RESUMO
OBJECTIVE AND DESIGN: This study is aimed at uncovering the signaling pathways activated by vasoactive intestinal peptide in human macrophages MATERIALS: Human peripheral blood mononuclear cell-derived macrophages were used for the in vitro investigation of the VIP-activated signaling pathways. METHODS AND TREATMENT: Time-course and dose-response experiments and siRNA were used in human macrophages co-challenged with various concentrations of VIP and different MAPK pharmacologic inhibitors to investigate signaling pathways activated by VIP. Flow analysis was performed to assess the levels of CD11b, CD35 and CD66. Luminescence spectrometry was used to measure the levels of the released hydrogen peroxide and the intracellular calcium levels in the media. RESULTS: Macrophages incubated with VIP showed increased phospho-AKT and phospho-ERK1/2 levels in a GTP-RhoA-GTPase-dependent manner. Similarly, VIP increased intracellular release of H2O2 and calcium via PLC and GTP-RhoA-GTPase, in addition to inducing the expression of CD11b, CD35, CD66 and MMP9. Furthermore, VIP activated P38 MAPK through the cAMP/PKA pathway but was independent of both PLC and RhoA signaling. The above-mentioned VIP effects were mediated via activation of the FPRL1 receptor. CONCLUSION: VIP/FPRL1/VPAC/GTP-RhoA-GTPase signaling modulated macrophages phenotype through activation of multiple signaling pathways including ERK1/2, AKT, P38, ROS, cAMP and calcium.
Assuntos
Macrófagos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Aerobic glycolysis, known as the "Warburg effect", is one of several hallmarks of cancer cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate can be down regulated by the re-expression of the embryonic isoform 2 of pyruvate kinase (PKM2). This mechanism allows the accumulation of glycolytic intermediates for the biosynthesis of macromolecules, such as proteins, lipids and nucleic acids. PKM2 is favored by the well-known PI3K/Akt/mTOR proliferative pathway. This pathway is induced by high glucose levels, and the mTOR kinase is the central activator of the Warburg effect. In this study, we investigated the role of glucose restriction (GR) and mTOR inhibition in reversing the Warburg effect in MDA-MB 231 and MCF-7 breast cancer cell lines. PKM2 expression was measured by western blot. Lactate production by cells was determined by a colorimetric assay. The concentration of glucose in the supernatant of cells was measured using the Trinder method. ATP level was evaluated by using a Colorimetric/Fluorometric ATP Assay Kit. Our results showed that MDA-MB 231 cells increased glucose consumption when the glucose concentration was 0 g/L (P <0.01). In MCF-7 cells, glucose deprivation reduced lactate secretion by 80% (P =0.0001) but tripled glucose consumption (P = 0.0041). ATP concentration increased approximately when MCF-7 cells were deprived of glucose (P = 0.02). GSK1059615 does not significantly modulate lactate secretion and glucose uptake in both cell lines. Glucose restriction contribute to the reduction of the Warburg effect through mTOR inhibition and regulation of PKM2 kinases.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Hormônios Tireóideos/metabolismo , Trifosfato de Adenosina/metabolismo , Aminopiridinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Colorimetria , Regulação para Baixo/efeitos dos fármacos , Citometria de Fluxo , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Piperidinas/farmacologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+ CD25 + CD127 low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 + CD25 + CD127 low Tregs in comparison to CD8 + CD25 - T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3'-untranslated region (3'-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Having identified potential miR target sites in the 3'-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-ß (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3'-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-ß expression. These results highlighted an important impact of the CD8 + Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Expressão Gênica/imunologia , Interleucina-10/metabolismo , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/imunologia , Humanos , MicroRNAs/genéticaRESUMO
OBJECTIVE AND DESIGN: The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte. MATERIALS OR SUBJECTS: Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP. TREATMENT: Time-course and dose-response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases. METHODS: Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media. RESULTS: fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H2O2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H2O2-induced activation of Akt and RhoA-GTPase. CONCLUSION: Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.
Assuntos
Peróxido de Hidrogênio/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Humanos , Leucócitos Mononucleares/metabolismo , RatosRESUMO
PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20â¯min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H2O2 production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.
Assuntos
Neoplasias da Mama/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Cálcio/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Células MCF-7 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Interferon gamma (IFN-É£) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-É£ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-É£ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-É£, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-É£ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-É£ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-É£ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.
RESUMO
EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt's lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10-20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5-10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Neoplasias Hematológicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/genética , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Cancer cells secrete VEGF, which plays a key role in their growth, invasion, extravasation and metastasis. Direct cancer cell-endothelial cell interaction, mediated by gap junctions, is of critical importance in the extravasation process. In this study, we evaluated avastin (Av), an anti-VEGF antibody; and oleamide (OL), a gap junction inhibitor, using MDA-MB-231 human breast cancer cells in vitro and a xenograft murine model in vivo. Results showed that Av/OL significantly decreased proliferation, induced cell cycle arrest and decreased migration and invasion of MDA-MB-231 cells in vitro. In addition, Av/OL significantly decreased homo and hetero-cellular communication interaction between MDA-MDA and MDA-endothelial cells, respectively. The expression levels of several factors including VEGF, HIF1α, CXCR4, Cx26, Cx43, and MMP9 were attenuated upon Av/OL treatment in vitro. On the other hand, avastin, but not oleamide, reduced tumor size of NSG mice injected subdermally (s.d.) with MDA-MB-231 cells, which was also associated with increased survival. Furthermore, Av but also OL, separately, significantly increased the survival rate, and reduced pulmonary and hepatic metastatic foci, of intravenously (i.v.) injected mice. Finally, OL reduced MMP9 protein expression levels, better than Av and in comparisons to control, in the lungs of MDA-MB-231 i.v. injected NSG mice. In conclusion, while avastin has anti-angiogenic, anti-tumor and anti-metastatic activities, oleamide has anti-metastatic activity, presumably at the extravasation level, providing further evidence for the role of gap junction intercellular communication (GJIC) in cancer cell extravasation.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Invasividade Neoplásica/prevenção & controle , Neovascularização Patológica/tratamento farmacológico , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Conexina 26 , Conexina 43/metabolismo , Conexinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Junções Comunicantes/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Neovascularização Patológica/metabolismo , Receptores CXCR4/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The G-protein coupled receptor (GPCR) fMLP receptor (FPR) and the two receptors tyrosine kinase (RTK), the nerve growth factor (NGF) receptor TrkA and the epidermal growth factor (EGF) receptor (EGFR) are involved in reactive oxygen species (ROS), matrix metalloproteinase-9 (MMP-9) production and CD11b membrane integrin upregulation. We show that in monocytes the three receptors crosstalk each other to modulate these pro-inflammatory mediators. Tyrphostin AG1478, the EGFR inhibitor, inhibits fMLP and NGF-associated ROS production, fMLP-associated CD11b upregulation and NGF-induced TrkA phosphorylation; K252a, the NGF receptor inhibitor, inhibits fMLP or EGF-associated ROS production, CD11b expression and EGF-induced EGFR phosphorylation; cyclosporine H, the FPR inhibitor inhibits EGF or NGF-associated ROS production, EGF-associated CD11b upregulation and prevents EGFR and TrkA phosphorylation by their respective ligand EGF and NGF. In response to fMLP, TrkA phosphorylation is inhibited by the EGFR inhibitor while EGFR phosphorylation is inhibited by the TrkA inhibitor. Receptor crosstalks are Src and ERK dependent. Down-regulation of each receptor by specific siRNA suppresses the ability of the two other receptors to promote ligand-mediated ERK phosphorylation and pro-inflammatory activities including ROS, MMP-9 production and CD11b upregulation. Thus, in monocytes GPCR ligands' activity involves activation of RTK while RTK-ligands activity engages GPCR-signalling molecules.
Assuntos
Receptores ErbB/metabolismo , Monócitos/metabolismo , Receptor Cross-Talk , Receptor trkA/metabolismo , Receptores de Formil Peptídeo/metabolismo , Antígeno CD11b/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/metabolismo , Monócitos/enzimologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fator de Crescimento Neural/farmacologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/genética , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/genéticaRESUMO
Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Sangue Fetal/imunologia , Fatores de Transcrição Forkhead/genética , Histona Desacetilases/genética , Ácido Valproico/farmacologia , Regiões 5' não Traduzidas/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Sequência Conservada , Primers do DNA , Histona Desacetilases/imunologia , Humanos , Recém-Nascido , MicroRNAs/genética , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , TransfecçãoRESUMO
Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor kappa B and beta-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electrophoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce T(H)1-type responses.
Assuntos
Vacinas Anticâncer , Técnicas de Cultura de Células/métodos , Núcleo Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , NF-kappa B/metabolismo , Transporte Ativo do Núcleo Celular , Apresentação de Antígeno , Antígenos de Diferenciação/metabolismo , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Células Cultivadas , Células Dendríticas/metabolismo , Humanos , Separação Imunomagnética , Imunofenotipagem , Interferon gama/metabolismo , Ativação Linfocitária , Análise em Microsséries , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The CD4+ T-cell malignancy induced by human T-cell leukemia virus type 1 (HTLV-I) infection and termed; Adult T-cell Leukemia lymphoma (ATLL), is caused by defects in the mechanisms underlying cell proliferation and cell death. In the CD4+ T-cells, calcium ions are central for both phenomena. ATLL is associated with a marked hypercalcemia in many patients. The consequence of a defect in the Ca2+ signaling pathway for lymphocyte activation is characterized by an impaired NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defense. Fresh ATLL cells lack the TCR/CD3 and CD7 molecules on their surface. Whereas CD7 is a calcium transporter, reduction in calcium influx in response to T-cell activation was reported as a functional consequence of TCR/CD3 expression deficiency. Understanding these changes and identifying the molecular players involved might provide further insights on how to improve ATLL treatment.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Cálcio/metabolismo , Transformação Celular Neoplásica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the beta2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.
Assuntos
Acetilcisteína/análogos & derivados , Antígenos CD18/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/sangue , Eritromicina/análogos & derivados , Metaloproteinase 9 da Matriz/sangue , Monócitos/fisiologia , Neutrófilos/fisiologia , Receptores de Complemento 3b/fisiologia , Receptores de Formil Peptídeo/sangue , Receptores de Lipoxinas/sangue , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/sangue , Peptídeo Intestinal Vasoativo/farmacologia , Acetilcisteína/sangue , Animais , Antígenos CD18/efeitos dos fármacos , Células CHO , Cálcio/fisiologia , Linhagem Celular , Cricetinae , Cricetulus , AMP Cíclico/fisiologia , Eritromicina/sangue , Humanos , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Receptores de Complemento 3b/efeitos dos fármacos , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Transdução de Sinais , TransfecçãoRESUMO
In human neutrophils, the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) acting via the G protein-coupled receptors vasoactive intestinal peptide/PACAP receptor 1 (VPAC-1) and formyl peptide receptor-like 1 (FPRL1) modulates Ca2+ and pro-inflammatory activities. We evaluated in human monocytes the importance of the Ca2+ signal and the participation of FPRL1 in PACAP-associated signaling pathways and pro-inflammatory activities. PACAP-evoked Ca2+ transient involved both Ca2+ influx and intracytoplasmic Ca2+ mobilisation. This was pertussis toxin, protein kinase A and adenylate cyclase dependent indicating the participation of Galphai and Galphas with mobilisation of both InsP3 sensitive and insensitive stores. Intra- or extracellular Ca2+ depletion resulted in the inhibition of PACAP-induced, Akt, ERK, p38 and NF-kappaB activations as well as a decrease in PACAP-associated reactive oxygen species (ROS) production and integrin CD11b membrane upregulation. The FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp (WRW4), decreased PACAP-evoked Ca2+ signal, Akt, ERK phosphorylation, ROS and CD11b upregulation without affecting p38 phosphorylation. NF-kappaB inhibitors prevented PACAP-induced Ca2+ mobilisation. Monocytes pre-treatment with fMLP but not with LPS desensitised cells to the pro-inflammatory effects of PACAP. Thus, both intra- and extracellular Ca2+ play a role in controlling pro-inflammatory functions stimulated by PACAP which acts through a VPAC-1, FPRL1/Galphai/PI3K/ERK pathway and a VPAC-1/Galphas/PKA/p38 pathway to fully activate monocytes.
Assuntos
Cálcio/metabolismo , Inflamação/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Antígeno CD11b/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/enzimologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.
Assuntos
Monócitos/fisiologia , Fator de Crescimento Neural/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptor trkA/fisiologia , Ativação Transcricional , Antígeno CD11b/metabolismo , Carbazóis/farmacologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Alcaloides Indólicos , Fosforilação , Receptor trkA/antagonistas & inibidores , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de SinaisRESUMO
Pituitary adenylate cyclase activating protein (PACAP) and its structurally related vasointestinal peptide (VIP) bind to three G-protein-coupled receptors named VPAC1 and VPAC2 for VIP/PACAP receptors and PAC1 for PACAP preferred receptors. We report that in freshly isolated human monocytes PACAP acts as a pro-inflammatory molecule. By RT-PCR, VPAC1 mRNA was the only receptor found to be expressed; VPAC1 protein was detected by Western blotting and visualized by immunohistochemistry. Signaling pathways activated by PACAP include the extracellular regulated kinase (ERK), the stress-activated MAPK p38, the focal adhesion kinase, Pyk2 and its associated cytoskeleton protein paxillin and the phosphatidylinositol 3-kinase (PI-3K). PACAP induces a transient peak in cytoplasmic calcium associated with an increase in reactive oxygen species production and upregulation in membrane expression of the integrin CD11b as well as the complement receptor 1. Control of the different pathways and functions stimulated by PACAP were evaluated using Phospholipase C (PLC), PI-3K, ERK and p38 MAPK inhibitors and led to the conclusion that PLC and to a lesser degree PI-3K activation are upstream events occurring in VPAC1 mediated PACAP stimulation of monocytes and are in contrast to ERK and p38 mandatory for the initiation of other cellular events associated with monocytes activation.