We describe here a novel biological function of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P): the activation of a serine protease, matriptase. Matriptase is a type II integral membrane serine protease, expressed on the surface of a variety of epithelial cells; it may play an important role in tissue remodeling. We have previously reported that the activation of matriptase is regulated by serum. We have now identified the bioactive component from serum. First, the activity was observed to co-purify with lipoproteins by conventional liquid chromatography and immunoaffinity chromatography. The ability of lipoproteins to induce the activation of matriptase was further confirmed with commercial preparations of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). Next, we observed that the bioactive component of LDL is associated with the phospholipid components of LDL. Fractionation of lipid components of LDL by thin layer chromatography (TLC) revealed that the bioactive component of LDL comigrates with S<em>1</em>P. Nanomolar concentrations of commercially obtained S<em>1</em>P were then observed to induce the rapid activation of matriptase on the surfaces of nontransformed human mammary epithelial cells. Other structurally related sphingolipids, including dihydro-S<em>1</em>P, ceramide <em>1</em>-<em>phosphates</em>, and <em>sphingosine</em> phosphocholine as well as lysophosphatidic acid, can also induce the activation of matriptase, but at significantly higher concentrations than S<em>1</em>P. Furthermore, S<em>1</em>P-dependent matriptase activation is dependent on Ca(2+) but not via G(i) protein-coupled receptors. Our results demonstrate that bioactive phospholipids can function as nonprotein activators of a cell surface protease, suggesting a possible mechanistic link between S<em>1</em>P and normal and possibly pathologic tissue remodeling.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S-<em>1</em>P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-<em>1</em>P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-alpha (TNF-alpha), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter <em>1</em>2-O-tetradecanoyl-phorbol-<em>1</em>3-acetate, which stimulates <em>sphingosine</em> kinase, the enzyme responsible for S-<em>1</em>P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethyl<em>sphingosine</em> (DMS), a specific inhibitor of <em>sphingosine</em> kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-alpha, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-<em>1</em>P cotreatment. Hence, S-<em>1</em>P, likely generated through a protein kinase C- mediated activation of <em>sphingosine</em> kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells.

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are serum-borne lipid mediators with potential proinflammatory and atherogenic properties. We studied the effects of LPA and S<em>1</em>P on [Ca(2+)](i), a second messenger of cellular activation, in human monocytic Mono Mac 6 (MM6) cells. LPA and S<em>1</em>P induced [Ca(2+)](i) transients with EC(50) values of 47 and 340 nM, respectively. Ca(2+) signals evoked by LPA and S<em>1</em>P originated mainly from the stimulation of Ca(2+) entry, were blocked by the phospholipase C inhibitor U73<em>1</em>22, and were inhibited by pertussis toxin. The LPA(<em>1</em>) and LPA(3) receptor antagonist dioctylglycerol pyro<em>phosphate</em> inhibited the LPA-induced Ca(2+) signal. Notably, serum and minimally modified LDL (mm-LDL) evoked [Ca(2+)](i) increases that were mediated entirely through activation of LPA receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the presence of the LPA and S<em>1</em>P receptor subtypes LPA(<em>1</em>), LPA(2,) S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), S<em>1</em>P(4) in MM6 cells, human monocytes and macrophages. Together these results indicate that LPA, mm-LDL and serum induce via activation of the LPA(<em>1</em>) receptor a G(i)/phospholipase C/Ca(2+) signalling pathway in monocytes. Our study is the first report showing the receptor-mediated activation of human monocytic cells by low nanomolar concentrations of LPA and S<em>1</em>P, and suggests a role of these lipid mediators in inflammation and atherogenesis.

OBJECTIVE

<em>Sphingosine</em> kinase (SphK) is a key enzyme in the synthesis of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK<em>1</em> in myocardial ischemia/reperfusion (IR) injury and IPC.

RESULTS

Wild-type and SphK<em>1</em> null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK<em>1</em> mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S<em>1</em>P levels were decreased by 4<em>1</em>%. SphK<em>1</em> null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK<em>1</em> mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK<em>1</em> null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.<em>1</em>+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by <em>1</em>0 min.

CONCLUSIONS

Deletion of the SphK<em>1</em> gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK<em>1</em>-S<em>1</em>P pathway is a critical mediator of IPC and cell survival.

Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-<em>1</em> (Ang-<em>1</em>)-regulated control of EC permeability is mediated by 2 different functional states of <em>sphingosine</em> kinase-<em>1</em> (SK-<em>1</em>). Mice depleted of SK-<em>1</em> have increased vascular leakiness, whereas mice transgenic for SK-<em>1</em> in ECs show attenuation of leakiness. Furthermore, Ang-<em>1</em> rapidly and transiently stimulates SK-<em>1</em> activity and phosphorylation, and induces an increase in intracellular <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) concentration. Overexpression of SK-<em>1</em> resulted in inhibition of permeability similar to that seen for Ang-<em>1</em>, whereas knockdown of SK-<em>1</em> by small interfering RNA blocked Ang-<em>1</em>-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-<em>1</em>, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-<em>1</em> mutant removed the capacity of Ang-<em>1</em> to inhibit permeability. These effects were independent of extracellular S<em>1</em>P as knockdown or inhibition of S<em>1</em>P<em>1</em>, S<em>1</em>P2, or S<em>1</em>P3, did not affect the Ang-<em>1</em> response. Thus, SK-<em>1</em> levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-<em>1</em>-induced inhibition of leakiness is mediated through activation of SK-<em>1</em>, defining a new signaling pathway in the Ang-<em>1</em> regulation of permeability.

The G-protein-coupled receptors of the endothelial differentiation gene (EDG) family mediate pro-angiogenic activities, such as endothelial cell proliferation, chemotaxis, and vessel morphogenesis. We synthesized and tested the effects of a 9-amino acid peptide (KRX-725), derived from the second intracellular loop of S<em>1</em>P3 (EDG3). KRX-725 mimics the effects of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), the natural ligand of S<em>1</em>P3, by triggering a Gi-dependent MEK-ERK (mitogen-activated protein kinase kinase and extracellular signal-regulated kinase) signal transduction pathway. Using aortic rings as an ex vivo model of angiogenesis, vascular sprouting was assessed in the presence of KRX-725 or S<em>1</em>P. KRX-725 induced extensive and dense vascular sprouts, which contain an elaborated organization of endothelial and smooth muscle layers, including lumen formation. When KRX-725 or S<em>1</em>P was combined with proangiogenic factors, such as basic fibroblast growth factor (bFGF), stem cell factor, or vascular endothelial growth factor, the effect was synergistic, leading to further enhancement of vascular sprouting. KRX-725 also initiated neovascularization in a mouse corneal pocket assay in vivo and showed synergism with bFGF. The specificity of KRX-725 was demonstrated via peptide-induced receptor internalization of S<em>1</em>P3 but not S<em>1</em>P<em>1</em>. The ability of a short peptide to stimulate extensive angiogenesis and to synergize with pro-angiogenic factors suggests that KRX-725 may serve as a useful agent in treating pathologic conditions such as peripheral vascular disease, cardiac ischemia, or tissue grafts.

Epidermal growth factor (EGF) treatment of A-43<em>1</em> cells induces a biphasic increase in the levels of inositol <em>phosphates</em>. The growth factor produces an initial, rapid increase in the level of inositol <em>1</em>,4,5-trisphosphate (Ins-<em>1</em>,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (<em>1</em>987) Biochem. Biophys. Res. Commun. <em>1</em>42, 688-695). The level of inositol <em>1</em>,3,4,5-tetrakisphosphate (Ins-<em>1</em>,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than <em>1</em> min) of Ins-<em>1</em>,4,5-P3 and Ins-<em>1</em>,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of <em>1</em>00 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol <em>phosphates</em> at later times after EGF addition (<em>1</em>-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-43<em>1</em> cells with Ca2+ ionophores (A23<em>1</em>87 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-<em>1</em>,4,5-P3, Ins-<em>1</em>,3,4,5-P4, and inositol <em>1</em>,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol <em>phosphates</em> was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. <em>1</em>2-O-Tetradecanoylphorbol-<em>1</em>3-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol <em>phosphates</em>. Tamoxifen and <em>sphingosine</em>, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol <em>phosphates</em>. Neither TPA nor tamoxifen significantly affects the <em>1</em>25I-EGF binding capacity of A-43<em>1</em> cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-43<em>1</em> cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S<em>1</em>P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S<em>1</em>P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S<em>1</em>P and EDG/S<em>1</em>P receptor expression were determined in rat VSM from cerebral artery and aorta. S<em>1</em>P induced constriction of cerebral artery, which was partly dependent on activation of p<em>1</em>60(ROCK) (Rho-kinase). S<em>1</em>P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S<em>1</em>P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S<em>1</em>P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol <em>1</em>,4,5-tris<em>phosphate</em>-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S<em>1</em>P. S<em>1</em>P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S<em>1</em>P receptor antibodies revealed that the expression of S<em>1</em>P3/EDG-3 and S<em>1</em>P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S<em>1</em>P(<em>1</em>)/EDG-<em>1</em> receptor expression was similar in both types of VSM. Therefore, the ability of S<em>1</em>P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S<em>1</em>P receptor subtypes.

The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP<em>1</em> is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP<em>1</em> is Mg(2+) dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and ceramide-<em>1</em>-<em>phosphate</em> in addition to PA and lyso-PA, it has been renamed lipid <em>phosphate</em> phosphohydrolase (LPP). LPP is Mg(2+) independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S<em>1</em>P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S<em>1</em>P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S<em>1</em>P receptors, Edg-<em>1</em> and Edg-3. S<em>1</em>P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0<em>1</em>26, an Erk kinase inhibitor. The S<em>1</em>P-evoked activation of Erk<em>1</em> was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk<em>1</em> activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S<em>1</em>P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S<em>1</em>P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S<em>1</em>P stimulated the production of inositol <em>phosphates</em> and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S<em>1</em>P. Altogether, these observations show that astrocytes are targets for S<em>1</em>P. Their proliferation in response to S<em>1</em>P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle-arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF-κB (RANK)-positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK-positive cells expressed c-Fms (receptors of macrophage colony-stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK-positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein-2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP-2-induced ectopic bone were bromodeoxyuridine-negative. RANK-positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)-positive macrophages. When BMP disks and control disks were implanted in RANK ligand-deficient mice, RANK-positive cells were observed in the BMP disks but not in the controls. F4/80-positive cells were distributed in both disks. Administration of FYT720, a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> agonist, promoted the egress of RANK-positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage-determined QOPs circulate in the blood and settle in the bone.

Escape of prostate cancer (PCa) cells from ionizing radiation-induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein <em>1</em> (AP-<em>1</em>) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy.

We have shown previously that LPPs (lipid <em>phosphate</em> phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (200<em>1</em>) J. Biol. Chem. 276, <em>1</em>3452-<em>1</em>3460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S<em>1</em>P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK<em>1</em> (<em>sphingosine</em> kinase <em>1</em>) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK<em>1</em> from being recruited to a perinuclear compartment upon induction of PLD<em>1</em> (phospholipase D<em>1</em>) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S<em>1</em>P respectively, that may govern the apoptotic status of the cell upon serum deprivation.

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p<em>1</em><em>1</em>5 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha(<em>1</em>3). Endogenous p<em>1</em><em>1</em>5 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p<em>1</em><em>1</em>5 RhoGEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p<em>1</em><em>1</em>5 RhoGEF exchange activity. Deletion of amino acids 252--288 in the linker region between the RGS domain and the Dbl homology domain or of the last <em>1</em>50 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p<em>1</em><em>1</em>5 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p<em>1</em><em>1</em>5 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p<em>1</em><em>1</em>5 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or <em>sphingosine</em> <em>1</em>-<em>phosphate</em> was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p<em>1</em><em>1</em>5 RhoGEF by G protein-coupled receptors.

Mammals are endowed with a complex set of mechanisms that sense mechanical forces imparted by blood flow to endothelial cells (ECs), smooth muscle cells, and circulating blood cells to elicit biochemical responses through a process referred to as mechanotransduction. These biochemical responses are critical for a host of other responses, including regulation of blood pressure, control of vascular permeability for maintaining adequate perfusion of tissues, and control of leukocyte recruitment during immunosurveillance and inflammation. This review focuses on the role of the endothelial surface proteoglycan/glycoprotein layer-the glycocalyx (GCX)-that lines all blood vessel walls and is an agent in mechanotransduction and the modulation of blood cell interactions with the EC surface. We first discuss the biochemical composition and ultrastructure of the GCX, highlighting recent developments that reveal gaps in our understanding of the relationship between composition and spatial organization. We then consider the roles of the GCX in mechanotransduction and in vascular permeability control and review the prominent interaction of plasma-borne <em>sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P), which has been shown to regulate both the composition of the GCX and the endothelial junctions. Finally, we consider the association of GCX degradation with inflammation and vascular disease and end with a final section on future research directions.

BACKGROUND

Apolipoprotein M (apoM) is present in 5% of high-density lipoprotein (HDL) particles in plasma. It is a carrier of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which is important for vascular barrier protection. The aim was to determine the plasma concentrations of apoM during sepsis and systemic inflammatory response syndrome (SIRS) and correlate them to levels of apolipoprotein A-I (apoA<em>1</em>), apolipoprotein B (apoB), HDL-, and low-density lipoprotein (LDL)-cholesterol.

METHODS

Plasma samples from patients with (<em>1</em>), severe sepsis with shock (n = 26); (2), severe sepsis without shock (n = 44); (3), sepsis (n = <em>1</em>00); (4), infections without SIRS (n = 43); and (5) SIRS without infection (n = 20) were analyzed. The concentrations of apoM, apoA<em>1</em>, and apoB were measured with enzyme-linked immunosorbent assays (ELISAs). Total, HDL-, and LDL-cholesterol concentrations were measured with a commercial HDL/LDL cholesterol test.

RESULTS

ApoM concentrations correlated negatively to acute-phase markers. Thus, apoM behaved as a negative acute-phase protein. Decreased values were observed in all patient groups (P < 0.000<em>1</em>), with the most drastic decreases observed in the severely sick patients. ApoM levels correlated strongly to those of apoA<em>1</em>, apoB, HDL, and LDL cholesterol. The HDL and LDL cholesterol levels were low in all patient groups, as compared with controls (P < 0.000<em>1</em>), in particular, HDL cholesterol. ApoA<em>1</em> and apoB concentrations were low only in the more severely affected patients.

CONCLUSIONS

During sepsis and SIRS, the plasma concentrations of apoM decrease dramatically, the degree of decrease reflecting the severity of the disease. As a carrier for barrier-protective S<em>1</em>P in HDL, the decrease in apoM could contribute to the increased vascular leakage observed in sepsis and SIRS.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S<em>1</em>P. All peritoneal B cells including B<em>1</em>a, B<em>1</em>b, and B2 B cells express comparable levels of the type <em>1</em> S<em>1</em>P receptor. Thus, treatment with FTY720, an S<em>1</em>P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heat-killed Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S<em>1</em>P in connecting peritoneal B cells with intestinal B-cell immunity.

BACKGROUND

The lysosphingolipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence suggests that many of the effects of HDL on cardiovascular function may be attributable to its S<em>1</em>P cargo.

METHODS

Here we have evaluated how levels of S<em>1</em>P and related sphingolipids in an HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S<em>1</em>P levels in the HDL-containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:≥ 73.5 mg/dL; males:≥ 6<em>1</em>.9 mg/dL) and verified IHD; subjects with high HDL-C and no IHD; individuals with low HDL-C (females:≤ 38.7 mg/dL; males:≤ 34.<em>1</em> mg/dL) and IHD, and subjects with low HDL-C and no IHD.

RESULTS

The results show a highly significant inverse relationship between the level of S<em>1</em>P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, an inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S<em>1</em>P and C24:<em>1</em>-ceramide, in the HDL-containing fraction of serum. Additionally, we demonstrated that the amount of S<em>1</em>P on HDL correlates with the magnitude of HDL-induced endothelial cell barrier signaling.

CONCLUSIONS

These findings indicate that compositional differences of sphingolipids in the HDL-containing fraction of human serum are related to the occurrence of IHD, and may contribute to the putative protective role of HDL in IHD.

Activation of the GPCR <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates key physiological processes. S<em>1</em>P<em>1</em> activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S<em>1</em>P<em>1</em> activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S<em>1</em>P<em>1</em> activation. These mice, known as S<em>1</em>P<em>1</em> GFP signaling mice, produce a S<em>1</em>P<em>1</em> fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S<em>1</em>P<em>1</em> recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S<em>1</em>P<em>1</em> was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S<em>1</em>P<em>1</em> agonist promoted S<em>1</em>P<em>1</em> activation in endothelial cells and hepatocytes. In S<em>1</em>P<em>1</em> GFP signaling mice, LPS-mediated systemic inflammation activated S<em>1</em>P<em>1</em> in endothelial cells and hepatocytes via hematopoietically derived S<em>1</em>P. These data demonstrate that S<em>1</em>P<em>1</em> GFP signaling mice can be used to evaluate S<em>1</em>P<em>1</em> activation and S<em>1</em>P<em>1</em>-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Novel isosteric analogs of the ceramidase inhibitors (<em>1</em>S,2R)-N-myristoylamino-phenylpropanol-<em>1</em> (d-e-MAPP) and (<em>1</em>R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-<em>1</em>,3 (B<em>1</em>3) with modified targeting and physicochemical properties were developed and evaluated for their effects on endogenous bioactive sphingolipids: ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Cer, Sph, and S<em>1</em>P) in MCF7 cells as determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Time- and dose-response studies on the effects of these compounds on Cer species and Sph levels, combined with structure-activity relationship (SAR) data, revealed 4 distinct classes of analogs which were predominantly defined by modifications of the N-acyl-hydrophobic interfaces: N-acyl-analogs (class A), urea-analogs (class B), N-alkyl-analogs (class C), and omega-cationic-N-acyl analogs (class D). Signature patterns recognized for two of the classes correspond to the cellular compartment of action of the new analogs, with class D acting as mitochondriotropic agents and class C compounds acting as lysosomotropic agents. The neutral agents, classes A and B, do not have this compartmental preference. Moreover, we observed a close correlation between the selective increase of C(<em>1</em>6)-, C(<em>1</em>4)-, and C(<em>1</em>8)-Cers and inhibitory effects on MCF7 cell growth. The results are discussed in the context of compartmentally targeted regulators of Sph, Cer species, and S<em>1</em>P in cancer cell death, emphasizing the role of C(<em>1</em>6)-Cer. These novel analogs should be useful in cell-based studies as specific regulators of Cer-Sph-S<em>1</em>P inter-metabolism, in vitro enzymatic studies, and for therapeutic development.

Lysophosphatidic acid (LPA) serves as the prototypic lysophospholipid mediator that acts through G-protein-coupled receptors to evoke a host of responses in numerous target cells. The hormone- and growth-factor-like activities of LPA, mediated by distinct G proteins, were discovered about <em>1</em>0 years ago. Since then, considerable progress has been made in our understanding of LPA receptor signaling, culminating in the recent identification of a growing family of heptahelical receptors specific for LPA and the structurally related lysolipid, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). In addition to stimulating Gi-Ras-mediated cell proliferation, LPA and S<em>1</em>P induce rapid G alpha <em>1</em>2/<em>1</em>3-RhoA-mediated cytoskeletal changes underlying such diverse responses as neurite retraction, cell rounding, and enhanced tumor cell invasiveness. LPA also triggers inhibition of gap-junctional communication. This overview focuses on how our understanding of LPA as an intercellular lipid mediator has developed during the last decade.

OBJECTIVE

We have previously described that bioactive lysophospholipids-lysophosphatidic acid (LPA), <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), and sphingosylphosphorylcholine (SPC)-are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development.

METHODS

The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC<em>1</em> ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8.

RESULTS

We describe here that LPA, S<em>1</em>P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to <em>1</em>2-fold) of IL-8 from ovarian cancer cells (HEY, OCC<em>1</em>, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (3<em>1</em>0 pg/ml/<em>1</em>0,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/<em>1</em>0,000 cells), compared with ovarian cancer cells (200-500 pg/ml/<em>1</em>0,000 cells). MCF7 cells responded to LPA, but not S<em>1</em>P and SPC, by increasing the secretion of IL-8. T47D and MCF<em>1</em>0A, an immortalized breast cell line, did not respond to LPA, S<em>1</em>P, or SPC to increase IL-8 secretion.

CONCLUSIONS

LPA, S<em>1</em>P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), the product of <em>sphingosine</em> kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse <em>sphingosine</em> kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human <em>sphingosine</em> kinase (huSPHK<em>1</em>). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK<em>1</em> cDNA specifically phosphorylates D-erythro-<em>sphingosine</em> and, to a lesser extent, D, L-erythro-dihydro<em>sphingosine</em>, and not at all the 'threo' isoforms of dihydro<em>sphingosine</em>; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-<em>phosphate</em> (PIP); or phosphatidylinositol-4, 5-bis<em>phosphate</em> (PIP(2)). huSPHK<em>1</em> shows typical Michaelis-Menten kinetics (V(max)=56microM and K(m)=5microM). The kinase is inhibited by D,L-threo-dihydro<em>sphingosine</em> (K(i)=3microM), and by N, N-dimethyl-<em>sphingosine</em> (K(i)=5microM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK<em>1</em> is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S<em>1</em>P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S<em>1</em>P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S<em>1</em>P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>) underlies sickling and disease progression by increasing S<em>1</em>P levels in the blood. Additionally, we observed elevated SPHK<em>1</em> activity in erythrocytes and increased S<em>1</em>P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK<em>1</em>-mediated production of S<em>1</em>P on sickling that was independent of S<em>1</em>P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK<em>1</em>-mediated elevation of S<em>1</em>P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.