em>1</em>P inhibition of tumor cell migration via Rho kinase-dependent pathway.

Sphingolipid (SPL) metabolism (Fig. <em>1</em>) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), <em>sphingosine</em> (Sph) and <em>sphingosine</em>l-<em>phosphate</em> (S<em>1</em>P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary. Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam<em>1</em>, an activator of the Rho-like GTPase Rac, and by constitutively active V<em>1</em>2Rac<em>1</em>. Here we show that activated V<em>1</em>2Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) (<em>1</em>0(-7)-<em>1</em>0(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S<em>1</em>P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V<em>1</em>4RhoA and V<em>1</em>2Rac<em>1</em> does not bypass the LPA/S<em>1</em>P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73<em>1</em>22, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam<em>1</em>/Rac or Cdc42 activation, and is dependent on LPA/S<em>1</em>P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.

GTPases of the Rho family are transducers of extracellular signals and control cellular processes such as organization of the actin cytoskeleton, motility, adhesion and gene regulation. The Rho signalling pathway is activated, for example, by bioactive sphingolipids such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) or by overexpression of Rho family members in tumorigenesis and metastases. Here, we show that stimulation of the Rho signalling pathway induces translocation of the transcriptional LIM-only coactivator FHL2 to the nucleus and subsequent activation of FHL2- and androgen receptor-dependent genes. Interestingly, prostate tumours overexpress Rho GTPases and display altered cellular localization of FHL2 concomitant with tumour dedifferentiation. SPP-induced FHL2 activation is mediated by Rho GTPases, but not by the GTPases Cdc42, Rac<em>1</em> or Ras, and depends on Rho-kinase. In addition, Rho signalling influences other transcriptional coactivators, thus pointing to a general regulatory role for Rho GTPases in cofactor function. In summary, our data propose a yet undescribed signalling pathway in which the coactivator FHL2 acts as a novel molecular transmitter of the Rho signalling pathway, thereby integrating extracellular cues into altered gene expression.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em>, a metabolite of sphingolipids which has previously been shown to stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (<em>1</em>99<em>1</em>) J. Cell Biol. <em>1</em><em>1</em>4, <em>1</em>55-<em>1</em>67), induced a transient increase in intracellular free calcium independent of extracellular calcium. The increase in calcium was completely abolished when intracellular calcium pools were depleted with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The dose-response for calcium release induced by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> correlated closely with the concentration required for stimulation of DNA synthesis. The magnitude of the calcium response decreased with successive challenges, although <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> did not attenuate the responses to either bradykinin or ionomycin. Conversely, prior stimulation of the cells with bradykinin had no effect on the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-induced calcium signal. Although <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> increased inositol (<em>1</em>,4,5)-tris<em>phosphate</em> levels, complete inhibition of inositol <em>phosphate</em> formation by pretreatment with <em>1</em>2-O-tetradecanoylphorbol-<em>1</em>3-acetate did not block <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-mediated calcium responses. Moreover, in permeabilized cells, heparin, an inositol (<em>1</em>,4,5)-tris<em>phosphate</em> antagonist, blocked Ca2+ release induced by inositol (<em>1</em>,4,5)-tris<em>phosphate</em>, but did not significantly alter the Ca2+ release induced by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> did not stimulate the release of arachidonic acid, another signaling molecule known to elevate [Ca2+]i without inositol lipid turnover or calcium influx. Our data suggest that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> mobilizes Ca2+ from internal stores primarily through a mechanism independent of inositol lipid hydrolysis and arachidonic acid release and that sphingolipid metabolism may be important in calcium homeostasis.

Sphingolipid metabolites, such as ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), are emerging as a new class of second messengers involved in cellular proliferation, differentiation, and apoptosis. Nerve growth factor (NGF), a neurotrophic factor for pheochromocytoma PC<em>1</em>2 cells, induced a biphasic increase in the activity of <em>sphingosine</em> kinase, the enzyme that catalyzes the formation of SPP. This activation was blocked by K252a, an inhibitor of tyrosine kinase A (trkA). A rapid <em>1</em>.7-fold increase was followed by a marked prolonged increase reaching a maximum of fourfold to fivefold stimulation with a concomitant increase in SPP levels and a corresponding decrease in endogenous <em>sphingosine</em> levels. Levels of ceramide, the precursor of <em>sphingosine</em>, were only slightly decreased by NGF in serum-containing medium. However, NGF decreased the elevation of ceramide induced by serum withdrawal. Treatment of PC<em>1</em>2 cells with SPP did not induce neurite outgrowth or neurofilament expression, yet it enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC<em>1</em>2 cells from apoptosis induced by serum withdrawal. To further substantiate a role for SPP in the cytoprotective actions of NGF, we found that N, N-dimethyl<em>sphingosine</em>, a competitive inhibitor of <em>sphingosine</em> kinase, also induced apoptosis and interfered with the survival effect of NGF. These effects were counteracted by exogenous SPP. Moreover, other structurally related compounds, such as dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> and lysophosphatidic acid, had no significant protective effects. Our results suggest that activation of <em>sphingosine</em> kinase and subsequent formation of SPP may play an important role in the differentiation and survival effects induced by NGF.

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized <em>1</em>-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor (S<em>1</em>P(<em>1</em>)), the serine/threonine kinase Akt, and the Rac<em>1</em> guanine nucleotide exchange factor Tiam<em>1</em> and phosphorylation of caveolin-<em>1</em>, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac<em>1</em> activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S<em>1</em>P(<em>1</em>) activation (threonine phosphorylation), whereas silencing S<em>1</em>P(<em>1</em>) receptor expression blocked OxPAPC-mediated Tiam<em>1</em> recruitment to CEMs, Rac<em>1</em> activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-<em>1</em> or S<em>1</em>P(<em>1</em>) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S<em>1</em>P(<em>1</em>) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.