<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of <em>sphingosine</em> kinase type <em>1</em>, which generated S<em>1</em>P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S<em>1</em>P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha<em>1</em>2/<em>1</em>3, and Gbetagamma subunits, the G proteins that S<em>1</em>P receptors (S<em>1</em>PRs) couple to and signal through. We found that intracellular S<em>1</em>P signaled "inside out" through its cell-surface receptors linked to G<em>1</em>2/<em>1</em>3-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S<em>1</em>P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S<em>1</em>PRs, we also demonstrated that, in contrast to exogenous S<em>1</em>P, intracellular S<em>1</em>P formed by overexpression of <em>sphingosine</em> kinase type <em>1</em> promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S<em>1</em>Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S<em>1</em>P, reminiscent of its action in yeast cells that lack S<em>1</em>PRs.

Radiation resistance in a subset of prostate tumors remains a challenge to prostate cancer radiotherapy. The current study on the effects of radiation on prostate cancer cells reveals that radiation programs an unpredicted resistance mechanism by upregulating acid ceramidase (AC). Irradiated cells demonstrated limited changes of ceramide levels while elevating levels of <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Conversely, AC overexpression further decreased sensitivity to radiation. We also observed that radiation-induced AC upregulation was sufficient to create cross-resistance to chemotherapy as demonstrated by decreased sensitivity to Taxol and C(6) ceramide compared to controls. Lower levels of caspase 3/7 activity were detected in cells pretreated with radiation, also indicating increased resistance. Finally, utilization of the small molecule AC inhibitor, LCL385, sensitized PPC-<em>1</em> cells to radiation and significantly decreased tumor xenograft growth. These data suggest a new mechanism of cancer cell resistance to radiation, through upregulation of AC that is, in part, mediated by application of the therapy itself. An improved understanding of radiotherapy and the application of combination therapy achieved in this study offer new opportunities for the modulation of radiation effects in the treatment of cancer.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) produced by <em>sphingosine</em> kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S<em>1</em>P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethyl<em>sphingosine</em> (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S<em>1</em>P release into BAL fluid and high expression of SPHK<em>1</em> around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S<em>1</em>P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as <em>sphingosine</em> kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethyl<em>sphingosine</em> (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) is one of the two known kinases, which generates <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK<em>1</em> and intracellular generation of S<em>1</em>P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK<em>1</em> provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK<em>1</em> overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (DHS<em>1</em>P) compared to S<em>1</em>P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK<em>1</em> with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS<em>1</em>P. On the contrary, S<em>1</em>P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of <em>sphingosine</em> from complex sphingolipids. As a result of SK<em>1</em> overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S<em>1</em>P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS<em>1</em>P or S<em>1</em>P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S<em>1</em>P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK<em>1</em>-mediated synthesis of DHS<em>1</em>P and S<em>1</em>P, whereas TNF-alpha enhanced only S<em>1</em>P production in HPAEC. These findings uncover a new functional role for SK<em>1</em>, which can control survival/death (DHS<em>1</em>P-S<em>1</em>P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS<em>1</em>P at a metabolic step preceding ceramide formation.

The role of "sphingolipid rheostat" by ceramide and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(-)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S<em>1</em>P treatment counteracted autophagy induction by AA(-) or C(2)-ceramide. AA(-) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S<em>1</em>P treatment suppressed mTOR inactivation and autophagy induction by AA(-). S<em>1</em>P exerts biological actions via cell surface receptors, and S<em>1</em>P(3) among five S<em>1</em>P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S<em>1</em>P(3) in suppressing autophagy induction. S<em>1</em>P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(-) or C(2)-ceramide. Whereas S<em>1</em>P treatment of S<em>1</em>P(3) overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(-) or C(2)-ceramide. These results indicate that S<em>1</em>P-S<em>1</em>P(3) plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(-)- or C(2)-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S<em>1</em>P(3) engagement by S<em>1</em>P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S<em>1</em>P-S<em>1</em>P(3) signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S<em>1</em>P signaling in regulation of microvascular permeability. S<em>1</em>P signaling is mediated by the S<em>1</em>P family of G protein-coupled receptors (S<em>1</em>P(<em>1</em>-5) receptors). S<em>1</em>P(<em>1</em>) and S<em>1</em>P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S<em>1</em>P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S<em>1</em>P(<em>1</em>)-mediated signaling by SEW287<em>1</em> and FTY720, two agonists of S<em>1</em>P(<em>1</em>), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 230<em>1</em>9 to antagonize S<em>1</em>P(<em>1</em>)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S<em>1</em>P(2) signaling by JTE-0<em>1</em>3, a specific antagonist of S<em>1</em>P(2), S<em>1</em>P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S<em>1</em>P(<em>1</em>)- and S<em>1</em>P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S<em>1</em>P(<em>1</em>) and S<em>1</em>P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.

Phosphorylated sphingolipids ceramide-<em>1</em>-<em>phosphate</em> (C<em>1</em>P) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have emerged as key regulators of cell growth, survival, migration and inflammation. C<em>1</em>P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C<em>1</em>P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C<em>1</em>P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with <em>phosphate</em> headgroups, namely C<em>1</em>P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD<em>1</em>, named here CPTP, is shown to specifically transfer C<em>1</em>P between membranes. Crystal structures establish C<em>1</em>P binding through a novel surface-localized, <em>phosphate</em> headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C<em>1</em>P steady-state levels and alters Golgi cisternae stack morphology. The resulting C<em>1</em>P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

The established role for phosphatidylinositol (3,4,5) tri<em>phosphate</em> (PI(3,4,5)P3) signaling pathways is to regulate cell metabolism. More recently it has emerged that PI(3,4,5)P3 signaling via mammalian target of rapamycin and Foxo transcription factors also controls lymphocyte trafficking by determining the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. In quiescent T cells, nonphosphorylated active Foxos maintain expression of KLF2, a transcription factor that regulates expression of the chemokine receptors CCR7 and <em>sphingosine</em> <em>1</em> <em>phosphate</em> receptor, and the adhesion receptor CD62L that together control T-cell transmigration into secondary lymphoid tissues. PI(3,4,5)P3 mediates activation of protein kinase B, which phosphorylates and inactivates Foxos, thereby terminating expression of KLF2 and its target genes. The correct localization of lymphocytes is essential for effective immune responses, and the ability of phosphoinositide 3-kinase and mammalian target of rapamycin to regulate expression of chemokine receptors and adhesion molecules puts these signaling molecules at the core of the molecular mechanisms that control lymphocyte trafficking.

Sphingolipids are abundant in the microvillar membrane of intestinal epithelial cells where they are essential for structural integrity and may act as receptors for toxins, virus and bacteria. Metabolism of dietary and membrane sphingolipids in the intestine generates ceramide, <em>sphingosine</em>, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, and ceramide-<em>1</em>-<em>phosphate</em>, via the action of alkaline sphingomyelinase, neutral ceramidase, <em>sphingosine</em>-<em>1</em>-kinase, and ceramide-<em>1</em>-kinase. These intermediary metabolites act as bioactive lipid messengers, influencing numerous cellular functions including growth, differentiation and apoptosis of both epithelial and immunocompetent cells in the gastrointestinal tract, and also the progress of inflammation and responsiveness of the mucosal cells to pathogens. This review summarizes background and recent progress in the metabolism of dietary and endogenous sphingolipids in the gut and its pathophysiological implications.

Hearing requires the transduction of vibrational forces by specialized epithelial cells in the cochlea known as hair cells. The human ear contains a finite number of terminally differentiated hair cells that, once lost by noise-induced damage or toxic insult, can never be regenerated. We report here that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) signaling, mainly via activation of its cognate receptor S<em>1</em>P2, is required for the maintenance of vestibular and cochlear hair cells in vivo. Two S<em>1</em>P receptors, S<em>1</em>P2 and S<em>1</em>P3, were found to be expressed in the cochlea by reverse transcription-PCR and in situ hybridization. Mice that are null for both these receptors uniformly display progressive cochlear and vestibular defects with hair cell loss, resulting in complete deafness by 4 weeks of age and, with complete penetrance, balance defects of increasing severity. This study reveals the previously unknown role of S<em>1</em>P signaling in the maintenance of cochlear and vestibular integrity and suggests a means for therapeutic intervention in degenerative hearing loss.

This article describes the regulation of cell signaling by lipid <em>phosphate</em> phosphatases (LPPs) that control the conversion of bioactive lipid <em>phosphates</em> to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid <em>phosphate</em> esters such as phosphatidate, lysophosphatidate, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and ceramide <em>1</em>-<em>phosphate</em>. In so doing, the LPPs could generate alternative signals from diacylglycerol, <em>sphingosine</em> and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> through their respective receptors.

High density lipoproteins (HDL) are major plasma carriers of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S<em>1</em>P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of Gi-coupled S<em>1</em>P receptors, as well as antagonists of the S<em>1</em>P receptor, S<em>1</em>P<em>1</em>, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk<em>1</em>/2 and Akt, signaling pathway intermediates that have been implicated in S<em>1</em>P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk<em>1</em>/2 and Akt activation and motility were suppressed by pertussis toxin and S<em>1</em>P<em>1</em> antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk<em>1</em>/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S<em>1</em>P component and signaling through the S<em>1</em>P<em>1</em>/Akt pathway.

Lipid <em>phosphate</em> phosphohydrolase (LPP)-<em>1</em> cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-<em>1</em> cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-<em>1</em> exhibited 3.<em>1</em>-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide <em>1</em>-<em>phosphate</em>, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and diacylglycerol pyro<em>phosphate</em> were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-<em>1</em> cDNA showed <em>1</em>.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide <em>1</em>-<em>phosphate</em> compared with vector control cells. Recombinant LPP-<em>1</em> was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-<em>1</em> expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-<em>1</em> cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)<em>1</em>/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor 2 (S<em>1</em>PR2) in rodent hepatocytes. In the current study, we demonstrate S<em>1</em>PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK<em>1</em>/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-0<em>1</em>3, a chemical antagonist of S<em>1</em>PR2, or by lentiviral short hairpin RNA silencing of S<em>1</em>PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/"duct-like" structures, which was blocked by treatment with JTE-0<em>1</em>3.

CONCLUSIONS

Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S<em>1</em>PR2.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an important bioactive sphingolipid involved in angiogenesis and lymphangiogenesis, 2 important processes that influence the growth, survival, and spread of tumors. S<em>1</em>P acts as an extracellular mediator through binding to 5 highly specific S<em>1</em>P receptors, S<em>1</em>P(<em>1</em>-5). <em>Sphingosine</em> kinase-<em>1</em> (SK<em>1</em>), one of 2 known <em>sphingosine</em> kinase enzymes responsible for S<em>1</em>P production, appears to be overexpressed in many tumors. Although a role for S<em>1</em>P in angiogenesis and lymphangiogenesis has been established, it is unclear whether S<em>1</em>P secreted from cancer cells has a paracrine function in a tumor environment. Here we investigated whether modulation of cellular SK<em>1</em> could initiate a paracrine angiogenic and lymphangiogenic switch. We found that SK<em>1</em> overexpression in HEK cells or its down-regulation in glioma or breast cancer cells modulated extracellular S<em>1</em>P levels accordingly, which in turn increased or decreased both migration and tube formation in cocultured vascular or lymphatic endothelial cells. In contrast, down-regulation of <em>sphingosine</em> kinase 2 in both glioma and breast cancer cells had no appreciable effect on cellular or secreted S<em>1</em>P levels. In addition, vascular endothelial growth factors VEGF and VEGF-C down-regulation in cancer cells appeared insufficient to block the angiogenic and lymphangiogenic switch triggered by these cells. Moreover, S<em>1</em>P initiated endothelial cell sprouting in 3-dimensional collagen matrices, which is representative of a multistep angiogenic process. Our data collectively demonstrate for the first time that SK<em>1</em> plays an essential role in regulating in vitro paracrine angiogenesis and lymphangiogenesis.

The mechanisms by which <em>sphingosine</em> kinase-<em>1</em> (SK-<em>1</em>)/<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-<em>1</em>/S<em>1</em>P enhances Bcr-Abl<em>1</em> protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl<em>1</em> stability was enhanced by ectopic SK-<em>1</em> expression. Conversely, siRNA-mediated SK-<em>1</em> knockdown in K562/IMA-3 cells, or its genetic loss in SK-<em>1</em>(-/-) MEFs, significantly reduced Bcr-Abl<em>1</em> stability. Regulation of Bcr-Abl<em>1</em> by SK-<em>1</em>/S<em>1</em>P was dependent on S<em>1</em>P receptor 2 (S<em>1</em>P2) signaling, which prevented Bcr-Abl<em>1</em> dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-<em>1</em>/S<em>1</em>P2 restored PP2A-dependent Bcr-Abl<em>1</em> dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T3<em>1</em>5I) Bcr-Abl<em>1</em> in situ. Accordingly, impaired SK-<em>1</em>/S<em>1</em>P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T3<em>1</em>5I-Bcr-Abl<em>1</em>-derived mouse allografts. Since SK-<em>1</em>/S<em>1</em>P/S<em>1</em>P2 signaling regulates Bcr-Abl<em>1</em> stability via modulation of PP2A, inhibition of SK-<em>1</em>/S<em>1</em>P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl<em>1</em> thereby overcoming drug resistance.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S<em>1</em>P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell-cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [<em>1</em>,2]. Given the wide variety of cellular and physiological processes in which S<em>1</em>P is involved, it is immediately obvious why the mechanisms governing S<em>1</em>P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the <em>sphingosine</em> kinase (SK)/S<em>1</em>P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S<em>1</em>P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Lipid mediators influence immunity in myriad ways. For example, circulating <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a key regulator of lymphocyte egress. Although the majority of plasma S<em>1</em>P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, the immunological functions of the ApoM-S<em>1</em>P complex are unknown. Here we show that ApoM-S<em>1</em>P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S<em>1</em>P<em>1</em> receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM (Apom(-/-)) had increased proliferation of Lin(-) Sca-<em>1</em>(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S<em>1</em>P<em>1</em> suppressed LSK and CLP cell proliferation in vivo. ApoM was stably associated with bone marrow CLPs, which showed active S<em>1</em>P<em>1</em> signalling in vivo. Moreover, ApoM-bound S<em>1</em>P, but not albumin-bound S<em>1</em>P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom(-/-) mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system and breakdown of the blood-brain barrier. Thus, the ApoM-S<em>1</em>P-S<em>1</em>P<em>1</em> signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S<em>1</em>P chaperones could be exploited for novel therapeutic opportunities.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has been implicated in tumor angiogenesis by acting through the G(i)-coupled chemotactic receptor S<em>1</em>P(<em>1</em>). Here, we report that the distinct receptor S<em>1</em>P(2) is responsible for mediating the G(<em>1</em>2/<em>1</em>3)/Rho-dependent inhibitory effects of S<em>1</em>P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S<em>1</em>P(2)(LacZ/+) mice, we found that S<em>1</em>P(2) was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD<em>1</em><em>1</em>b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B<em>1</em>6 melanoma cells implanted in S<em>1</em>P(2)-deficient (S<em>1</em>P(2)(-/-)) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S<em>1</em>P(2)(-/-) ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S<em>1</em>P(2)(-/-) ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S<em>1</em>P(2)(-/-) mice were also more efficient at recruiting CD<em>1</em><em>1</em>b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S<em>1</em>P(2) acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S<em>1</em>P(<em>1</em>), which stimulates tumor angiogenesis, S<em>1</em>P(2) on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) can prevent endothelial cell apoptosis. We investigated the molecular mechanisms and signaling pathways by which S<em>1</em>P protects endothelial cells from serum deprivation-induced apoptosis. We show here that human umbilical vein endothelial cells (HUVECs) undergo apoptosis associated with increased DEVDase activity, caspase-3 activation, cytochrome c release, and DNA fragmentation after 24 h of serum deprivation. These apoptotic markers were suppressed by the addition of S<em>1</em>P, the NO donor S-nitroso-N-acetylpenicillamine (<em>1</em>00 micrometer), or caspase-3 inhibitor z-VAD-fmk. The protective effects of S<em>1</em>P were reversed by the nitric-oxide synthase (NOS) inhibitor N-monomethyl-l-arginine, but not by the soluble guanylyl cyclase inhibitor <em>1</em>H-(<em>1</em>,2,4)oxadiazolo[4,3-a]-quanoxaline-<em>1</em>-one, suggesting that NO, but not cGMP, is responsible for S<em>1</em>P protection from apoptosis. Furthermore, S<em>1</em>P increased NO production by enhancing Ca(2+)-sensitive NOS activity without changes in the eNOS protein level. S<em>1</em>P-mediated cell survival and NO production were suppressed significantly by pretreatment with antisense oligonucleotide of EDG-<em>1</em> and partially by EDG-3 antisense. S<em>1</em>P-mediated NO production was suppressed by the addition of pertussis toxin, an inhibitor of G(i) proteins, the specific inhibitor of phospholipase C (PLC), and the Ca(2+) chelator BAPTA-AM. These findings indicate that S<em>1</em>P protects HUVECs from apoptosis through the activation of eNOS activity mainly through an EDG-<em>1</em> and -3/G(i)/PLC/Ca(2+) signaling pathway.

The sphingolipids <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and ceramide are important bioactive lipids with many cellular effects. Intracellular ceramide accumulation causes insulin resistance, but <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) prevents ceramide accumulation, in part, by promoting its metabolism into S<em>1</em>P. Despite this, the role of SphK<em>1</em> in regulating insulin action has been largely overlooked. Transgenic (Tg) mice that overexpress SphK<em>1</em> were fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK<em>1</em> Tg mice fed an HFD displayed increased SphK activity in skeletal muscle, which was associated with an attenuated intramuscular ceramide accumulation compared with wild-type (WT) littermates. This was associated with a concomitant reduction in the phosphorylation of c-jun amino-terminal kinase, a serine threonine kinase associated with insulin resistance. Accordingly, skeletal muscle and whole-body insulin sensitivity were improved in SphK<em>1</em> Tg, compared with WT mice, when fed an HFD. We have identified that the enzyme SphK<em>1</em> is an important regulator of lipid partitioning and insulin action in skeletal muscle under conditions of increased lipid supply.

Based on the results of research conducted over last two decades, lysophospholipids (LPLs) were observed to be not only structural components of cellular membranes but also biologically active molecules influencing a broad variety of processes such as carcinogenesis, neurogenesis, immunity, vascular development or regulation of metabolic diseases. With a growing interest in the involvement of extracellular lysophospholipids in both normal physiology and pathology, it has become evident that those small molecules may have therapeutic potential. While lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have been studied in detail, other LPLs such as lysophosphatidylglycerol (LPG), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE) or even lysophosphatidylcholine (LPC) have not been elucidated to such a high degree. Although information concerning the latter LPLs is sparse as compared to LPA and S<em>1</em>P, within the last couple of years much progress has been made. Recently published data suggest that these compounds may regulate fundamental cellular activities by modulating multiple molecular targets, e.g. by binding to specific receptors and/or altering the structure and fluidity of lipid rafts. Therefore, the present review is devoted to novel bioactive glycerol-based lysophospholipids and recent findings concerning their functions and possible signaling pathways regulating physiological and pathological processes.

Sphingolipids are a major component of membrane lipids and their metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S<em>1</em>P production, <em>sphingosine</em> kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S<em>1</em>P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Delta4-unsaturated long-chain sphingoid bases <em>sphingosine</em> (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydro<em>sphingosine</em> and phyto<em>sphingosine</em> (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK<em>1</em> efficiently phosphorylated Sph, dihydro<em>sphingosine</em>, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-<em>1</em>00 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phyto<em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, similar to S<em>1</em>P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein alpha-subunit gene, GPA<em>1</em>.