Pulmonary metastasis of breast cancer requires recruitment and expansion of T regulatory cells (Tregs) that promote escape from host protective immune cells. However, it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells, designated tumor-evoked Bregs (tBregs), phenotypically resemble activated but poorly proliferative mature B2 cells (CD19+ CD25High CD69High) that express constitutively active Stat3 and B7-H1High CD81High CD86High CD62LLowIgMInt. Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGFβ-dependent conversion of FoxP3+ Tregs from resting CD4+ T cells. In the absence of tBregs, 4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications, since they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis.

Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA 1 , LPA 2 , and LPA 3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA 4 /GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA 1 , LPA 2 , or LPA 3 , we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhibited the LPA-induced response in the decreasing order of LPA 1 Ն LPA 3 Ͼ Ͼ LPA 2 , whereas DGPP 8:0 preferentially inhibited the LPA 3 -induced actions. Ki16425 inhibited LPA-induced guanosine 5Ј-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced longterm responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA 1 and LPA 3 ; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.Lysophosphatidic acid (LPA) has been shown to elicit diverse biological actions, including Ca 2ϩ mobilization, change in cAMP accumulation, change in cell shape and motility in association with actin rearrangement, and proliferation in a variety of cell types (Moolenaar, 1999;Contos et al., 2000;Ye et al., 2002). Extracellular LPA has also been shown to be involved in certain diseases, such as atherosclerosis and cancer (Xu et al., 1995(Xu et al., , 2001Siess et al., 1999;Maschberger et al., 2000). LPA was first thought to be released from activated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called autotaxin (Sano et al., 2002;Tokumura et al., 2002;Umezu-Goto et al., 2002). The concentration of plasma LPA is about 100 nM, and its serum concentration can be as high as 5 M (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., This work was supported in part by a research grant grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by research gr...

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, Gprotein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as protonsensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0 -6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1-and GPR4-expressing cells but not for prostaglandin E 2 -and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pHdependent action, although to a lesser extent. Psychosinesensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against proteinsensing receptors, including TDAG8, GPR4, and OGR1. TDAG81 was initially cloned as an orphan GPCR, which is up-regulated during the programmed cell death of T lymphocytes (1-3). This gene product has recently been reported (4) to be a receptor for psychosine, a lysosphingolipid, which induces the formation of multinuclear cells. OGR1, which shares 41% identical amino acids with TDAG8, was initially reported (5) to be a receptor for sphingosylphosphorylcholine (SPC). GPR4 also shares homology with TDAG8 and was identified as a receptor for lysolipids, including lysophosphatidylcholine (LPC) and SPC (6). It has recently been reported (7), however, that OGR1 and GPR4 sense extracellular protons through histidine residues of receptors and are coupled to G-proteins to stimulate intracellular signaling pathways. Thus, OGR1 stimulation causes inositol phosphate production, and the subsequent mobilization of intracellular calcium and GPR4 stimulation induces cAMP accumulation, probably reflecting the activation of adenylyl cyclase in response to an extracellular pH change (7). These results raise the possibility that TDAG8 may also respond to extracellular pH change and stimulate intracellular signaling pathways.If TDAG8 is prov...

Objective-Plasma high-density lipoprotein (HDL) level is inversely correlated with the risk of atherosclerosis. However, the cellular mechanism by which HDL exerts antiatherogenic actions is not well understood. In this study, we focus on the lipid components of HDL as mediators of the lipoprotein-induced antiatherogenic actions. Methods and Results-HDL and sphingosine 1-phosphate (S1P) stimulated the migration and survival of human umbilical vein endothelial cells. These responses to HDL and S1P were almost completely inhibited by pertussis toxin and other specific inhibitors for intracellular signaling pathways, although the inhibition profiles of migration and survival were different. The HDL-stimulated migration and survival of the cells were markedly inhibited by antisense oligonucleotides against the S1P receptors EDG-1/S1P 1 and EDG-3/S1P 3 . Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S1P 1 . The S1P-rich fraction and chromatographically purified S1P from HDL stimulated cell migration, but the rest of the fraction did not, as was the case of the cell survival. Key Words: high-density lipoprotein Ⅲ sphingosine 1-phosphate Ⅲ migration Ⅲ EDG Ⅲ endothelial cell P lasma lipoproteins are responsible for the transport of cholesterol to cells and the control of cholesterol synthesis. 1-3 Low-density lipoprotein (LDL) provides cholesterol to cells through LDL receptors, whereas high-density lipoprotein (HDL) has been shown to remove excess cholesterol from the cells. The so-called reverse transport of cholesterol is thought to be an important mechanism for the antiatherogenic actions of HDL. 1,2 Recent studies have shown that HDL induces cytoprotective actions, 3,4 proliferation, 5 and migration in endothelial cells, 2,6 activities presumably independent of cholesterol metabolism, 2,3 although the mechanism by which HDL induces these antiatherogenic actions has not been well characterized. It has been reported recently that HDL activates endothelial nitric oxide (NO) production through the scavenger receptor-BI (SR-BI). 7 NO production has been shown to be involved in the cytoprotective action of endothelial cells. 8 In endothelial cells, sphingosine 1-phosphate (S1P) has been shown to regulate a wide range of cellular activities associated with angiogenesis, wound healing, apoptosis, and atherosclerosis. 3,4,8 -18 S1P promotes cell migration, 10 -13,16 -18 DNA synthesis, 10 cell survival, 4,9 cell barrier integrity, 15 NO production, 8,14,16,17 and the expression of several cell adhesion molecules. 3 We recently reported that S1P accumulates in the lipoprotein fraction, especially the HDL fraction, and that HDL-associated S1P mediates the cytoprotective actions of HDL in human umbilical vein endothelial cells (HUVECs). 4,19 Nofer et al 20 reported that sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF) were major components of HDL responsible for these cytoprotective actions. Thus, lipoproteinassociated lipids may also be involved in some HDL-induced...

Cytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA 1 and LPA 3 ), but not by an LPA 3 -selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA 1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA 1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA 1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells.

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATPbinding cassette transporter A1 (ABCA1). Astrocytes from Abca1)/) mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1. Keywords: apolipoprotein E, astrocyte, ATP-binding cassette transporter A1, high-density lipoprotein, sphingosine 1-phosphate. Sphingosine 1-phosphate (S1P), a sphingolipid metabolite, is a pleiotropic lipid mediator involved in a variety of cell activities, including morphology, motility, and growth. Five subtypes of S1P-specific receptors, S1P 1-5 , have been isolated so far (Ishii et al. 2004). In neural cells, these receptors are expressed in a cell-specific manner (Van Brocklyn et al. 1999;Sato et al. 2000) and involved in the regulation of neural cell functions; for example, a rapid process retraction through S1P 5 in oligodendrocytes (Jaillard et al. 2005), rounding of the cell body in PC12 and N1E115 cells (Postma et al. 1996;Sato et al. 1997) possibly through S1P 2 (Van Brocklyn et al. 1999), and regulation of motility in astrocytes and glioma cells . In addition, S1P induces the proliferation in astroglial cells, which was associated with the activation of extracellular signal-regulated kinase (ERK), activation of the phospholipase C, and Ca 2+ mobilization (Tas and Koschel 1998;Sato et al. 1999;Pebay et al. 2001). S1P 1 is more important than S1P 2 for the stimulation of ERK, while S1P 2 may be responsible for the activation of phospholipase C and Ca 2+ mobilization (Sato et al. 2000;Malchinkhuu et al. 2003). Thus, S1P and its receptors may play important roles in maintaining the functions of the CNS.We have recently demonstrated that S1P is concentrated in lipoproteins, such as high-density lipoprotein (HDL), in circulating blood (Murata et al. 2000b), and that the lipid mediates lipoprotein-induced anti-atherogenic actions, including cell survival, cell migration, and inhibition of adhesion molecule expression through S1P receptors in endothelial cells Okajima 2002). Thus, lipoproteins seem to serve as carriers for extracellular S1P in circulating blood. In astrocytes as well, plasma HDL ...

Inflammation is a double-edged sword that can promote or suppress cancer progression. Here we report that thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, is also expressed in human and murine cancers. Our studies with murine cancer cells indicate that TSLP plays an essential role in cancer escape, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP receptor on CD4+ T cells, promoting Th2-skewed immune responses and production of immunosuppressive factors such as IL-10 and IL-13. Expression of TSLP therefore may be a useful prognostic marker and its targeting could have therapeutic potential.

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P 1 to inhibition through S1P 2 . LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA 1 receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signalregulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominantnegative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/ Rac1/JNK pathways are equally important for LPA 1 receptor-mediated migration.