RESUMO
A role for adenosine in immunosenescence was investigated in T cells from older (≥65 yr) and younger (24-45 yr) healthy humans. Adenosine concentrations in cultures of activated T cells were significantly higher (P<0.0001) for older (145±47 nM, mean±sd) than younger (58±5.5 nM) subjects. Expression of the activation coreceptor CD28 was suppressed significantly by 0.1 to 1 µM exogenous adenosine, with greater effects of 1 µM (P<0.01) on T cells of younger (mean suppression of 67 and 65% for CD4 and CD8 T cells, respectively) than older (means of 42 and 46%) subjects. T-cell chemotaxis to CCL21 was suppressed significantly by 0.3 and 1 µM exogenous adenosine, with mean maximum decreases of 39 and 49%, respectively, for younger subjects and 28 and 31% for older subjects. Generation of IL-2 and IFN-γ by T cells of younger and older subjects was suppressed substantially only at adenosine levels of 3 µM or higher. Lower baseline expression of CD28 and chemotaxis to CCL21 and S1P for T cells from older subjects attributable to endogenous adenosine were reversed completely by two different A(2A) adenosine receptor antagonists without affecting T cells of younger subjects. Adenosine is an endogenous T-cell immunosuppressor in older humans, and A(2A) antagonists reverse adenosine-induced T-cell deficiencies of aging.
Assuntos
Adenosina/imunologia , Adenosina/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apirase/imunologia , Apirase/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Fenetilaminas/farmacologia , Pirimidinas/farmacologia , Linfócitos T/metabolismo , Triazóis/farmacologia , Adulto JovemRESUMO
Inflammation is a double-edged sword that can promote or suppress cancer progression. In this study, 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.
Assuntos
Neoplasias da Mama/imunologia , Citocinas/metabolismo , Neoplasias Mamárias Animais/imunologia , Animais , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/genética , Progressão da Doença , Feminino , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno , Células Th2/imunologia , Evasão Tumoral , Linfopoietina do Estroma do TimoRESUMO
Action mechanism of lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1ß resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1ß precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1ß treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1ß treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1ß was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1ß-treated cells. These results suggest that IL-1ß inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.
Assuntos
Astrócitos/metabolismo , Movimento Celular/fisiologia , Interleucina-1beta/fisiologia , Lisofosfolipídeos/farmacologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Interleucina-1beta/antagonistas & inibidores , Ratos , Ratos Wistar , Receptores de Interleucina-1/fisiologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Malignant ascites from pancreatic cancer patients has been reported to stimulate migration of pancreatic cancer cells through lysophosphatidic acid (LPA) and LPA(1) receptors. Indeed, ascites- and LPA-induced migration was inhibited by Ki16425, an LPA(1) and LPA(3) antagonist, in Panc-1 cells. Unexpectedly, however, in the presence of Ki16425, ascites and LPA inhibited cell migration in response to epidermal growth factor (EGF). The inhibitory migratory response to ascites and LPA was also observed in the cells treated with pertussis toxin (PTX), a G(i) protein inhibitor, and attenuated by a small interfering RNA (siRNA) specific to the LPA(2) receptor. The inhibitory LPA action was reversed by the regulators of G-protein signaling domain of p115RhoGEF, dominant-negative RhoA or C3 toxin. Indeed, LPA activated RhoA, which was attenuated by the siRNA against the LPA(2) receptor. Moreover, LP-105, an LPA(2) agonist, also inhibited EGF-induced migration in the PTX-treated cells. A similar inhibitory migration response through LPA(2) receptors was also observed in YAPC-PD, BxPC-3, CFPAC-1 and PK-1 pancreatic cancer cell lines. LPA also inhibited the invasion of Panc-1 cells in the PTX-treated cells in the in vitro Matrigel invasion assay. We conclude that LPA(2) receptors are coupled to the G(12/13) protein/Rho-signaling pathway, leading to the inhibition of EGF-induced migration and invasion of pancreatic cancer cells.
Assuntos
Ascite/metabolismo , Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Neoplasias Pancreáticas/metabolismo , Receptores de Ácidos Lisofosfatídicos/fisiologia , Ascite/patologia , Linhagem Celular Tumoral , Colágeno , Combinação de Medicamentos , Fator de Crescimento Epidérmico/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Isoxazóis/farmacologia , Laminina , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Toxina Pertussis/farmacologia , Propionatos/farmacologia , Proteoglicanas , RNA Interferente Pequeno/genética , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P(2) receptor, a carboxyl-terminal region of Galpha12 or Galpha13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P(2) receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P(2) receptors/G(12/13)-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P(2) receptor-mediated action in glioma cells.
Assuntos
Movimento Celular , Glioma/patologia , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Glioma/enzimologia , Humanos , Lisofosfolipídeos/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
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 ATP-binding 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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Astrócitos/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adenoviridae/genética , Animais , Apolipoproteínas E/biossíntese , Western Blotting , Bucladesina/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados , Vetores Genéticos , Lipoproteínas/biossíntese , Lipoproteínas HDL/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Esfingosina/metabolismo , Esfingosina/farmacologia , Estimulação Química , Tretinoína/farmacologiaRESUMO
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 signal-regulated 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 dominant-negative 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.
Assuntos
Movimento Celular/fisiologia , Glioma/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisofosfolipídeos/fisiologia , Esfingosina/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular Tumoral , Glioma/enzimologia , Glioma/metabolismo , Toxina Pertussis/farmacologia , Ratos , Esfingosina/fisiologiaRESUMO
High-density lipoprotein (HDL) is a well-established anti-risk factor against atherosclerosis, but the mechanism of its anti-atherogenic actions is not fully understood. Here, we examined the role of the HDL-associated sphingosine 1-phosphate (S1P), a lysolipid mediator, in the lipoprotein-induced actions in rat vascular smooth muscle cells (VSMCs). Both HDL and S1P inhibited platelet-derived growth factor-induced migration of VSMCs. The inhibitory effect was associated with an inhibition of cell spreading and these responses were reversed by a desensitization of VSMCs with S1P. HDL and S1P also inhibited migration of Chinese hamster ovary cells and this effect was enhanced by overexpressing S1P2 receptor. Finally, we showed that, even though S1P promoted DNA synthesis, HDL and S1P did not increase cell number of VSMCs. These findings suggest a novel mechanism for anti-atherogenic actions of HDL through its S1P component.
Assuntos
Lipoproteínas HDL/farmacologia , Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Animais , Células CHO , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , DNA/biossíntese , Lipoproteínas HDL/química , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos WistarRESUMO
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/S1P1 and EDG-3/S1P3. Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S1P1. 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. CONCLUSIONS: HDL-induced endothelial cell migration and survival may be mediated by the lipoprotein component S1P and the lipid receptors S1P1 and S1P3.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/fisiologia , Humanos , Lipoproteína(a)/farmacologia , Lipoproteínas LDL/farmacologia , Lisofosfolipídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisofosfolipídeos , Transdução de Sinais/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is an incurable and progressive neurodegenerative senile disorder associated with the brain accumulation of Aß plaques. Although vaccines that reduce Aß plaques can control AD, the rationale for their use at the onset of the disease remains debatable. Old humans and mice usually respond poorly to vaccines due to presumably age-related immunological impairments. Here, we report that by modifying vaccines, the poor responsiveness of old mice can be reversed. Unlike the Aß peptide vaccine, DNA immunizations with the amino-terminal Aß(1-11) fragment exposed on the surface of HBsAg particles elicit high levels of anti-Aß antibody both in young and old mice. Importantly, in AD model 3xTgAD mice, the vaccine reduced Aß plaques, ameliorated cognitive impairments and, surprisingly, significantly increased life span. Hence, we propose that vaccines targeting Aß(1-11) can efficiently combat AD-induced pathological alterations and provide survival benefit in patients with AD.
Assuntos
Doença de Alzheimer/prevenção & controle , Vacinas contra Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Fragmentos de Peptídeos/imunologia , Fatores Etários , Doença de Alzheimer/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologia , Vacinas de DNA/imunologiaRESUMO
Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) 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 (tBreg), phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-ß-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, as 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.
Assuntos
Linfócitos B/citologia , Neoplasias da Mama/metabolismo , Linfócitos T CD4-Positivos/citologia , Neoplasias Pulmonares/patologia , Linfócitos T Reguladores/citologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Transplante de NeoplasiasRESUMO
Low-density lipoprotein (LDL) and lysophosphatidic acid (LPA), one of the lipid components of lipoprotein, induced the DNA synthesis of coronary artery smooth muscle cells (CASMCs). The LDL- and LPA-induced DNA synthesis was markedly inhibited by the LPA receptor antagonist Ki16425, pertussis toxin, small interfering RNAs targeted for LPA1 receptors, and a potent calcineurin inhibitor cyclosporine A. It has been reported that LDL and LPA induced a migration response in a manner sensitive to Ki16425, pertussis toxin, and a LPA1 receptor-specific small interfering RNA. However, cyclosporine A was ineffective in inhibiting the migration response. Instead, an epidermal growth factor (EGF) receptor tyrosine kinase inhibitor markedly suppressed the migration response to LDL and LPA without having any significant effect on DNA synthesis. Thus, the LDL-induced stimulation of DNA synthesis and migration in CASMCs is mediated by its component LPA through LPA1 receptors and G(i/o)-proteins. Ca2+/calcineurin pathways and transactivation of EGF receptors mediate LPA1-receptor-induced DNA synthesis and migration, respectively.
Assuntos
Movimento Celular/fisiologia , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , DNA/biossíntese , Lipoproteínas LDL/fisiologia , Lisofosfolipídeos/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Transdução de Sinais/fisiologia , Calcineurina/fisiologia , Células Cultivadas , Receptores ErbB/fisiologia , Humanos , Receptores de Ácidos Lisofosfatídicos/agonistas , Ativação Transcricional/fisiologiaRESUMO
The clarification of mechanisms that negatively regulate the invasive behavior of human glioma cells is of great importance in order to find new methods of treatment. In this study, we have focused on the negative regulation of lysophosphatidic acid (LPA)-induced migration in glioma cells. Using small interference RNA and dominant-negative gene strategies in addition to pharmacological tools, we found that isoproterenol (ISO) and sphingosine-1-phosphate (S1P) negatively but differently regulate the LPA-induced migration. ISO-induced suppression of the migration of glioma cells occurs via beta(2)-adrenergic receptor/cAMP/Epac/Rap1B/inhibition of Rac, whereas S1P has been shown to suppress the migration of the cells through S1P(2) receptor/Rho-mediated down-regulation of Rac1. The expression of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is required for the inhibitory ISO-induced and Rap1B-mediated actions on the migration, Rac1 activation, and Akt activation in response to LPA. Thus, the PTEN-mediated down-regulation of phosphatidylinositol 3-kinase activity may be involved in the regulation of Rap1B-dependent inhibition of Rac1 activity. These findings suggest that there are at least two distinct inhibitory pathways, which are mediated by the S1P(2) receptor and beta(2)-adrenergic receptor, to control the migratory, hence invasive, behavior of glioma cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Glioma/enzimologia , Glioma/patologia , Isoproterenol/farmacologia , Lisofosfolipídeos/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
High-density lipoprotein (HDL)-associated sphingosine 1-phosphate mediates a variety of lipoprotein-induced actions in vascular cell systems. However, it remains unknown whether extracellular S1P is associated with lipoproteins to exert biological actions in central nervous system. Human cerebrospinal fluid (CSF) induced rat astrocyte migration in a manner sensitive to S1P receptor antagonist VPC23019 and the migration activity was recovered in S1P fraction by thin-layer chromatography. Density-gradient separation of CSF revealed that the major S1P activity was detected in the HDL fraction. In conditioned medium of rat astrocytes cultured with sphingosine, the S1P activity was recovered again in the HDL fraction. The HDL fraction also induced migration of astrocytes and process retraction of oligodendrocytes in a manner similar to S1P. We concluded that S1P is accumulated in HDL-like lipoproteins in CSF and mediates some of lipoprotein-induced neural cell functions in central nervous system.
Assuntos
Líquido Cefalorraquidiano , Lipoproteínas HDL/farmacologia , Lisofosfolipídeos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Esfingosina/análogos & derivados , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Humanos , Neurônios/citologia , Ratos , Esfingosina/metabolismoRESUMO
Abstract Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long-acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long-acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso-PLD). Although the lyso-PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso-PLD activity and an approximately 120-kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso-PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso-PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.
Assuntos
Glucose-6-Fosfato Isomerase/líquido cefalorraquidiano , Glucose-6-Fosfato Isomerase/fisiologia , Glicoproteínas/líquido cefalorraquidiano , Glicoproteínas/fisiologia , Complexos Multienzimáticos/líquido cefalorraquidiano , Complexos Multienzimáticos/fisiologia , Neuritos/metabolismo , Animais , Células Cultivadas , Cães , Humanos , Isoxazóis/farmacologia , Lisofosfolipídeos/farmacologia , Masculino , Neuritos/química , Neuritos/efeitos dos fármacos , Células PC12 , Fosfodiesterase I , Diester Fosfórico Hidrolases , Propionatos/farmacologia , Pirofosfatases , Ratos , Ratos Wistar , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
It has been suggested that lipoproteins in the central nervous system are involved in the regulation of several neural functions independent of cholesterol metabolism as well as those related to lipid metabolism. We recently demonstrated that lipoproteins are carriers for sphingosine 1-phosphate (S1P). This raised the possibility that S1P mediates the neural cell functions induced by lipoproteins. In the current study, we examined the effects of plasma high-density lipoprotein (HDL) on astroglial cell functions, focusing especially on the role of the lipoprotein-associated S1P. In rat type I astrocytes or C6 glioma cells, similar to S1P, HDL stimulated DNA synthesis and mRNA expression of fibroblast growth factor-2, a potent neurotrophic factor, which was associated with the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner. The data from fractionation studies of HDL indicated that S1P may be a major component for the activation of ERK. In C6 glioma cells, HDL also induced phospholipase C-dependent intracellular Ca(2+) mobilization. Desensitization of the C6 glioma cells with S1P abolished these HDL-induced actions. Furthermore, overexpression of S1P receptors in C6 glioma cells led to a significant enhancement of HDL-induced ERK activation and Ca(2+) mobilization. Thus, at least some HDL-induced actions may be mediated by cell-surface S1P receptors in astroglial cells. These results imply that S1P might partially mediate lipoprotein-induced cholesterol metabolism-independent neural cell functions in the central nervous system.
Assuntos
Astrócitos/metabolismo , Lipoproteínas HDL/metabolismo , Lisofosfolipídeos , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animais , Astrócitos/citologia , Cálcio/metabolismo , DNA/metabolismo , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Lipoproteínas VLDL/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Lisofosfolipídeos , Células Tumorais CultivadasRESUMO
Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA1, LPA2, and LPA3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA4/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 LPA1, LPA2, or LPA3, 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 LPA1 >/= LPA3 >> LPA2, whereas DGPP 8:0 preferentially inhibited the LPA3-induced actions. Ki16425 inhibited LPA-induced guanosine 5'-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 long-term responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA1 and LPA3; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.
Assuntos
Isoxazóis/farmacologia , Propionatos/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores Acoplados a Proteínas G , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HL-60 , Humanos , Isoxazóis/química , Propionatos/química , Receptores de Superfície Celular/fisiologia , Receptores de Ácidos LisofosfatídicosRESUMO
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 (LPA1 and LPA3), but not by an LPA3-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 LPA1 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 LPA1 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 LPA1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells.
Assuntos
Ascite/metabolismo , Lisofosfolipídeos/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animais , Northern Blotting , Adesão Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Cromatografia em Camada Fina , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Isoxazóis/farmacologia , Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Monoacilglicerol Lipases/farmacologia , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Toxina Pertussis/farmacologia , Propionatos/farmacologia , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
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, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing 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 pH-dependent action, although to a lesser extent. Psychosine-sensitive 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 protein-sensing receptors, including TDAG8, GPR4, and OGR1.