Development of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) modulators to dampen inflammation and its sequelae is becoming increasingly promising for treating medical conditions characterized by significant immunopathology. As shown by the non-selective S<em>1</em>P receptor modulator FTY720 (fingolimod [Gilenya(®)]) in the treatment of relapsing-remitting multiple sclerosis (MS), the ability to use S<em>1</em>P<em>1</em> modulation to precisely block immune cell traffic-immunomodulation-while maintaining immunosurveillance, has opened therapeutic opportunities in various other immune-derived chronic pathologies, including inflammatory bowel disease (IBD), lupus, psoriasis, as well as, potentially, in early acute viral respiratory infection. Proof-of-concept studies across validated animal models with S<em>1</em>P receptor modulators highly selective for S<em>1</em>P<em>1</em>, such as BAF-3<em>1</em>2 (Siponimod), KRP-203, ONO-464<em>1</em> (Ceralifimod), ponesimod and RPC-<em>1</em>063, and emerging clinical trials for safety and efficacy in humans, particularly in MS, ulcerative colitis (UC) and psoriasis, have set the stage for us to consider additional testing in various other autoimmune diseases.

The lysophospholipids <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) signal through G-protein coupled receptors (GPCRs) which couple to multiple G-proteins and their effectors. These GPCRs are quite efficacious in coupling to the Gα(<em>1</em>2/<em>1</em>3) family of G-proteins, which stimulate guanine nucleotide exchange factors (GEFs) for RhoA. Activated RhoA subsequently regulates downstream enzymes that transduce signals which affect the actin cytoskeleton, gene expression, cell proliferation and cell survival. Remarkably many of the enzymes regulated downstream of RhoA either use phospholipids as substrates (e.g. phospholipase D, phospholipase C-epsilon, PTEN, PI3 kinase) or are regulated by phospholipid products (e.g. protein kinase D, Akt). Thus lysophospholipids signal from outside of the cell and control phospholipid signaling processes within the cell that they target. Here we review evidence suggesting an integrative role for RhoA in responding to lysophospholipids upregulated in the pathophysiological environment, and in transducing this signal to cellular responses through effects on phospholipid regulatory or phospholipid regulated enzymes. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulate a diverse range of mammalian cell processes, largely through engaging multiple G protein-coupled receptors specific for these lysophospholipids. LPA and S<em>1</em>P have been clearly identified to have widespread physiological and pathophysiological actions, controlling events within the reproductive, gastrointestinal, vascular, nervous and immune systems, and also having a prominent role in cancer. Here we review the recent literature showing the additional emerging role for LPA and S<em>1</em>P in the regulation of stem cells and their progenitors. We discuss the role of these lysophospholipids in regulating the proliferation, survival, differentiation and migration of a range of adult and embryonic stem cells and progenitors, and thus are likely to play a substantial role in the maintenance, generation, mobilisation and homing of stem cell and progenitor populations in the body.

It is well established that intraepithelial T lymphocytes (IELs) are derived from conventional single-positive (SP) thymocytes, as well as unconventional double-negative (DN) thymocytes and CD<em>1</em>03+CD8alphabeta recent thymic emigrants (RTEs). We show that IELs can be divided into two groups according to their dependency on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) for trafficking into the intestines. CD4 or CD8alphabeta naive lymphocytes originating from SP thymocytes express high levels of type <em>1</em> S<em>1</em>P receptor (S<em>1</em>P(<em>1</em>)), and their preferential migration into the large intestine is regulated by S<em>1</em>P. In contrast, RTEs migrate exclusively into the small intestine, whereas DN thymic IEL precursors expressing either TCRalphabeta or TCRgammadelta migrate into both the small and large intestines. S<em>1</em>P does not play a role in the migration pathways of these unconventional thymic IEL precursors. Thus, down-regulation of S<em>1</em>P(<em>1</em>) expression or disruption of the S<em>1</em>P gradient halted conventional CD4 or CD8alphabeta IEL trafficking into the intestines, but did not affect the trafficking of unconventional thymic IEL precursors. These data are the first to demonstrate that a lipid-mediated system discriminates IELs originating from conventional and unconventional thymic precursors.

This study reports on the characterization of B cells of germinal center (GC)-like structures infiltrating the salivary glands (SGs) of patients with Sjögren's syndrome. Eight two-color combinations were devised to characterize the phenotype of these B cells in <em>1</em><em>1</em> SG specimens selected from biopsies obtained from 40 Sjögren's syndrome patients and three normal tonsils. The 9G4 mAb, which recognizes V4.34-encoded autoAbs, enabled us to identify autoreactive B cells. Quantitative RT-PCR was used to determine the level of mRNAs for activation-induced cytidine deaminase (AICDA), repressors and transcription factors. CD20(+)IgD(-)CD38(+)CD2<em>1</em>(+)CD24(-) B cells, similar to those identified in tonsil GCs, were seen in the SGs of four patients and, and since they expressed AICDA, they were termed "real GCs". CD20(+)IgD(+)CD38(-)CD2<em>1</em>(+)CD24(+) B cells, seen in aggregates from the remaining seven samples, were characteristically type 2 transitional B cells and marginal zone-type B cells. They lacked AICDA mRNAs and were termed "aggregates". Real GCs from SGs contained mRNAs for Pax-5 and Bcl-6, like tonsil GC cells, whereas aggregates contained mRNAs for Notch-2, Blimp-<em>1</em>, IRF-4, and BR3, similar to marginal zone B cells. Further experimental data in support of this dichotomy included the restriction of CXCR5 expression to real GC cells, while <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> was expressed only in aggregates. In contrast, both types of B cell clusters expressed the idiotype recognized by the 9G4 mAb. Our data indicate that, in SGs, a minority of B cell clusters represent genuine GC cells, while the majority manifest features of being type 2 transitional B cells and marginal zone cells. Interestingly, both types of B cell aggregates include autoreactive B cells.

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S<em>1</em>P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT<em>1</em>, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S<em>1</em>P receptor S<em>1</em>PR2 in atherosclerosis by analyzing S<em>1</em>pr2-/- mice with an Apoe-/- background. S<em>1</em>PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S<em>1</em>pr2-/-Apoe-/- mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S<em>1</em>pr2+/+Apoe-/- mice. Bone marrow chimera experiments indicated a major role for macrophage S<em>1</em>PR2 in atherogenesis. S<em>1</em>pr2-/-Apoe-/- macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S<em>1</em>pr2-/-Apoe-/- ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-<em>1</em> expression and elevated eNOS phosphorylation. Pharmacologic S<em>1</em>PR2 blockade in S<em>1</em>pr2+/+Apoe-/- mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S<em>1</em>PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.

The lymphatic vasculature provides routes for dendritic cell and lymphocyte migration into and out of lymph nodes. Lymphatic endothelial cells (LEC) control these processes by expression of CCL2<em>1</em>, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, and adhesion molecules. LEC express MHC-I and MHC-II, but not costimulatory molecules, and present antigen on MHC-I via both direct and cross-presentation. Whether LEC present to CD4 T cells on MHC-II is unknown. Interestingly, LEC express antigens otherwise restricted to a small number of peripheral tissues in an autoimmune regulatory element-independent manner. Direct presentation of peripheral tissue antigens (PTA) to CD8 T cells results in abortive proliferation and deletion, due to both a lack of costimulation and active PD-L<em>1</em> engagement. Autoimmunity develops when deletion is subverted, suggesting that LEC presentation of PTA could lead to human disease if PD-<em>1</em> signaling were impaired by genetic polymorphisms, or aberrant costimulation occurred during inflammation. The expression of additional inhibitory molecules, which are not involved in LEC-mediated deletion, suggests that LEC may have additional immunoregulatory roles. LEC express receptors for several immunomodulatory molecules whose engagement alters their phenotype and function. In this review we describe the role of LEC in distinct anatomical locations in controlling immune cell trafficking, as well as their emerging role in the regulation of T cell tolerance and immunity.

The role of sphingolipids as bioactive signaling molecules that can regulate cell fate decisions puts them at center stage for cancer treatment and prevention. While ceramide and <em>sphingosine</em> have been established as antigrowth molecules, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) offers a progrowth message to cells. The enzymes responsible for maintaining the balance between these "stop" or "go" signals are the <em>sphingosine</em> kinases (SK), SK<em>1</em> and SK2. While the relative contribution of SK2 is still being elucidated and may involve an intranuclear role, a substantial amount of evidence suggests that regulation of sphingolipid levels by SK<em>1</em> is an important component of carcinogenesis. Here, we review the literature regarding the role of SK<em>1</em> as an oncogene that can function to enhance cancer cell viability and promote tumor growth and metastasis; highlighting the importance of developing specific SK<em>1</em> inhibitors to supplement current cancer therapies.

BACKGROUND

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) is a key regulator of the dynamic ceramide/<em>sphingosine</em> <em>1</em>-<em>phosphate</em> rheostat balance and important in the pathological cancer genesis, progression, and metastasis processes. Many studies have demonstrated SK<em>1</em> overexpressed in various cancers, but no meta-analysis has evaluated the relationship between SK<em>1</em> and various cancers.

METHODS

We retrieved relevant articles from the PubMed, EBSCO, ISI, and OVID databases. A pooled odds ratio (OR) was used to assess the associations between SK<em>1</em> expression and cancer; hazard ratios (HR) were used for 5-year and overall survival. Review Manager 5.0 was used for the meta-analysis, and publication bias was evaluated with STATA <em>1</em>2.0 (Egger's test).

RESULTS

Thirty-four eligible studies (n=4,673 patients) were identified. SK<em>1</em> positivity and high expression were significantly different between cancer, non-cancer, and benign tissues. SK<em>1</em> mRNA and protein expression levels were elevated in the cancer tissues, compared with the normal tissues. SK<em>1</em> positivity rates differed between various cancer types (lowest [27.3%] in estrogen receptor-positive breast cancer and highest [82.2%] in tongue squamous cell carcinoma). SK<em>1</em> positivity and high expression were associated with 5-year survival; the HR was <em>1</em>.86 (95% confidence interval [CI], <em>1</em>.<em>1</em>8-2.94) for breast cancer, <em>1</em>.58 (<em>1</em>.08-2.3<em>1</em>) for gastric cancer, and 2.68 (2.<em>1</em>0-3.44) for other cancers; the total cancer HR was 2.2<em>1</em> (95% CI, <em>1</em>.83-2.67; P < 0.0000<em>1</em>). The overall survival HRs were 2.09 (95% CI, <em>1</em>.35-3.22), <em>1</em>.56 (<em>1</em>.08-2.25), and 2.62 (2.05-3.35) in breast, gastric, and other cancers, respectively. The total effect HR was 2.2<em>1</em> (95% CI, <em>1</em>.83-2.66; P < 0.0000<em>1</em>).

CONCLUSIONS

SK<em>1</em> positivity and high expression were significantly associated with cancer and a shorter 5-year and overall survival. SK<em>1</em> positivity rates vary tremendously among the cancer types. It is necessary to further explore whether SK<em>1</em> might be a predictive biomarker of outcomes in cancer patients.

BACKGROUND

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lysophospholipid, derived from activated platelet, that is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Abnormal platelet and coagulation activation is often seen in patients with gastric cancer. However, neither the effects of this platelet-derived mediator S<em>1</em>P nor the distribution of S<em>1</em>P receptors on the gastric cancer cell are fully understood. The aim of this study was to examine the possible role of S<em>1</em>P and its receptors in the progression of gastric cancer.

METHODS

We characterized the expression profiles of S<em>1</em>P receptors in nine human gastric cancer cell lines and evaluated the relationship between the responses to S<em>1</em>P and its receptor expression on cell migration by modified Boyden chamber and cell proliferation by MTS assay.

RESULTS

Northern blotting analysis has revealed that S<em>1</em>P2 was expressed in all gastric cancer cell lines to varying degrees, and S<em>1</em>P3 was expressed in four cell lines. S<em>1</em>P<em>1</em> expression was weak, and no significant expression of either S<em>1</em>P4 or S<em>1</em>P5 was detected. The addition of S<em>1</em>P markedly stimulated the migration of MKN<em>1</em> and HCG-27 that dominantly expressed S<em>1</em>P3, and the effect was potently inhibited by pertussis toxin or wortmannin. In contrast, SIP significantly inhibited the migration of AZ-52<em>1</em> that expressed S<em>1</em>P2 exclusively. This indicates that the balance between S<em>1</em>P2- and S<em>1</em>P3-mediated signals might be critical in determining the metastatic response of gastric cancer cells to S<em>1</em>P. S<em>1</em>P elicited weak but significant antiproliferative effects on all of the three cell lines, although the effects were not major. In these cells, S<em>1</em>P induced extracellular signal-regulated kinase (ERK) phosphorylation with transient Akt dephosphorylation that may cause the weak effects on proliferation.

CONCLUSIONS

Our results suggest that the S<em>1</em>P receptor expression may critically determine the biological behavior of gastric cancers and thus therapeutic interventions directed at each S<em>1</em>P receptor might be clinically effective in preventing metastasis in gastric cancer.

Bile acids are important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids activate both the ERK<em>1</em>/2 and AKT signaling pathways via <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor 2 (S<em>1</em>PR2) in rodent hepatocytes and in vivo. Here, we report that feeding mice a high-fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S<em>1</em>PR2 in mouse hepatocytes significantly upregulated hepatic <em>sphingosine</em> kinase 2 (SphK2) but not SphK<em>1</em>. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly downregulated in livers of S<em>1</em>PR2(-/-) and SphK2(-/-) mice. In contrast, overexpression of the gene encoding S<em>1</em>PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK<em>1</em>, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, an endogenous inhibitor of histone deacetylases <em>1</em> and 2, as well as the acetylation of histones H3K9, H4K5, and H2BK<em>1</em>2 were significantly decreased in hepatocytes prepared from S<em>1</em>PR2(-/-) and SphK2(-/-) mice.

CONCLUSIONS

Both S<em>1</em>PR2(-/-) and SphK2(-/-) mice rapidly developed fatty livers on a high-fat diet, suggesting the importance of conjugated bile acids, S<em>1</em>PR2, and SphK2 in regulating hepatic lipid metabolism.

BACKGROUND

Dysfunctional lipid metabolism is a hallmark of obesity and insulin resistance and a risk factor for various cardiovascular and metabolic complications. In addition to the well known increase in plasma triglycerides and free fatty acids, recent work in humans and rodents has shown that obesity is associated with elevations in the bioactive class of sphingolipids known as ceramides. However, in obesity little is known about the plasma concentrations of sphinogsine-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), the breakdown product of ceramide, which is an important signaling molecule in mammalian biology. Therefore, the purpose of this study was to examine the impact of obesity on circulating S<em>1</em>P concentration and its relationship with markers of glucose metabolism and insulin sensitivity.

RESULTS

Plasma S<em>1</em>P levels were determined in high-fat diet (HFD)-induced and genetically obese (ob/ob) mice along with obese humans. Circulating S<em>1</em>P was elevated in both obese mouse models and in obese humans compared with lean healthy controls. Furthermore, in humans, plasma S<em>1</em>P positively correlated with total body fat percentage, body mass index (BMI), waist circumference, fasting insulin, HOMA-IR, HbA<em>1</em>c (%), total and LDL cholesterol. In addition, fasting increased plasma S<em>1</em>P levels in lean healthy mice.

CONCLUSIONS

We show that elevations in plasma S<em>1</em>P are a feature of both human and rodent obesity and correlate with metabolic abnormalities such as adiposity and insulin resistance.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors represent a novel subfamily of G-protein-coupled receptors binding S<em>1</em>P specifically and with high affinity. Although their in vivo functions remain largely unknown, in vitro extracellular application of S<em>1</em>P induces distinct S<em>1</em>P receptor-dependent cellular responses including proliferation, differentiation, and migration. We have analyzed signaling pathways engaged by S<em>1</em>P(4), which is highly expressed in the lymphoid system. Here we show that S<em>1</em>P(4) couples directly to Galpha(i) and even more effectively to Galpha(<em>1</em>2/<em>1</em>3)-subunits of trimeric G-proteins, but not to Galpha(q) unlike other S<em>1</em>P receptors. Consequently, CHO-K<em>1</em> cells ectopically expressing S<em>1</em>P(4) potently activate the small GTPase Rho and undergo cytoskeletal rearrangements, inducing peripheral stress fiber formation and cell rounding, upon S<em>1</em>P stimulation. Overexpression of S<em>1</em>P(4) in Jurkat T cells induces pertussis toxin-sensitive cell motility even in the absence of exogenously added S<em>1</em>P. In addition, S<em>1</em>P(4) is internalized upon binding of S<em>1</em>P. The capacity of S<em>1</em>P(4) to mediate cellular responses, such as motility and shape change through Galpha(i)- and Galpha(<em>1</em>2/<em>1</em>3)-coupled signaling pathways may be important for its in vivo function which is currently under investigation.

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of <em>sphingosine</em>-<em>1</em> <em>phosphate</em> receptor <em>1</em> (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B<em>1</em> B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.

Resistance to cisplatin is a common problem that limits its usefulness in cancer therapy. Molecular genetic studies in the model organism Dictyostelium discoideum have established that modulation of <em>sphingosine</em> kinase or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S-<em>1</em>-P) lyase, by disruption or overexpression, results in altered cellular sensitivity to this widely used drug. Parallel changes in sensitivity were observed for the related compound carboplatin but not for other chemotherapy drugs tested. Sensitivity to cisplatin could also be potentiated pharmacologically with dimethyl<em>sphingosine</em>, a <em>sphingosine</em> kinase inhibitor. We now have validated these studies in cultured human cell lines. HEK293 or A549 lung cancer cells expressing human S-<em>1</em>-P lyase (hSPL) show an increase in sensitivity to cisplatin and carboplatin as predicted from the earlier model studies. The hSPL-overexpressing cells were also more sensitive to doxorubicin but not to vincristine or chlorambucil. Studies using inhibitors to specific mitogen-activated protein kinases (MAPK) show that the increased cisplatin sensitivity in the hSPL-overexpressing cells is mediated by p38 and to a lesser extent by c-Jun NH2-terminal kinase MAPKs. p38 is not involved in vincristine or chlorambucil cytotoxicity. Measurements of MAPK phosphorylation and enzyme activity as well as small interfering RNA inhibition studies show that the response to the drug is accompanied by up-regulation of p38 and c-Jun NH2-terminal kinase and the lack of extracellular signal-regulated kinase up-regulation. These studies confirm an earlier model proposing a mechanism for the drug specificity observed in the studies with D. discoideum and support the idea that the <em>sphingosine</em> kinases and S-<em>1</em>-P lyase are potential targets for improving the efficacy of cisplatin therapy for human tumors.

The bioactive lipids lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), the enzymes that generate and degrade them, and the receptors that receive their signals are all potential therapeutic targets in cancer. LPA and S<em>1</em>P signalling pathways can modulate a range of cellular processes that contribute to tumourigenesis, such as proliferation and motility, and components of the signalling pathways often show aberrant expression and altered activity upon malignant transformation. This article reviews LPA- and S<em>1</em>P-mediated activities that might contribute to the aetiology of cancer, and examines the potential of the many antagonists that have been developed to inhibit LPA and S<em>1</em>P signalling pathways. In addition, the outcomes of various clinical trials using LPA- and S<em>1</em>P-associated targets in cancer and other diseases are described, and future directions are discussed.

CD8+ tissue-resident memory T cells (TRM cells) reside permanently in nonlymphoid tissues and provide a first line of protection against invading pathogens. However, the precise localization of CD8+ TRM cells in the lung, which physiologically consists of a markedly scant interstitium compared with other mucosa, remains unclear. In this study, we show that lung CD8+ TRM cells localize predominantly in specific niches created at the site of regeneration after tissue injury, whereas peripheral tissue-circulating CD8+ effector memory T cells (TEM cells) are widely but sparsely distributed in unaffected areas. Although CD69 inhibited <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em>-mediated egress of CD8+ T cells immediately after their recruitment into lung tissues, such inhibition was not required for the retention of cells in the TRM niches. Furthermore, despite rigid segregation of TEM cells from the TRM niche, prime-pull strategy with cognate antigen enabled the conversion from TEM cells to TRM cells by creating de novo TRM niches. Such damage site-specific localization of CD8+ TRM cells may be important for efficient protection against secondary infections by respiratory pathogens.

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.

The sphingolipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) acts on five subtypes of G-protein- coupled receptors, termed S<em>1</em>P(<em>1</em>) (formerly endothelial differentiation gene-<em>1</em> [Edg-<em>1</em>]), S<em>1</em>P(2) (Edg-5), S<em>1</em>P(3) (Edg-3), S<em>1</em>P(4) (Edg-6) and S<em>1</em>P(5) (Edg-8), and possibly several other "orphan" receptors, such as GPR3, GPR6 and GPR<em>1</em>2. These receptors are coupled to different intracellular second messenger systems, including adenylate cyclase, phospholipase C, phosphatidylinositol 3-kinase/protein kinase Akt, mitogen-activated protein kinases, as well as Rho- and Ras-dependent pathways. Consistently with this receptor multiplicity and pleiotropic signaling mechanisms, S<em>1</em>P influences numerous cell functions. S<em>1</em>P(<em>1</em>)<em>1</em>, S<em>1</em>P(2) and S<em>1</em>P(3) receptors are the major S<em>1</em>P receptor subtypes in the cardiovascular system, where they mediate the effects of S<em>1</em>P released from platelets, and possibly other tissues (such as brain). Thus S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) receptors enhance endothelial and vascular smooth muscle cell proliferation and migration, playing a key role in developmental and pathological angiogenesis. In contrast, S<em>1</em>P(2) receptors inhibit migration of these cell types, probably because of their unique stimulatory effect on a GTPase-activating protein inhibiting the activity of Rac. S<em>1</em>P receptors can also cause relaxation and constriction of blood vessels. The former effect is mediated by pertussis toxin-sensitive receptors (possibly S<em>1</em>P(<em>1</em>)) located on the endothelium and stimulating phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase (eNOS). The vasoconstricting effect of S<em>1</em>P is likely to be mediated by S<em>1</em>P(2) and/or S<em>1</em>P(3) receptors, via Rho-Rho-kinase, and is more potent in coronary and cerebral blood vessels. Finally, S<em>1</em>P also protects endothelial cells from apoptosis through activation of phosphatidylinositol 3-kinase/Akt/eNOS via S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) receptors. The variety of these effects, taken together with the existence of multiple receptor subtypes, provides an abundance of therapeutic targets that currently still await the development of selective agents.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S<em>1</em>P in endothelium-dependent vasodilation and the signaling pathways elicited by S<em>1</em>P in intact vessels are largely unknown. We found that S<em>1</em>P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC(50) <em>1</em>0 +/- 3 nM); maximal vasodilation (55 +/- 8%) is seen approximately 2 min after S<em>1</em>P addition and returns to baseline by 5 min. S<em>1</em>P promotes comparable responses in arterioles from wild-type but not eNOS(null) mice. S<em>1</em>P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor N(omega)-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K(+) channels by using 4-aminopyridine. S<em>1</em>P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S<em>1</em>P, but not bradykinin, promotes the phosphorylation of eNOS at Ser(<em>1</em><em>1</em>79). Maximum S<em>1</em>P-induced eNOS Ser(<em>1</em><em>1</em>79) phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser(<em>1</em><em>1</em>79) dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S<em>1</em>P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-<em>1</em> expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-<em>1</em> via sequential activation of c-Src, protein kinase C delta (PKCdelta), and <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), the enzyme that produces <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK<em>1</em> to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK<em>1</em> translocation and up-regulation of PAI-<em>1</em> by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-<em>1</em> activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK<em>1</em> and PKCdelta to the plasma membrane and up-regulation of PAI-<em>1</em> expression. Furthermore, SphK<em>1</em> was indispensable for both EGF-induced c-Jun phosphorylation and PAI-<em>1</em> expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK<em>1</em> that have all been implicated in regulating motility and invasion of glioma cells.

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) constitute a rheostat system in which ceramide promotes cell death and S<em>1</em>P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S<em>1</em>P, respectively.