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Background: Mitochondrial (Mito) dysfunction in IBD reduces mucosal O2 consumption and increases O2 delivery to the microbiome. Increased enteric O2 promotes blooms of facultative anaerobes (eg. Proteobacteria ) and restricts obligate anaerobes (eg. Firmicutes ). Dysbiotic metabolites negatively affect host metabolism and immunity. Our novel compound (AuPhos) upregulates intestinal epithelial cell (IEC) mito function, attenuates colitis and corrects dysbiosis in humanized Il10-/- mice. We posit that AuPhos corrects IBD-associated dysbiotic metabolism. Methods: Primary effect of AuPhos on mucosal Mito respiration and healing process was studied in ex vivo treated human colonic biopsies and piroxicam-accelerated (Px) Il10-/- mice. Secondary effect on microbiome was tested in DSS-colitis WT B6 and germ-free 129.SvEv WT or Il10-/- mice reconstituted with human IBD stool (Hu- Il10-/- ). Mice were treated orally with AuPhos (10- or 25- mg/kg; q3d) or vehicle, stool samples collected for fecal lipocalin-2 (f-LCN2) assay and microbiome analyses using 16S rRNA sequencing. AuPhos effect on microbial metabolites was determined using untargeted global metabolomics. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Effect of AuPhos on enteric oxygenation was assessed by E. coli Nissle 1917 WT (aerobic respiration-proficient) and cytochrome oxidase (cydA) mutant (aerobic respiration-deficient). Results: Metagenomic (16S) analysis revealed AuPhos reduced relative abundances of Proteobacteria and increased blooms of Firmicutes in uninflamed B6 WT, DSS-colitis, Hu-WT and Hu- Il10-/- mice. AuPhos also increased hypoxyprobe-1 staining in surface IECs suggesting enhanced O2 utilization. AuPhos-induced anaerobiosis was confirmed by a significant increase in cydA mutant compared to WT (O2-utlizing) E.coli . Ex vivo treatment of human biopsies with AuPhos showed significant increase in Mito mass, and complexes I and IV. Further, gene expression analysis of AuPhos-treated biopsies showed increase in stem cell markers (Lgr4, Lgr5, Lrig1), with concomitant decreases in pro-inflammatory markers (IL1ß,MCP1, RankL). Histological investigation of AuPhos-fed Px- Il10-/- mice showed significantly decreased colitis score in AuPhos-treated Px- Il10-/- mice, with decrease in mRNA of pro-inflammatory cytokines and increase in Mito complexes ( ND5 , ATP6 ). AuPhos significantly altered microbial metabolites associated with SCFA synthesis, FAO, TCA cycle, tryptophan and polyamine biosynthesis pathways. AuPhos increased pyruvate, 4-hydroxybutyrate, 2-hydroxyglutarate and succinate, suggesting an upregulation of pyruvate and glutarate pathways of butyrate production. AuPhos reduced IBD-associated primary bile acids (BA) with concomitant increase in secondary BA (SBA). AuPhos treatment significantly decreased acylcarnitines and increased L-carnitine reflective of enhanced FAO. AuPhos increases TCA cycle intermediates and creatine, energy reservoir substrates indicating enhanced OxPHOS. Besides, AuPhos also upregulates tryptophan metabolism, decreases Kynurenine and its derivatives, and increases polyamine biosynthesis pathway (Putresceine and Spermine). Conclusion: These findings indicate that AuPhos-enhanced IEC mitochondrial function reduces enteric O2 delivery, which corrects disease-associated metabolomics by restoring short-chain fatty acids, SBA, AA and IEC energy metabolism.
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Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active ß-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.
Assuntos
Hepatócitos/citologia , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Quinases Semelhantes a Duplacortina , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Xenoenxertos , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Esferoides Celulares , alfa-Fetoproteínas/metabolismoRESUMO
Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination, ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.
Assuntos
Colite/metabolismo , Neoplasias Colorretais/metabolismo , Ativação Transcricional , beta Catenina/genética , beta Catenina/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Colite/genética , Neoplasias Colorretais/genética , Células HCT116 , Células HT29 , Humanos , Camundongos , Peso Molecular , Mutação , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição 4/metabolismoRESUMO
AIM: To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate pß-cateninS552 as a biomarker of recurrent dysplasia. METHODS: We examined the effects of dietary myo-inositol treatment on inflammation, pß-cateninS552 and pAkt levels by histology and western blot in IL-10-/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear pß-cateninS552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia. RESULTS: In mice, pß-cateninS552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, pß-cateninS552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease. CONCLUSION: Enumerating crypts with increased numbers of pß-cateninS552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials.
Assuntos
Biomarcadores Tumorais/metabolismo , Colite Ulcerativa/tratamento farmacológico , Neoplasias Colorretais/diagnóstico , Inositol/farmacologia , beta Catenina/metabolismo , Animais , Biomarcadores Tumorais/análise , Biópsia , Núcleo Celular/metabolismo , Proliferação de Células , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Colo/citologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias Colorretais/prevenção & controle , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Inositol/uso terapêutico , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Projetos Piloto , Placebos , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , beta Catenina/análiseRESUMO
TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/ß-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/ß-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-typeâTnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/ß-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/ß-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ß-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/ß-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.
Assuntos
Células-Tronco Adultas/fisiologia , Anticorpos Monoclonais/uso terapêutico , Colite/imunologia , Células Epiteliais/fisiologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Sulfato de Dextrana , Humanos , Doenças Inflamatórias Intestinais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Proteínas Wnt/metabolismo , Cicatrização , beta Catenina/metabolismoRESUMO
Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of ß-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome.
Assuntos
Adaptação Fisiológica/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Síndrome do Intestino Curto/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Western Blotting , Classe Ia de Fosfatidilinositol 3-Quinase , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Modelos Animais de Doenças , Enterocolite Necrosante/cirurgia , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Proteínas Inibidoras de Apoptose/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/biossíntese , Síndrome do Intestino Curto/etiologia , SurvivinaRESUMO
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
A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.
RESUMO
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
BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs. METHODS: Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53â»/â», Bidâ»/â», Bimâ»/â», Bax3â»/â», Bakâ»/â», PUMAâ»/â», and Noxaâ»/â» mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis. RESULTS: Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53â»/â» and PUMAâ»/â» mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53â»/â» mice indicated that PUMA-mediated apoptosis was independent of p53. CONCLUSIONS: In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Colite/patologia , Colite/fisiopatologia , Mucosa Intestinal/patologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Estudos de Casos e Controles , Caspase 3/metabolismo , Caspase 9/metabolismo , Colite/induzido quimicamente , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/fisiologia , Linfócitos T/patologia , Linfócitos T/fisiologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
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
BACKGROUND & AIMS: Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation. METHODS: The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined. RESULTS: Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. CONCLUSIONS: PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.
Assuntos
Colite/enzimologia , Colo/enzimologia , Mucosa Intestinal/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , beta Catenina/metabolismo , Animais , Biópsia , Proliferação de Células , Colite/complicações , Colite/genética , Colite/imunologia , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Colonoscopia , Modelos Animais de Doenças , Humanos , Interleucina-10/deficiência , Interleucina-10/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Células-Tronco/patologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fatores de Tempo , Proteínas Wnt/metabolismoRESUMO
In inflammatory bowel disease (IBD), aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-kappaB) in IBD-induced enhanced permeability, epithelial-specific IkappaBalpha mutant (NF-kappaB super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice, following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore, epithelial NF-kappaB super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-kappaB-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-kappaB-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-kappaB activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-kappaB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen.
Assuntos
Células Epiteliais/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Mucosa/patologia , NF-kappa B/metabolismo , Linfócitos T/imunologia , Junções Íntimas/metabolismo , Animais , Diarreia/imunologia , Diarreia/metabolismo , Células Epiteliais/patologia , Humanos , Proteínas I-kappa B/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patologia , Substâncias Macromoleculares/metabolismo , Camundongos , Camundongos Transgênicos , Mucosa/metabolismo , Mutação/genética , Inibidor de NF-kappaB alfa , Especificidade de Órgãos , Permeabilidade , Reologia , Junções Íntimas/patologiaRESUMO
BACKGROUND & AIMS: Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID). METHODS: Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon. RESULTS: Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling. CONCLUSIONS: The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.