RESUMO
Wnt/ß-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated ß-catenin (pß-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in ß-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with ß-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial ß-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces ß-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the ß-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Colite/metabolismo , Neoplasias do Colo/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Colite/genética , Colite/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , beta Catenina/genéticaRESUMO
Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS(-/-) mice. IEC p53 levels and apoptosis were reduced in IL-10(-/-) colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF-treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF-treated CUC patients.
Assuntos
Apoptose , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Colite/patologia , Enterócitos/enzimologia , Enterócitos/patologia , Células Epiteliais/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial ß-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate ß-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-ß-catenin(552)). Our aim was to determine whether epithelial PI3K/Akt activation is required for ß-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and ß-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-ß-catenin(552)-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of ß-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1ß) or chemokine (CXCL1, CXCL5, CXCL9, and CXCL10) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated ß-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Células Epiteliais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/imunologia , beta Catenina/metabolismo , Animais , Colite/imunologia , Colite/metabolismo , Colite/patologia , Infecções por Enterobacteriaceae/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/patologia , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. EXPERIMENTAL DESIGN: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10(-/-) mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. RESULTS: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. CONCLUSION: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10(-/-) mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002.
Assuntos
Colite/imunologia , Neoplasias Colorretais/imunologia , Células Epiteliais/enzimologia , Macrófagos/enzimologia , Mastócitos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Anticarcinógenos/farmacologia , Degranulação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cromonas/farmacologia , Colite/complicações , Colite/tratamento farmacológico , Colite/patologia , Colo/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Inibidores de Ciclo-Oxigenase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células HT29 , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Invasividade Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Piroxicam/farmacologia , Transdução de Sinais , Triptases/metabolismoRESUMO
BACKGROUND: Activated effector T cells contribute to tissue injury observed in inflammatory bowel disease. T cells are recruited to effector sites after activation in peripheral lymph nodes directs expression of tissue-specific homing receptors. One such mechanism for effector T cell recruitment employs activation-induced fucosylation of P-selectin glycoprotein ligand (PSGL)-1 that mediates binding to endothelial P-selectin. Here we examine the differential role of PSGL-1 in recruiting effector T-cell subsets in colitis. METHODS: C57BL/6 wildtype and PSGL-1 mice received 2.5% dextran sodium sulfate (DSS) for 6 days and were euthanized 7 and 14 days after the initiation of DSS. Disease activity was monitored throughout. Histologic colitis scores, colonic CD4+ accumulation, and cytokine production were assessed at days 7 and 14. Recruitment of T-helper (Th) subsets was assessed by enumerating adoptively transferred Th1 or Th17 CD4+ cells 2 days after transfer to DSS-treated mice. RESULTS: DSS colitis increases CD4+ T cells in colonic tissue and induces Th1 (interferon gamma [IFN-γ], tumor necrosis factor [TNF]) and Th17 (interleukin [IL]-17, IL-22) cytokines. Loss of PSGL-1 attenuates DSS colitis, decreases colonic CD4+ T cell numbers, and reduces both Th1 and Th17 cytokine production. Colitis increases recruitment of Th1 (19-fold) and Th17 (2.5-fold) cells. PSGL-1 deficiency in transferred T cells abrogates colonic recruitment of Th1 cells in DSS colitis, whereas Th17 recruitment is unaffected. CONCLUSIONS: PSGL-1 selectively controls Th1 recruitment in colitis. Whereas Th17 recruitment is independent of PSGL-1, generation of colonic Th17 cytokine requires initial Th1 recruitment. Therefore, attenuating PSGL-1 binding may prevent colonic recruitment of disease-causing Th1 cells that promote local Th17 generation.
Assuntos
Movimento Celular/fisiologia , Colite/imunologia , Glicoproteínas de Membrana/fisiologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Contagem de Células , Colite/induzido quimicamente , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de DoençaRESUMO
PURPOSE: Immunocytokine (IC) hu14.18-IL2 is a fusion protein of humanized antidisialoganglioside (GD2) antibody (hu14.18) and interleukin (IL)-2. Sixty-one melanoma and neuroblastoma patients received IC in phase I/Ib studies. Patient sera were examined in ELISA to determine if an anti-IC antibody response occurred during treatment. EXPERIMENTAL DESIGN: Serum was assayed for anti-idiotypic antibody (anti-id Ab) based on ability to bridge biotinylated hu14.18 to plate-bound hu14.18 and ability to inhibit binding of hu14.18 to GD2 antigen and/or murine anti-idiotypic antibody. ELISA was also used to detect antibodies to the Fc-IL2 end of hu14.18-IL2. RESULTS: Thirty-two patients (52%) developed an anti-idiotypic antibody response (absorbance, >0.7) in the bridge ELISA. Twelve patients (20%) had an intermediate response, whereas 17 patients (28%) were negative (adsorbance, <0.3). The development of antibody to hu14.18-IL2 detected in the bridge ELISA was not related to the dose of hu14.18-IL2. Twenty of 33 adult patients (61%) demonstrated an anti-idiotypic antibody response based on binding inhibition ELISA. The anti-idiotypic response was inversely correlated (P < 0.002) with IC measured during the second course of treatment, indicating that development of anti-idiotypic antibodies interfered with detection of circulating hu14.18-IL2. All patients developed some inhibitory activity in the binding inhibition assay designed to detect antibodies to the Fc-IL2 region of the IC. There was a positive correlation between the peak serum level of IC in course 1 and the anti-Fc-IL2 response. CONCLUSIONS: Patients treated with hu14.18-IL2 developed anti-idiotypic antibodies and anti Fc-IL2 antibodies. No association was seen between development of anti-IC antibodies and clinical toxicity.