RESUMO
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single ß-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-κB and inducible nitric oxide synthase (iNOS). In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor mammalian Sin3A (mSin3A). Furthermore, mSin3A, histone deacetylase (HDAC)1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A, and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor trichostatin A (TSA) on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases, p300, and cAMP response element-binding protein-binding protein (CBP) enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-κB activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.
Assuntos
Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Repressoras/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Camundongos , N-Acetilglucosaminiltransferases/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição GênicaRESUMO
Expression of inducible nitric oxide synthase (iNOS) protein by lipopolysaccharide (LPS) in BV2 microglia cells increased in a biphasic manner. Glucosamine (GlcN) selectively suppressed the late- but not early-stage iNOS response to LPS. Prolonged induction of iNOS expression by LPS was inhibited by cycloheximide, suggesting that de novo protein synthesis was required. Late-phase activation of nuclear factor-kappaB (NF-κB) activity required for sustained iNOS induction. Nuclear translocation and DNA binding of NF-κB, and Rel proteins expressions were inhibited by GlcN at later time points but not upon immediate early-stage activation by LPS. We show that GlcN selectively inhibits sustained iNOS induction by inhibiting Rel protein expression at both the mRNA and protein levels; such expression is required for prolonged iNOS induction by LPS. Our results provide mechanistic evidence that GlcN regulates inflammation, represented by iNOS. The implication of these results is that GlcN may be a potent transcriptional regulator of iNOS and other genes involved in the general inflammation process.
Assuntos
Glucosamina/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Oncogênicas v-rel/antagonistas & inibidores , Animais , Western Blotting , Linhagem Celular , Indução Enzimática/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/biossíntese , NF-kappa B/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Oncogênicas v-rel/biossíntese , Proteínas Oncogênicas v-rel/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
DDR1 (discoidin domain receptor tyrosine kinase 1) kinase s highly expressed in a variety of human cancers and occasionally mutated in lung cancer and leukemia. It is now clear that aberrant signaling through the DDR1 receptor is closely associated with various steps of tumorigenesis, although little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. Besides the role of DDR1 in tumorigenesis, we previously identified DDR1 kinase as a transcriptional target of tumor suppressor p53. DDR1 is functionally activated as determined by its tyrosine phosphorylation, in response to p53-dependent DNA damage. In this study, we report the characterization of the Notch1 protein as an interacting partner of DDR1 receptor, as determined by tandem affinity protein purification. Upon ligand-mediated DDR1 kinase activation, Notch1 was activated, bound to DDR1, and activated canonical Notch1 targets, including Hes1 and Hey2. Moreover, DDR1 ligand (collagen I) treatment significantly increased the active form of Notch1 receptor in the nuclear fraction, whereas DDR1 knockdown cells show little or no increase of the active form of Notch1 in the nuclear fraction, suggesting a novel intracellular mechanism underlying autocrine activation of wild-type Notch signaling through DDR1. DDR1 activation suppressed genotoxic-mediated cell death, whereas Notch1 inhibition by a γ-secretase inhibitor, DAPT, enhanced cell death in response to stress. Moreover, the DDR1 knockdown cancer cells showed the reduced transformed phenotypes in vitro and in vivo xenograft studies. The results suggest that DDR1 exerts prosurvival effect, at least in part, through the functional interaction with Notch1.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/fisiologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Dano ao DNA/fisiologia , Dipeptídeos/farmacologia , Receptor com Domínio Discoidina 1 , Ativação Enzimática/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fosforilação/fisiologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptor Notch1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição HES-1 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Microglia contributes significantly to brain tumor mass, particularly in astrocytic gliomas. Here, we examine the cytotoxic effects of soluble components secreted from microglia culture on glioma cells. Microglia conditioned culture medium (MCM) actively stimulated apoptotic death of glioma cells, and the effects of MCM prepared from LPS- or IFN-gamma-activated microglia were more pronounced. The cytotoxic effects were glioma-specific in that primary cultured rat astrocytes were not affected by MCM. A donor of peroxynitrite induced glioma-specific cell death. In addition, NO synthase inhibitor suppressed glioma cell death induced by activated MCM, indicating that NO is one of the key molecules responsible for glioma cytotoxicity mediated by activated MCM. However, since unstimulated resting microglia produces low or very limited level of NO, MCM may contain other critical molecule(s) that induce glioma apoptosis. To identify the proteins secreted in MCM, proteomic analysis was performed on control or activated medium. Among over 200 protein spots detected by Coomassie blue staining, we identified 26 constitutive and 28 LPS- or IFN-gamma-regulated MCM proteins. Several cathepsin proteases were markedly expressed, which were reduced upon activation. In particular, suppression of cathepsin B by the chemical inhibitors significantly reversed MCM-induced glioma cell death, implying a critical role of this protease in cytotoxicity. Our findings provide evidence on the functional implications of specific microglial-secreted proteins in glioma cytotoxicity, as well as a basis to develop a proteomic databank of both basal and activation-related proteins in microglia.
Assuntos
Apoptose , Catepsina B/metabolismo , Glioma/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Animais , Antivirais/farmacologia , Catepsina B/antagonistas & inibidores , Meios de Cultivo Condicionados/farmacologia , Inibidores Enzimáticos/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the neuroprotective effect of glucosamine (GlcN) in a rat middle cerebral artery occlusion model. At the highest dose used, intraperitoneal GlcN reduced infarct volume to 14.3% ± 7.4% that of untreated controls and afforded a reduction in motor impairment and neurological deficits. Neuroprotective effects were not reproduced by other amine sugars or acetylated-GlcN, and GlcN suppressed postischemic microglial activation. Moreover, GlcN suppressed lipopolysaccharide (LPS)-induced upregulation of proinflammatory mediators both in vivo and in culture systems using microglial or macrophage cells. The anti-inflammatory effects of GlcN were mainly attributable to its ability to inhibit nuclear factor kappaB (NF-κB) activation. GlcN inhibited LPS-induced nuclear translocation and DNA binding of p65 to both NF-κB consensus sequence and NF-κB binding sequence of inducible nitric oxide synthase promoter. In addition, we found that GlcN strongly repressed p65 transactivation in BV2 cells using Gal4-p65 chimeras system. P65 displayed increased O-GlcNAcylation in response to LPS; this effect was also reversed by GlcN. The LPS-induced increase in p65 O-GlcNAcylation was paralleled by an increase in interaction with O-GlcNAc transferase, which was reversed by GlcN. Finally, our results suggest that GlcN or its derivatives may serve as novel neuroprotective or anti-inflammatory agents.
Assuntos
Encefalite/tratamento farmacológico , Encefalite/etiologia , Glucosamina/uso terapêutico , Infarto da Artéria Cerebral Média/complicações , Fármacos Neuroprotetores/uso terapêutico , Animais , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/etiologia , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina/métodos , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Ratos , Índice de Gravidade de Doença , Sais de Tetrazólio , Transfecção/métodosRESUMO
Radiotherapy remains a major therapeutic option for patients with advanced lung cancer. Nevertheless, the effects of irradiation on malignant biological behaviours (e.g. migration and transformation of cancer cells) have yet to be clarified. We conducted an in vitro study to investigate the radiation-induced alterations including morphology, adhesion, and cell motility of A549 human lung cancer cells. These changes, which are associated with epithelial-mesenchymal transdifferentiation (EMT), seem to be linked to radiation-induced fibrosis, which represents one of the most common long-term adverse effects of curative radiotherapy. In addition, loss of intercellular adhesion and increased cell motility may be involved in post-radiotherapy-associated metastasis. We showed that stress fibres and focal adhesions are increased and that cell-cell junctions are decreased in response to ionising radiation. Radiation also significantly increased cell motility. The p38-specific inhibitor, SB203580, reduced the radiation-promoted migration of A549 cells, whereas SP600125, a JNK MAPK-specific inhibitor, inhibited both inherent and radiation-mediated cell motility. Consistent with this observation, radiation up-regulated the phosphorylation of p38 MAPK. Current approaches to cancer treatment involving more intensive radiotherapy regimens have been suggested to be associated with a higher incidence of local or distant metastasis. Therefore, a subset of patients may benefit from a combination of radiotherapy with inhibitors of EMT or cell migration.
Assuntos
Adenocarcinoma/patologia , Movimento Celular/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Células Epiteliais/efeitos da radiação , Neoplasias Pulmonares/patologia , Actinas/metabolismo , Adesão Celular/efeitos da radiação , Células Epiteliais/patologia , Quinase 1 de Adesão Focal/metabolismo , Humanos , Paxilina/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Radiação Ionizante , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The Wnt/ß-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of ß-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic ß-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/ß-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/ß-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in vitro and in mouse cancer models. MSAB binds to ß-catenin, promoting its degradation, and specifically downregulates Wnt/ß-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/ß-catenin signaling pathway.
Assuntos
Benzoatos/farmacologia , Oncogenes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , meta-Aminobenzoatos/farmacologia , Animais , Benzoatos/química , Linhagem Celular Tumoral , Camundongos , Sulfonamidas/química , Ensaios Antitumorais Modelo de Xenoenxerto , meta-Aminobenzoatos/químicaRESUMO
TP53 is the most frequently mutated gene in human cancer, and small-molecule reactivation of mutant p53 function represents an important anticancer strategy. A cell-based, high-throughput small-molecule screen identified chetomin (CTM) as a mutant p53 R175H reactivator. CTM enabled p53 to transactivate target genes, restored MDM2 negative regulation, and selectively inhibited the growth of cancer cells harboring mutant p53 R175H in vitro and in vivo. We found that CTM binds to Hsp40 and increases the binding capacity of Hsp40 to the p53 R175H mutant protein, causing a potential conformational change to a wild-type-like p53. Thus, CTM acts as a specific reactivator of the p53 R175H mutant form through Hsp40. These results provide new insights into the mechanism of reactivation of this specific p53 mutant.
Assuntos
Antineoplásicos/farmacologia , Dissulfetos/farmacologia , Proteínas de Choque Térmico HSP40/metabolismo , Alcaloides Indólicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Dissulfetos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Ensaios de Triagem em Larga Escala , Humanos , Alcaloides Indólicos/química , Camundongos , Camundongos Nus , Mutação , Bibliotecas de Moléculas Pequenas/química , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.
Assuntos
Apoptose/imunologia , Proteínas de Membrana/metabolismo , Fagocitose/imunologia , Fosfatidilserinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Antígenos B7 , Linhagem Celular Tumoral , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Transdução de SinaisRESUMO
AIMS: The objective of this study is to investigate glucosamine (GlcN) as a transcriptional regulator of iNOS and other genes in association with the dynamic O-GlcNAcylation of RNA polymerase II (RNAPII). MAIN METHODS: The LPS- and/or GlcN-stimulated transcriptional activities of various Gal4-binding site/TATA-box-containing reporter constructs were measured. KEY FINDINGS: Basal transcriptional activities of nuclear factor-κB (NF-κB) and nitric oxide synthase (iNOS) reporter plasmids are inhibited by GlcN in RAW264.7 cells. Furthermore, GlcN suppressed whereas lipopolysaccharide (LPS) stimulated the basal activity of Gal4-binding site/TATA-box-containing reporter constructs. LPS reduced the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of RNAPII, but enhanced the binding of this enzyme to the iNOS promoter. In contrast, GlcN enhanced RNAPII O-GlcNAcylation, but inhibited iNOS promoter binding. Furthermore, the basal activities of reporter plasmids containing activator protein 1 (AP1), E2F, or cyclic AMP response element (CRE) binding sites were consistently inhibited by GlcN in a dose-dependent manner. However, GlcN did not inhibit the phorbol 12-myristate 13-acetate- (PMA-) or forskolin-induced transcriptional activities of AP1 and CRE. The transcriptional activity of transforming growth factor alpha (TGF-α) was slightly increased by both LPS and GlcN. SIGNIFICANCE: In conclusion, our data demonstrate that LPS activates, whereas GlcN suppresses, basal activities of transcription through the regulation of RNAPII O-GlcNAcylation and DNA binding.
Assuntos
DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucosamina/metabolismo , Lipopolissacarídeos/metabolismo , RNA Polimerase II/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Análise de Variância , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Fatores de Transcrição E2F/metabolismo , Galactosiltransferases , Glucosamina/genética , Glucosamina/fisiologia , Immunoblotting , Luciferases , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Elementos Reguladores de Transcrição/genética , Fator de Transcrição AP-1/metabolismo , Aglutininas do Germe de TrigoRESUMO
BACKGROUND AND PURPOSE: Previously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on LPS-induced inducible NOS (iNOS) through the inhibition of NF-κB activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation. EXPERIMENTAL APPROACH: BV2 cells were stimulated with LPS with or without GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interactions were examined by co-immunoprecipitation. KEY RESULTS: LPS stimulated the activation of c-Rel, increased the O-GlcNAcylation of c-Rel and enhanced the binding of c-Rel to the NF-κB site in the iNOS promoter; GlcN attenuated these effects of LPS. O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel was increased by LPS and reduced by GlcN. LPS increased the interaction of c-Rel with O-GlcNAc transferase (OGT) and p50/p105, and GlcN suppressed these interactions. Knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, but did not affect either the binding of c-Rel to the iNOS promoter or the transcriptional activity of c-Rel. CONCLUSIONS AND IMPLICATIONS: In BV2 microglial cells, the anti-inflammatory effect of GlcN is mediated by prevention of the prolonged activation of transcription factors, c-Rel and NF-κB. Further clarification of the mechanism by which GlcN exerts this effect will facilitate the development of pharmacological strategies for preventing excessive NO formation when targeting inflammatory diseases of the periphery or CNS.
Assuntos
Acilação/efeitos dos fármacos , Glucosamina/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Glucosamina/química , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras GenéticasRESUMO
Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismoRESUMO
Microglia are the primary central nervous system immune effector cells. Microglial activation is linked to interactions with extracellular cytokines and the extracellular matrix (ECM). Astrocytomas are characterized by their diffuse nature, which is regulated by insoluble ECM components produced by the tumor cells that are largely absent from normal central nervous system tissue. The present study examined the influence of astrocytoma (C6 rat glioma) insoluble matrix components on interferon-gamma (IFN-gamma)-mediated inducible nitric-oxide synthase (iNOS) induction in microglial cells. We found that IFN-gamma-stimulated iNOS induction and nitric oxide release was greater in microglia cultured on C6 glioma cell-derived matrices compared with microglia cultured on primary rat astrocyte-derived matrices. Culture of microglia on C6 glioma cell-derived matrices also led to activation of STAT1, augmentation of IFN-gamma-induced STAT-3 activation, and an increase in IFN-gamma-activated site (GAS)-luciferase reporter activity. In addition, culture of microglia on C6 glioma cell-derived matrices activated NF-kappaB DNA binding activity and transcriptional activity. The results suggest that insoluble matrix components derived from malignant glioma cells can regulate microglia activation. These factors may include ECM components, such as fibronectin, collagen, laminin, vitronectin, and other nondiffusible compounds, and laminin seems to a critical regulator of this process. Microglia activation and subsequent brain inflammation may influence tumor growth, treatment, and metastasis. Better understanding of the regulation of microglial activation by astrocytoma-derived insoluble matrix components may be important in the development of immune-based treatment strategies against malignant brain tumors.
Assuntos
Interferon gama/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/biossíntese , Animais , Sequência de Bases , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Primers do DNA/genética , Indução Enzimática/efeitos dos fármacos , Matriz Extracelular/metabolismo , Glioma/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
This study examines the influence of insoluble matrix components of glioma (astrocytoma) cells on LPS-mediated inducible nitric oxide (NO)/NO synthase (iNOS) induction in microglia cells. Insoluble matrix components prepared from C6 rat glioma cells strongly suppressed iNOS induction and subsequent NO release induced by LPS. Matrices prepared from several glioma cell lines displayed similar inhibitory effects on LPS-induced NO/iNOS induction, whereas matrices from primary cultured rat astrocytes had a minimal influence. Of the various purified ECM materials examined, collagen suppressed LPS-mediated iNOS/NO induction in microglia. C6 matrices potentiated LPS-induced NF-kappaB DNA binding/transcriptional activity, suggesting that the suppression of LPS-induced iNOS by C6 matrices is NF-kappaB independent. C6 matrices inhibited LPS-mediated activation of p38 and JNK MAP kinases. This study shows that non-diffusible factors derived from astrocytoma cells in the brain are critically involved in the suppression of microglial cell activation. Our results indicate that activation of microglia can be regulated by various cellular and pathological environmental conditions, not only through cell-cell contact or soluble factors, but also via insoluble matrix components.
Assuntos
Extratos Celulares/farmacologia , Glioma/química , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Solubilidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In the present study we sought to examine cell-cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1beta were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1beta was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1beta was shown to be a key regulator of iNOS induction. An IL-1beta-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1beta stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1beta had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-kappaB) expression plasmids induced iNOS, and IL-1beta further enhanced the MEKK1 response. Furthermore, IL-1beta-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-kappaB by specific chemical inhibitors. Both IL-1beta and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-kappaB pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia-glioma or microglia-astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases.
Assuntos
Interleucina-1beta/metabolismo , Microglia/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrocitoma , Western Blotting/métodos , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção/métodosRESUMO
Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma.
Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular/efeitos da radiação , Glioma/patologia , Proteínas rac de Ligação ao GTP/genética , Animais , MAP Quinase Quinase 4/metabolismo , Invasividade Neoplásica , Tolerância a Radiação , Radiação Ionizante , Ratos , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac de Ligação ao GTP/fisiologiaRESUMO
The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.
Assuntos
Astrócitos/efeitos da radiação , Gliose/etiologia , Microglia/efeitos da radiação , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/metabolismo , Dinoprostona/efeitos da radiação , Ensaio de Desvio de Mobilidade Eletroforética , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Nitrobenzenos/farmacologia , RNA Mensageiro/análise , Radiação Ionizante , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologiaRESUMO
Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.