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1.
Stem Cell Res ; 55: 102499, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34399164

RESUMO

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3ß inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.


Assuntos
Adenoma , Células Epiteliais , Animais , Mucosa Intestinal , Intestinos , Camundongos , Fatores de Iniciação de Peptídeos , Via de Sinalização Wnt
2.
J Crohns Colitis ; 11(7): 831-839, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28158397

RESUMO

BACKGROUND AND AIM: T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. METHODS: Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRß repertoire was analysed by next-generation sequencing of biopsy RNA. RESULTS: Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. CONCLUSIONS: The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy.


Assuntos
Doença de Crohn/imunologia , Doença de Crohn/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Adalimumab/uso terapêutico , Adulto , Anti-Inflamatórios/uso terapêutico , Biópsia , Budesonida/uso terapêutico , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Células Clonais/efeitos dos fármacos , Colo/patologia , Doença de Crohn/tratamento farmacológico , Feminino , Fármacos Gastrointestinais/uso terapêutico , Humanos , Íleo/patologia , Inflamação/imunologia , Inflamação/patologia , Infliximab/uso terapêutico , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Linfócitos T/efeitos dos fármacos , Adulto Jovem
3.
Aliment Pharmacol Ther ; 45(8): 1128-1134, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28230306

RESUMO

BACKGROUND: Loss of response to anti-tumour necrosis factor (TNF) therapy in patients with inflammatory bowel disease (IBD) is often caused by anti-drug antibody formation with neutralisation of drug effect. Addition of an immunomodulator has been suggested to reduce immunogenicity, leading to regained response. AIM: To investigate whether addition of an immunomodulator to anti-TNF monotherapy could lead to anti-drug antibody suppression and regained clinical response in IBD patients. METHODS: We retrospectively collected measurements of infliximab or adalimumab serum concentrations and anti-drug antibodies to identify anti-drug positive patients with loss response who were given an immunomodulator. RESULTS: Anti-drug antibodies against infliximab and adalimumab were detected in 98/376 (26%) and in 61/226 (27%) patients, respectively. Immunomodulators were given to 17/159 patients. Clinical response was recaptured in 6/10 patients receiving a thiopurine and in all (7/7) patients receiving methotrexate. In 7/8 patients on infliximab, serum concentrations increased (median 2.84 µg/mL; IQR: 1.19-4.98) and in 6/9 patients on adalimumab (median 3.10 µg/mL; IQR: 1.45-4.45). This was accompanied by a decrease in anti-drug antibodies to undetectable levels (median 11 months for both anti-TNF agents). In 23 patients, no immunomodulator was added but anti-TNF interval was shortened (17/23) or dosage was increased (6/23), which resulted in a clinical response in 10/17 and 2/6 patients, respectively. CONCLUSION: In 77% of IBD patients with loss of response due to immunogenicity, addition of immunomodulator resulted in undetectable anti-drug antibody levels, increased serum drug concentrations and regained clinical response. This strategy should be considered in this patient population before switching to other agents.


Assuntos
Adalimumab/imunologia , Anticorpos Monoclonais/sangue , Fatores Imunológicos/administração & dosagem , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/imunologia , Metotrexato/uso terapêutico , Adalimumab/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Adulto , Idoso , Quimioterapia Combinada , Feminino , Humanos , Doenças Inflamatórias Intestinais/imunologia , Infliximab/administração & dosagem , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto Jovem
4.
Mucosal Immunol ; 10(2): 352-360, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27435106

RESUMO

Thiopurines are commonly used drugs in the therapy of Crohn's disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn's disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn's disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn's disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Doença de Crohn/imunologia , Células Dendríticas/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Alelos , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Estruturas da Membrana Celular/patologia , Movimento Celular , Células Cultivadas , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Citoesqueleto/metabolismo , Células Dendríticas/patologia , Feminino , Predisposição Genética para Doença , Humanos , Mercaptopurina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo Genético , RNA Interferente Pequeno/genética , Risco
5.
Oncogene ; 36(24): 3397-3405, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27819675

RESUMO

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear ß-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear ß-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of ß-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Células Epiteliais/citologia , Proteínas de Choque Térmico/genética , Células-Tronco/citologia , Animais , Diferenciação Celular , Proliferação de Células , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Células-Tronco/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
6.
Am J Gastroenterol ; 111(2): 163-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26416189

RESUMO

The human appendix has long been considered as a vestigial organ, an organ that has lost its function during evolution. In recent years, however, reports have emerged that link the appendix to numerous immunological functions in humans. Evidence has been presented for an important role of the appendix in maintaining intestinal health. This theory suggests that the appendix may be a reservoir or 'safe house' from which the commensal gut flora can rapidly be reestablished if it is eradicated from the colon. However, the appendix may also have a role in the development of inflammatory bowel disease (IBD). Several large epidemiological cohort studies have demonstrated the preventive effect of appendectomy on the development of ulcerative colitis, a finding that has been confirmed in murine colitis models. In addition, current studies are examining the possible therapeutic effect of an appendectomy to modulate disease course in patients with ulcerative colitis. This literature review assesses the current knowledge about the clinical and immunological aspects of the vermiform appendix in IBD and suggests that the idea of the appendix as a vestigial remnant should be discarded.


Assuntos
Apêndice/imunologia , Colite Ulcerativa/imunologia , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Apendicectomia , Apêndice/microbiologia , Linfócitos B/imunologia , Colite Ulcerativa/epidemiologia , Doença de Crohn/epidemiologia , Doença de Crohn/imunologia , Progressão da Doença , Disbiose/epidemiologia , Humanos , Imunoglobulina A/imunologia , Células T Matadoras Naturais/imunologia , Fatores de Proteção , Índice de Gravidade de Doença
7.
Br J Cancer ; 112(1): 122-30, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25393365

RESUMO

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Colorretais/metabolismo , Proteína Smad4/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt , Proteínas Morfogenéticas Ósseas/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Transdução de Sinais , Transfecção , Proteína Supressora de Tumor p53/genética , beta Catenina/genética , beta Catenina/metabolismo
8.
Cell Death Differ ; 21(6): 956-66, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24583641

RESUMO

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.


Assuntos
Apoptose/genética , Enterócitos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Enterócitos/efeitos dos fármacos , Enterócitos/efeitos da radiação , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Radiação Ionizante
9.
Oncogene ; 32(9): 1202-6, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22469986

RESUMO

Development of colon cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens (PAMPs) or cellular damage (DAMPs). These molecules can promote tumourigenesis by activation of the innate immune system, but the individual contribution of ligands and their receptors remains elusive. The receptor for advanced glycation end products (Rage) is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment such as Hmgb1 and S100 protein. Here we show that Rage signalling has a critical role in sporadic development of intestinal adenomas, as Apc(Min/+) Rage(-/-) mice are protected against tumourigenesis.


Assuntos
Adenoma/metabolismo , Neoplasias Intestinais/metabolismo , Receptores Imunológicos/metabolismo , Animais , Camundongos , Receptor para Produtos Finais de Glicação Avançada , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais
10.
Biochim Biophys Acta ; 1822(1): 9-13, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21146606

RESUMO

The link between inflammation and colorectal cancer development is becoming increasingly clear. It had long been recognized that patients with inflammatory bowel disease are at an increased risk of colon cancer. Evidence from experimental animals now also implicates the innate immune system in the development of sporadically occurring intestinal adenomas, the precursors to colorectal cancer. Here we discuss the interaction between the immune system and the adenoma to carcinoma sequence with a special emphasis on the role of mast cells which may play a key role in adenoma development. This article is part of a Special Issue entitled: Mast cells in inflammation.


Assuntos
Adenoma/patologia , Neoplasias Colorretais/patologia , Mastócitos/patologia , Imunidade Adaptativa , Adenoma/imunologia , Animais , Transformação Celular Neoplásica/imunologia , Neoplasias Colorretais/imunologia , Humanos , Imunidade Inata , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mastócitos/imunologia
11.
J Pathol ; 215(4): 411-20, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18498120

RESUMO

Helicobacter pylori infection induces intestinal metaplasia of the stomach, a preneoplastic lesion associated with an increased risk for gastric cancer development. Intestinal metaplasia is induced by the intestine-specific transcription factor CDX2 but the mechanisms responsible for this ectopic expression have never been described. We hypothesized that the BMP/SMAD pathway has a role in CDX2 regulation, in this context, for the following reasons: (1) the BMP pathway is crucial for normal intestinal differentiation and (2) there is an influx of BMP2 and BMP4-producing cells to the stomach upon Helicobacter pylori infection. We evaluated the expression of key elements of the BMP pathway in human stomach specimens with IM. Growth factor treatments, with BMP2 and BMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was done using RNAi. We showed overexpression in IM of BMP2/4, BMPR1A, and SMAD4 in 56% of IM foci, and pSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In vitro, treatment of AGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as the intestinal differentiation markers MUC2 and LI-cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2, and LI-cadherin in AGS. Treatment of the SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wild-type cells. A 9.3 kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in a cell-dependent manner. In conclusion, we identified for the first time that the BMP pathway is active in intestinal metaplasia and that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiation through the canonical signal transducers.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias Gástricas/genética , Fator de Crescimento Transformador beta/metabolismo , Western Blotting/métodos , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Fator de Transcrição CDX2 , Carcinoma/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Homeodomínio/análise , Humanos , Imuno-Histoquímica , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína Smad1/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Neoplasias Gástricas/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia
12.
J Crohns Colitis ; 2(2): 99-106, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21172199

RESUMO

Hematopoietic stem cell transplantation and mesenchymal stromal cell therapy are currently under investigation as novel therapies for inflammatory bowel diseases. Hematopoietic stem cells (HSC) are thought to repopulate the immune system and reset the immunological response to luminal antigens. Mesenchymal stromal cells (MSC) are cells that have the capacity to differentiate into wide variety of distinct cell lineages and suppress immune responses in vitro and in vivo. Recent results from animal models and early human experience in graft-versus-host disease but also Crohn's Disease suggest that ex vivo expanded MSCs may have clinically useful immunomodulatory effects.

13.
Oncogene ; 25(49): 6447-56, 2006 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-16878161

RESUMO

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/beta-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of beta-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of beta-catenin were assessed by immunoblotting, and also the localization of beta-catenin was shown by green fluorescent protein-tagged beta-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/beta-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/beta-catenin pathway activity in these cells.


Assuntos
Aspirina/farmacologia , Fosfoproteínas Fosfatases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Anti-Inflamatórios não Esteroides/farmacologia , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Células HCT116 , Humanos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2 , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fatores de Transcrição TCF/metabolismo , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética
15.
J Pathol ; 209(2): 190-7, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16550632

RESUMO

Morphogens regulate epithelial cell fate decisions in the adult gastrointestinal tract. The authors hypothesized that influx of inflammatory cells into the lamina propria may disturb the normal expression gradients of morphogens (morphogenetic landscape) in gastrointestinal epithelia. Changes in the activity of the bone morphogenetic protein (BMP) pathway in normal and Helicobacter pylori-infected gastric mucosa were therefore examined. It is shown that BMP receptors, the activated (phosphorylated) form of the intracellular BMP signal transduction protein SMAD1, and BMP target ID2 all localize to gastric epithelial cells that are at the end of the axis of epithelial renewal in normal mucosa. Colonization of human gastric mucosa with H. pylori was associated with an increase in BMP2 expression due to influx of inflammatory cells that produce BMP2. Furthermore, whereas no BMP4 was detected in the normal antrum, focal infiltrates of BMP4-expressing cells were found in the H. pylori-infected stomach. This influx of BMP-expressing cells was associated with an increase in epithelial BMP signalling. Interestingly, a shift in activity of the BMP pathway was observed towards the precursor cell compartment (isthmus) of the gastric units. Thus, H. pylori infection results in an influx of inflammatory cells that disturb the normal activity gradient of a morphogenetic pathway with an established role in epithelial cell fate regulation. The data suggest that morphological changes in epithelial histology may result from alterations in the morphogenetic landscape secondary to changes in the cellular composition of the lamina propria.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Transdução de Sinais/fisiologia , Gastropatias/metabolismo , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/análise , Proteínas Morfogenéticas Ósseas/análise , Contagem de Células , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Humanos , Imuno-Histoquímica/métodos , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteína Smad1/metabolismo , Fator de Crescimento Transformador beta/análise
17.
Gut ; 51(5): 628-33, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12377798

RESUMO

BACKGROUND: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel's diverticulum. METHODS: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. RESULTS: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. CONCLUSION: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.


Assuntos
Fundo Gástrico/química , Divertículo Ileal/metabolismo , RNA Mensageiro/análise , Transativadores/análise , Adulto , Diferenciação Celular , Esôfago/metabolismo , Esôfago/patologia , Fundo Gástrico/citologia , Proteínas Hedgehog , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Metaplasia , Transativadores/genética
19.
Gastroenterology ; 121(2): 317-28, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11487541

RESUMO

BACKGROUND & AIMS: Gastric epithelial renewal is an asymmetric process. A stem cell located halfway up the tubular unit gives rise to both a basal gland region and a luminal pit compartment, but the mechanisms responsible for the maintenance of this asymmetry are obscure. We investigated whether Sonic hedgehog (Shh), an established polarizing signal protein during development, is expressed and functional in the adult human and murine stomach. METHODS: Expression of Shh and putative transcriptional targets was investigated using immunoblot and immunohistochemistry. Mice were treated with the Shh inhibitor cyclopamine and examined for expression levels of Shh targets and proliferation of gastric epithelial cells. RESULTS: Shh was expressed in the stomach. In cyclopamine-treated mice, we observed decreased expression of HNF3beta, Islet (Isl)-1 and BMP4, 3 putative Shh target genes. Inhibition of Shh markedly enhanced gastric epithelial proliferation and affected the cell cycle of gastric epithelial gland cells, whereas pit cells remained unaffected. CONCLUSIONS: Shh controls the expression of at least 3 factors important for epithelial differentiation and is a negative regulator of gastric gland cell proliferation. Shh is a candidate polarizing signal in the maintenance of gastric pit-gland asymmetry.


Assuntos
Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Proteínas do Tecido Nervoso , Proteínas/metabolismo , Transativadores , Fatores de Transcrição , Fator de Crescimento Transformador beta , Fatores Etários , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/biossíntese , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Células Epiteliais/química , Células Epiteliais/enzimologia , Mucosa Gástrica/química , ATPase Trocadora de Hidrogênio-Potássio/análise , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas Hedgehog , Fator 3-beta Nuclear de Hepatócito , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/biossíntese , Humanos , Imuno-Histoquímica , Proteínas com Homeodomínio LIM , Camundongos , Proteínas Nucleares/análise , Proteínas Nucleares/biossíntese , Proteínas/análise , Proteínas/antagonistas & inibidores , Especificidade da Espécie , Alcaloides de Veratrum/farmacologia
20.
Gastroenterology ; 121(1): 79-90, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11438496

RESUMO

BACKGROUND AND AIMS: Obesity increases the risk of colon cancer, whereas physical activity reduces the risk. Plasma levels of leptin increase in proportion to the level of obesity and are reduced by physical activity. Leptin acts as a growth factor for several cell types and thus may provide a biological explanation for the observed epidemiological risk factors. The aim of this study was to investigate whether leptin is a growth factor for colonic epithelial cells. METHODS: The presence of the leptin receptor in human colon cancer cell lines was assessed using reverse-transcription polymerase chain reaction and immunoblotting, and its presence in human colonic tissue was assessed by immunohistochemistry. The effects of leptin in vitro on HT29 cells were assessed by assessing p42/44 mitogen-activated protein kinase phosphorylation, thymidine incorporation, and cell numbers and in vivo in C57BL/6 mice by colonic bromodeoxyuridine incorporation. RESULTS: The leptin receptor is expressed in human colon cancer cell lines and human colonic tissue. Stimulation with leptin leads to phosphorylation of p42/44 mitogen-activated protein kinase and increases proliferation in vitro and in vivo. CONCLUSIONS: Leptin is a growth factor in colonic epithelial cells and one that may provide a biological explanation for the observed associations between obesity, physical activity, and colon cancer.


Assuntos
Adenocarcinoma/patologia , Proteínas de Transporte/efeitos dos fármacos , Neoplasias do Colo/patologia , Leptina/farmacologia , Receptores de Superfície Celular , Adenocarcinoma/etiologia , Adenocarcinoma/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Proteínas de Transporte/isolamento & purificação , Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Substâncias de Crescimento/farmacologia , Humanos , Leptina/isolamento & purificação , Camundongos , Obesidade/complicações , Proteínas Quinases/metabolismo , Receptores para Leptina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/metabolismo , Células Tumorais Cultivadas
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