RESUMO
BACKGROUND: Extracellular nucleotides released in conditions of cell stress alert the immune system from tissue injury or inflammation. We hypothesized that the P2X7 receptor (P2X7-R) could regulate key elements in inflammatory bowel disease pathogenesis. METHODS: Colonoscopy samples obtained from patients with Crohn's disease (CD), ulcerative colitis, and controls were used to analyze P2X7-R expression by RT and real-time PCR, immunohistochemistry, and confocal microscopy. Inflammatory response was determined by the levels of cytokines by enzyme-linked immunosorbent assay in cultures of intestinal explants. Apoptosis was determined by the TUNEL assay. P2X7-R C57BL/6 mice were treated with trinitrobenzene sulfonic acid or dextran sulfate sodium (DSS) for inducing colitis. RESULTS: P2X7-R was expressed in higher levels in inflamed CD epithelium and lamina propria, where it colocalizes more with dendritic cells and macrophages. Basal levels of P2X7-R mRNA were higher in CD inflamed mucosa compared with noninflamed CD and controls and were upregulated after interferon-γ in controls. Apoptotic rates were higher in CD epithelium and lamina propria compared with ulcerative colitis and controls. Levels of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-17 were higher, whereas IL-10 was lower in CD compared with controls. Levels of tumor necrosis factor-α-α and interleukin-1ß increased after adenosine-triphosphate and decreased after KN62 treatment in CD. P2X7-R animals did not develop trinitrobenzene sulfonic acid or DSS colitis. CONCLUSIONS: The upregulation of P2X7-R in CD inflamed mucosa is consistent with the involvement of purinoceptors in inflammation and apoptosis. These observations may implicate purinergic signaling in the pathogenesis of intestinal inflammation, and the P2X7-R may represent a novel therapeutic target in CD.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Mucosa Intestinal/metabolismo , Receptores Purinérgicos P2X7/fisiologia , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Animais , Apoptose , Western Blotting , Estudos de Casos e Controles , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colonoscopia , Doença de Crohn/genética , Doença de Crohn/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Seguimentos , Humanos , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted ß-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas Hedgehog/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Butiratos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Neoplasias do Colo/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Células HT29 , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/genética , Humanos , Interleucina-8/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transativadores/genética , Transativadores/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Introdução: A via hedgehog (Hh) está envolvida na embriogênese e em processos fisiológicos incluindo sobrevivência e proliferação celular. Objetivos: células de carcinoma de cólon humano HT-29 foram utilizadas parainvestigar a via de sinalização Hh e as funções biológicas nas células epiteliais colônicas. Métodos: Foram realizadas culturas de células HT-29 sob diferentes condições e exposição a vários estímulos. A expressão dos componentes da via de sinalização Hh, proteínas e genes relacionados, foram avaliados por munofluorescência e por reação de cadeia de polimerase (PCR) em tempo real. Viabilidade, proliferação celular e apoptose foram avaliados, pelo método de 3-(4,5-Dimetiltiazol-2-yl)- ,5-difeniltetrazolium bromida, tetrazol (MTT), por incorporação de Brom odeoxiuridina (BrdU) e pela reação de Annexina - V/7- AAD, respectivamente. A produção de quimiocinas no sobrenadante de culturas de células HT -29 foi avaliada por reação imunoenzimática (ELISA). Resultados: Níveis de RNA mensageiro de Indian e Sonic Hh e o s fatores de transcrição Gli-1 e Gli-2 aumentaram após tratamento com agonistas Hh e butirato,masdiminuiramapós exposição a iclopamina. A expressão de proteínas orfogenéticas 4 e 7 (BMP4 e BMP7)diminui após o bloqueio da via Hh com ciclopamina. A expressão da proteína Gli-1 aumenta após exposição com agonistas e diminui após ciclopamina. A exposição a agonistas Hh promove a redução da β-catenina e causa sua redistribuição subcelular. Níveis de interleucina 8 (IL-8) e proteína quimiotática de monócitos (MCP-1) diminuem após exposição a agonistas Hh quando comparados a ciclopamina, lipopolissacarídeos (LPS), interferon (IFN-γ) ou fator de crescimento epitelial (EGF). A proliferação e a viabilidade celular diminuem após o bloqueio da via Hh. Agonistas Hh revogaram a apoptose induzida pelo anticorpo anti-CD95. Conclusão: A via Hh é um controlador fundamental das células epiteliais colônicas...
Background & Aims: The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. Methods: The human colon carcinoma HT-29 cell line was used to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components, related proteins and genes were assessed by immunofluorescence and protein chain reaction real -time (PCR). Viability, cell proliferation and apoptosis were measured by the 3-(4,5 Dimethylthiazol -2-yl)-,5 diphenyltetrazolium bromide, tetrazole (MTT) assay, Bromodeoxiuridine (BrdU) uptake and Annexin-V/7-AAD staining, respectively. Chemokines production was measured by enzyme-linked immunosorbent assay (ELISA)in culture supernatants. ResultsIndian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine. Bone morphogen 4 and 7 (BMP4 and BMP7) expression decreased after Hh blockade with cyclopamine. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of interleukin 8 (IL-8) and monocyte chemotactic protein (MCP-1) decreased upon exposure to Hh agonists compared to cyclopamine, lipopolyssacharide (LPS), interferon(IFN-γ) or epidermal growth factor (EGF). Cellular proliferation and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Conclusions: Hh pathway is a key controller of colonic epithelial cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response...