Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients.

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.

Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC(1)R and VPAC(2)R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC(1)R is expressed constitutively, whereas VPAC(2)R is induced by immune stimuli. VPAC(2)R-null (VPAC(2)R(-/-)) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3(+)) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC(2)R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were > or =70% lower in VPAC(2)R-null mice than in wild-type controls. Cytokine production by splenic CD4(+) T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-gamma (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC(2)R-null mice than wild-type controls. Loss of VIP-VPAC(2)R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.

Allergic diathesis in transgenic mice with constitutive T cell expression of inducible vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) and its G-protein-coupled receptors (VPAC1 and VPAC2 Rs) are prominent in the immune system. In T cells, VPAC1 R is expressed constitutively whereas VPAC2 R is induced only after stimulation of the T cell receptor (TCR) or exposure to some cytokines. VPAC1 R and VPAC2 R also transduce different effects of VIP on T cells. Constitutive expression of VPAC2 R selectively in CD4+ T cells (helper-inducer Th cells) of transgenic (TG) C57BL/6 mice directed by the lck tyrosine kinase promoter is now shown to evoke production of more Th2-type interleukins 4 and 5, and less Th1-type interferon gamma after TCR activation. VPAC2 R TG mice consequently have significant elevations of blood IgE, IgG1, and eosinophils. VPAC2 R TG mice also show increased IgE antibody responses, which mediate heightened cutaneous allergic reactions, and have depressed delayed-type hypersensitivity. VIP enhancement of the ratio of Th2 cell to Th1 cell cytokines thus evokes an allergic state in normally nonallergic mice, which suggests the possibility of neuropeptide contributions to immune phenotypic alterations in human hypersensitivity diseases.

The lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid enhance survival during hypoxia in neonatal rat cardiac myocytes.

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S1P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/1% CO2 (normoxia) or in an atmosphere of 99% N2/1%CO2 (hypoxia) at 37 degrees C for 18-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-1.0% of the cells were viable after 18-20 h. Severe hypoxia reduced viability to 61.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either 10 microM S1P or 1 microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (1 microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, 100 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S1P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethylsphingosine (DMS), which inhibits sphingosine kinase synthesis of S1P. Incubation of neonatal rat cardiac myocytes with 10 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during 18-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S1P or GM-1, a ganglioside that activates sphingosine kinase to increase intracellular levels of S1P. We conclude that both S1P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S1P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S1P are effective in preventing cell death induced by inhibition of sphingosine kinase.

Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells.

BACKGROUND: Lysophosphatidic acid (LPA) stimulates ovarian tumor growth at concentrations present in ascitic fluid. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and plays a pivotal role in the formation of ovarian cancer-associated ascites. We examined whether LPA promotes ovarian tumor growth by increasing angiogenesis via VEGF. METHODS: VEGF expression was examined in a simian virus 40 T-antigen-immortalized ovarian surface epithelial cell line (IOSE-29) and in ovarian cancer cell lines (OVCAR-3, SKOV-3, and CAOV-3) treated with LPA. VEGF promoter activity was measured in OVCAR-3 cells after transfection or cotransfection with c-Fos and c-Jun, components of AP1 transcription factor, potential binding sites for which are present in the VEGF promoter. The expression of the LPA receptors Edg2 and Edg4 was also assessed. All statistical tests were two-sided. RESULTS: LPA treatment increased steady-state VEGF messenger RNA (mRNA) levels in OVCAR-3 cells in a time- and dose-dependent fashion and stimulated VEGF promoter activity without prolonging mRNA half-life in these cells, but LPA had little effect on IOSE-29 cells. Forced overexpression of c-Jun and c-Fos in OVCAR-3 cells stimulated VEGF promoter activity fourfold. LPA also elevated VEGF protein levels by 1.5-fold in SKOV-3 cells (P =.0148), 1.9-fold in CAOV-3 cells (P<.001), and threefold in OVCAR-3 cells (P<.0001). Both Edg2 and Edg4 were detected in ovarian cancer cells; however, only Edg2 was present in normal ovarian surface epithelial cells and IOSE-29 cells. CONCLUSIONS: LPA stimulates ovarian tumor growth, at least in part, via induction of VEGF expression through transcriptional activation. However, this LPA response is not evident in normal ovarian surface epithelial cells. Our data suggest that Edg4, but not Edg2, plays a role in LPA stimulation of ovarian tumor growth.

Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids.

The activities of cell-derived lysophospholipid (LPL) growth factors on cellular proliferation and a range of proliferation-independent functions are regulated at multiple levels. This section focuses first on the capacity of the actin-severing protein gelsolin to bind lysophosphatidic acid (LPA), but not sphingosine 1-phosphate (S1P), and either sequester LPA or present it to responsive cells. Expression of members of the family of endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) for LPLs is controlled developmentally and by cell-activating stimuli. Edg R transduction of cellular effects of LPLs involves both direct actions on target cells and induction of generation of proteins with relevant actions capable of amplifying or diminishing primary direct effects of LPLs. These general mechanisms are evident in Edg R mediation of proliferation, cytokine secretion and suppression of apoptosis. The availability of functionally-active anti-Edg R antibodies and Edg R-specific pharmacological probes, establishment of Edg R transgenes and gene knockouts, and identification of natural genetic anomalies of LPL metabolism and recognition by Edg Rs will permit elucidation of the in vivo activities of LPA and S1P normally and in disease states.

Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10(-9) to 10(-6) M LPA and by 30-300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [(3)H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.

Altered expression and functional profile of lysophosphatidic acid receptors in mitogen-activated human blood T lymphocytes.

Lysophosphatidic acid (LPA) from platelets and mononuclear phagocytes regulates T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs). Human blood unactivated CD4+ T cells express predominant ly Edg-4 LPA R over marginal levels of Edg-2 LPA R, as assessed by semiquantitative PCR and Western blots. After mitogen activation, the CD4+ T cells express Ed g-2 Rs at approximately one half the level of Edg-4 Rs. Secretion of IL-2 by unactivated Edg-4 R-predominant CD4+ T cells incubated with anti-CD3 plus anti-CD28 antibodies was suppressed significantly and by up to 60% by 10-10 M to 10-6 M LPA, whereas secretion of IL-2 by mitogen-activated Edg-2 R and Edg-4 R codominant CD4+ T cells was enhanced by up to twofold by the same concentrations of LPA. The possibility that the two Edg Rs transduce different LPA signals to CD4+ T cells was supported by findings that IL-2 secretion was inhibited by mouse anti-Edg-4 R monoclonal antibody, but enhanced by mouse anti-Edg-2 R monoclonal antibody. The separate effects of each LPA R were studied in Jurkat T cell transfectants expressing principally human Edg-2 Rs (Jurkat-T-2) or Edg-4 Rs (Jurkat-T-4) and stimulated with anti-CD3 plus phorbol myristate acetate. LPA and anti-Edg-4 R antibody suppressed IL-2 secretion by stimulated Jurkat-T-4 cells, whereas LPA and anti-Edg-2 R antibody enhanced IL-2 secretion by stimulated Jurkat-T-2 cells. Activation-induced alterations in the relative levels of Edg-2 and -4 Rs on CD4+ T cells thus reverse the effects of LPA on T cell receptor-stimulated generation of IL-2.

Inhibition of expression of the type I G protein-coupled receptor for vasoactive intestinal peptide (VPAC1) by hammerhead ribozymes.

Vasoactive intestinal peptide (VIP) is a neuromediator expressed widely in the nervous, gastrointestinal, respiratory, and immune systems. Two G protein-coupled receptors (GPCRs), designated VPAC1 and VPAC2, bind VIP with high affinity and transduce increases in [cyclic AMP](i) and [Ca(2+)](i). As there are no potent VPAC1- or VPAC2-selective antagonists, a hammerhead ribozyme (Rz) strategy capable of in vivo application was adopted to inactivate individual domains of VPAC1. Three Rzs were designed to cleave mRNA encoding the amino terminus, the third intracellular loop, and the cytoplasmic tail of human VPAC1 and were introduced by transfection into HEK-293 cells expressing recombinant human VPAC1. Each Rz specifically degraded VPAC1 mRNA and down-regulated VPAC1 protein and VIP-binding activity, as assessed by ribonuclease protection assays, Western blots, and binding of (125)I-VIP. Rz-mediated down-regulation of VPAC1 was associated with up to 75% suppression of VIP signaling of increases in [cyclic AMP](i) and [IP3](i), and of cyclic AMP response element-luciferase reports. The Rz specific for the amino terminus inhibited VPAC1 expression and signaling to the greatest extent. VIP-evoked cellular responses thus appear to be proportional to the level of VPAC1 expression. Specific Rzs may be powerful tools for manipulating tissue-specific contributions of GPCRs in vitro and in vivo.

Gelsolin binding and cellular presentation of lysophosphatidic acid.

Lysophosphatidic acid (LPA) in biological fluids binds to serum albumin and other proteins that enhance its effects on cellular functions. The actin-severing protein gelsolin binds LPA with an affinity (K(d) = 6 nm) similar to that of the G protein-coupled LPA receptors encoded by endothelial differentiation genes 2, 4, and 7 (Edg-2, -4, and -7 receptors) and greater than that of serum albumin (K(d) = 360 nm). At concentrations of 10% or less of that in plasma, which are observed in fluids of injured tissues, purified and recombinant gelsolin augment LPA stimulation of nuclear signals and protein synthesis in rat cardiac myocytes (RCMs) that express Edg-2 and -4 receptors. At concentrations of 20% or more of that in plasma, gelsolin suppresses LPA stimulation of RCMs. The lack of effect of gelsolin on RCM responses to monoclonal anti-Edg-4 receptor antibody plus a phorbol ester without LPA attests to its specificity for LPA delivery and the absence of post-receptor effects. Inhibition of gelsolin binding and cellular delivery of LPA by l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP2) and peptides constituting the two PIP2 binding domains of gelsolin suggests competition between LPA and PIP2 for the same sites. Thus, delivery of LPA to RCMs is affinity-coupled to Edg receptors by gelsolin in a PIP2-regulated process.

Cutting edge: differential constitutive expression of functional receptors for lysophosphatidic acid by human blood lymphocytes.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and macrophages mediate T cell functions. Endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) are specific for S1P (Edg-1, -3, -5, and -8 Rs) and LPA (Edg-2, -4, and -7 Rs). Human T cell tumors express many Edg Rs for both LPA and S1P. In contrast, human blood CD4+ T cells express predominantly Edg-4, and CD8+ T cells show only traces of Edg-2 and -5, by quantification of mRNA and Edg R Ags. LPA at 10-10-10-6 M suppressed significantly the secretion of IL-2 from anti-CD3 plus anti-CD28 Ab-challenged CD4+ T cells, but not CD8+ T cells. Monoclonal anti-Edg-4 R Ab, like LPA, suppressed stimulated IL-2 secretion from CD4+ T cells, but not CD8+ T cells. Constitutive expression of Edg-4 by CD4+, but not CD8+, human T cells accounts for differential functional responsiveness of the T cell subsets to LPA.

Mechanisms of lysolipid phosphate effects on cellular survival and proliferation.

The specificity of cellular effects of lysolipid phosphate (LLP) growth factors is determined by binding to endothelial differentiation gene-encoded G protein-coupled receptors (EDG Rs), which transduce diverse proliferative and effector signals. The primary determinants of cellular responses to LLPs are the generative and biodegradative events, which establish steady-state concentrations of each LLP at cell surfaces, and the relative frequency of expression of each EDG R. There are major differences among types of cells in the net effective generation of the LLPs, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), and in their profile of expression of EDG Rs. The less well characterized secondary determinants of cellular specificity of LLPs are high-affinity binding proteins with carrier and cell-presentation functions, cell-selective regulators of expression of EDG Rs, and cellular factors that govern coupling of EDG Rs to G protein transductional pathways. The roles of components of the LLP-EDG R system in normal physiology and disease processes will be definitively elucidated only after development of animal models with biologically meaningful alterations in genes encoding EDG Rs and the discovery of potent and selective pharmacological probes.

Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent lipid growth factors with similar abilities to stimulate cytoskeleton-based cellular functions. Their effects are mediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes (edgs). We hypothesize that large quantities of LPA and S1P generated by activated platelets may influence endothelial cell functions. Using an in vitro wound healing assay, we observed that LPA and S1P stimulated closure of wounded monolayers of human umbilical vein endothelial cells and adult bovine aortic endothelial cells, which express LPA receptor Edg2, and S1P receptors Edg1 and Edg3. The two major components of wound healing, cell migration and proliferation, were stimulated individually by both lipids. LPA and S1P also stimulated intracellular Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. Pertussis toxin partially blocked the effects of both lipids on endothelial cell migration, MAPK phosphorylation, and Ca(2+) mobilization, implicating G(i)/(o)-coupled Edg receptor signaling in endothelial cells. LPA and S1P did not cross-desensitize each other in Ca(2+) responses, suggesting involvement of distinct receptors. Thus LPA and S1P affect endothelial cell functions through signaling pathways activated by distinct GPCRs and may contribute to the healing of wounded vasculatures.

Vasoactive intestinal peptide mediation of development and functions of T lymphocytes.

The first phase in investigating neural regulation of immunity has delineated anatomical connections, shared mediators and receptors for mediators with distinctive effects, and the immune functional consequences of altering relevant neural activities. Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are represented prominently in immune organs. They have potent novel effects on many aspects of immunity, are derived from and serve as autacoids in some sets of immune cells, and they participate in both physiological and pathological immune responses. The present phase of neuroimmune research has begun to elucidate the genetic determinants of expression and functions of neuromediators in immunity. Our evolving understanding of the novel mechanisms for adaptation and specificity in the VIP/PACAP neuroimmune network suggests the importance of immunoselective transcriptional control of expression of VIP/PACAP receptors in T cells, a dominant role for numerous cytokines, and the critical involvement of small subsets of VIP-/PACAP-responsive thymocytes and T cells.

Stem cell factor influences neuro-immune interactions: the response of mast cells to pituitary adenylate cyclase activating polypeptide is altered by stem cell factor.

Mast cells degranulation can be elicited by a number of biologically important neuropeptides, but the mechanisms involved in mast cell-neuropeptide interactions have not been fully elucidated. Stem cell factor (SCF), also known as c-kit or kit ligand, induces multiple effects on mast cells, including proliferation, differentiation, maturation, and prevents apoptosis. We investigated the ability of SCF to affect mast cell responsiveness to the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PACAP 1-27, PACAP1-38, or VIP failed to induced preformed mediator release from mouse bone-marrow-cultured mast cells (BMCMC) derived in concanavalin A-stimulated spleen conditioned medium (CM). By contrast, BMCMC grown in SCF-containing medium or freshly isolated peritoneal mast cells exhibited significant 3H-hydroxytrypamine (5-HT) release in response to PACAP peptides or VIP. Deoxyglucose and the mitochondrial inhibitor antimycin significantly inhibited PACAP-induced 5-HT release indicating that the central event induced by PACAP peptides was exocytosis. The G(alpha)i inhibitor, pertussis toxin, significantly diminished PACAP-induced 5-HT release from BMCMCs in SCF suggesting the involvement of heterotrimeric G-proteins. Western blot analysis using antibodies directed against the human VIP type I/PACAP type II receptor demonstrated a 70-72 kD immunoreactive protein expressed in greater amounts in BMCMC grown in SCF compared with BMCMC in CM. We conclude that SCF induces a mast cell population that is responsive to PACAPs and VIP involving a heterotrimeric G-protein-dependent mechanism.

Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer.

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-1, -3, and -5 bind sphingosine 1-phosphate (S1P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S1P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S1P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.

Dual mechanisms for lysophospholipid induction of proliferation of human breast carcinoma cells.

Endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) Edg-1, Edg-3, and Edg-5 bind sphingosine 1-phosphate (S1P), and Edg-2 and Edg-4 Rs bind lysophosphatidic acid (LPA). LPA and S1P initiate ras- and rho-dependent signaling of cellular growth. Cultured lines of human breast cancer cells (BCCs) express Edg-3 > Edg-4 > Edg-5 > or = Edg-2, without detectable Edg-1, by both assessment of mRNA and Western blots with rabbit and monoclonal mouse anti-Edg R antibodies. BCC proliferation was stimulated significantly by 10(-9) M to 10(-6) M LPA and S1P. Luciferase constructs containing the serum response element (SRE) of growth-related gene promoters reported mean activation of BCCs by LPA and S1P of up to 85-fold. LPA and S1P stimulated BCC secretion of type II insulin-like growth factor (IGF-II) by 2-7-fold, to levels at which exogenous IGF-II stimulated increased proliferation and SRE activation of BCCs. All BCC responses to LPA and S1P were suppressed similarly by pertussis toxin, mitogen-activated protein kinase kinase inhibitors, and C3 exoenzyme inactivation of rho, suggesting mediation by Edg Rs. Monoclonal anti-IGF-II and anti-IGFR1 antibodies suppressed proliferation and SRE reports of BCCs to LPA and S1P by means of up to 65%. Edg Rs thus transduce LPA and S1P enhancement of BCC growth, both directly through SRE and indirectly by enhancing the contribution of IGF-II.

Lysophospholipid enhancement of human T cell sensitivity to diphtheria toxin by increased expression of heparin-binding epidermal growth factor.

The effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on T cell expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), the diphtheria toxin (DT) receptor, were investigated in the Tsup-1 cultured line of human CD4+ 8+ 3low T lymphoblastoma cells. Tsup-1 cells bear endothelial differentiation gene (edg)-2 and -4-encoded G protein-coupled receptors (GPCRs) for LPA and Edg-3 and -5 GPCRs for S1P. Suppression by DT of Tsup-1 cell protein synthesis was enhanced by LPA and S1P, with lipid structural specificity similar to that required for their recognition by Edg receptors. LPA and S1P increased the Tsup-1 cell level of immunoreactive HB-EGF, and neutralizing antibodies to HB-EGF inhibited LPA and S1P enhancement of Tsup-1 cell susceptibility to DT. Stabilized transfection of Tsup-1 cells with a combination of plasmids encoding Edg-2 plus -4 antisense mRNA suppressed the levels of Edg-2 and -4, but not Edg-3 and -5, in Western blots and reduced in parallel the increments in HB-EGF and susceptibility to DT evoked by LPA but not S1P. Similar transfection with Edg-3 plus -5 antisense plasmids suppressed Tsup-1 cell levels of immunoreactive Edg-3 and -5, but not Edg-2 or -4, and concurrently reduced S1P-, but not LPA-, induced Tsup-1 cell increases in both HB-EGF and susceptibility to DT. Edg GPCR-mediated LPA and S1P enhancement of T cell sensitivity to DT, thus, may be attributable to increased expression of the DT receptor HB-EGF.