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.