RESUMO
We have previously shown that the sequential transcription factors Xbp1âMist1 (Bhlha15) govern the ultrastructural maturation of the secretory apparatus in enzyme-secreting zymogenic chief cells (ZCs) in the gastric unit. Here we sought to identify transcriptional regulators upstream of X-box binding protein 1 (XBP1) and MIST1. We used immunohistochemistry to characterize Hnf4α(flox/flox) adult mouse stomachs after tamoxifen-induced deletion of Hnf4α We used qRT-PCR, Western blotting, and chromatin immunoprecipitation to define the molecular interaction between hepatocyte nuclear factor 4 alpha (HNF4α) and Xbp1 in mouse stomach and human gastric cells. We show that HNF4α protein is expressed in pit (foveolar) cells, mucous neck cells, and zymogenic chief cells (ZCs) of the corpus gastric unit. Loss of HNF4α in adult mouse stomach led to reduced ZC size and ER content, phenocopying previously characterized effects of Xbp1 deletion. However, HNF4α(Δ/Δ) stomachs also exhibited additional phenotypes including increased proliferation in the isthmal stem cell zone and altered mucous neck cell migration, indicating a role of HNF4α in progenitor cells as well as in ZCs. HNF4α directly occupies the Xbp1 promoter locus in mouse stomach, and forced HNF4α expression increased abundance of XBP1 mRNA in human gastric cancer cells. Finally, as expected, loss of HNF4α caused decreased Xbp1 and Mist1 expression in mouse stomachs. We show that HNF4α regulates homeostatic proliferation in the gastric epithelium and is both necessary and sufficient for the upstream regulation of the Xbp1âMist1 axis in maintenance of ZC secretory architecture.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem Celular , Células Epiteliais/patologia , Mucosa Gástrica/patologia , Regulação da Expressão Gênica , Genótipo , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Homeostase , Humanos , Camundongos Knockout , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais , Transfecção , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44(+) population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK â CD44 â STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.
Assuntos
Proliferação de Células , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Receptores de Hialuronatos/metabolismo , Células Parietais Gástricas/metabolismo , Células-Tronco/metabolismo , Animais , Feminino , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Humanos , Receptores de Hialuronatos/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Células Parietais Gástricas/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células-Tronco/patologiaRESUMO
BACKGROUND: Intestinal metaplasia (IM), a premalignant lesion, is associated with an increased risk of gastric cancer. Although estrogen exposure, including tamoxifen, has been studied in correlation with gastric cancer, little has been investigated about its effects on IM. AIMS: Therefore, we investigated whether chronic tamoxifen use was associated with the risk of IM in human stomach. METHODS: We evaluated 512 gastric biopsies from 433 female breast cancer patients that underwent endoscopic gastroduodenoscopy (EGD) ≥6 months after breast surgery. Histopathological findings were scored according to the updated Sydney classification. Demographic and clinical characteristics were also included to identify predictive factors for IM. RESULTS: In a multivariate logistic regression analysis, age at EGD (odds ratio [OR], 1.04; P = 0.002), biopsies from antrum (OR 2.08; P < 0.001), and Helicobacter pylori positivity (OR 1.68; P = 0.016) were significantly associated with an increased risk of IM, whereas chronic tamoxifen use (≥3 months) was associated with a decreased risk of IM (OR 0.59; P = 0.025). After stratifying by biopsy site, association between tamoxifen use and IM persisted for corpus (OR 0.42; P = 0.026) but not for antrum (OR 0.74; P = 0.327). In analysis limited to patients with follow-up EGD, chronic tamoxifen use also correlated with improved IM score compared to no tamoxifen use (improved, 77.8 vs. 22.2%; no change, 65.4 vs. 34.6%; worsened, 30.0 vs. 70.0%; P = 0.019). CONCLUSIONS: This study suggests that chronic tamoxifen use can decrease the risk of IM in human stomach. The effect of tamoxifen is predominantly observed in the corpus.
Assuntos
Epitélio/efeitos dos fármacos , Metaplasia/prevenção & controle , Estômago/efeitos dos fármacos , Tamoxifeno/administração & dosagem , Tamoxifeno/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Esquema de Medicação , Epitélio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Análise de Regressão , Fatores de Risco , Estômago/patologia , Adulto JovemRESUMO
In a screen for genes expressed specifically in gastric mucous neck cells, we identified GKN3, the recently discovered third member of the gastrokine family. We present confirmatory mouse data and novel porcine data showing that mouse GKN3 expression is confined to mucous cells of the corpus neck and antrum base and is prominently expressed in metaplastic lesions. GKN3 was proposed originally to be expressed in some human populations and a pseudogene in others. To investigate that hypothesis, we studied human GKN3 evolution in the context of its paralogous genomic neighbors, GKN1 and GKN2. Haplotype analysis revealed that GKN3 mimics GKN2 in patterns of exonic SNP allocation, whereas GKN1 appeared to be more stringently selected. GKN3 showed signatures of both directional selection and population based selective sweeps in humans. One such selective sweep includes SNP rs10187256, originally identified as an ancestral tryptophan to premature STOP codon mutation. The derived (nonancestral) allele went to fixation in Asia. We show that another SNP, rs75578132, identified 5 bp downstream of rs10187256, exhibits a second selective sweep in almost all Europeans, some Latinos, and some Africans, possibly resulting from a reintroduction of European genes during African colonization. Finally, we identify a mutation that would destroy the splice donor site in the putative exon3-intron3 boundary, which occurs in all human genomes examined to date. Our results highlight a stomach-specific human genetic locus, which has undergone various selective sweeps across European, Asian, and African populations and thus reflects geographic and ethnic patterns in genome evolution.
Assuntos
Proteínas de Transporte/genética , Evolução Molecular , Loci Gênicos/genética , Proteínas de Membrana/genética , Pseudogenes/genética , Grupos Raciais/genética , Seleção Genética/genética , Animais , Proteínas de Transporte/metabolismo , Biologia Computacional , Primers do DNA/genética , Imunofluorescência , Mucosa Gástrica/metabolismo , Genética Populacional , Genótipo , Haplótipos/genética , Humanos , Funções Verossimilhança , Macaca mulatta/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/genética , Análise em Microsséries , Microscopia Confocal , Modelos Genéticos , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Especificidade da Espécie , Sus scrofa/genéticaRESUMO
Tamoxifen, a selective estrogen receptor modulator, is widely used in research and clinically in patients. We find that treatment of normal mice with a single ≥3 mg/20 g body weight dose of tamoxifen leads to apoptosis of >90% of all gastric parietal cells (PCs) and metaplasia of zymogenic chief cells within 3 days. Remarkably, gastric histology returns to nearly normal by 3 weeks. Tamoxifen toxicity occurs by oral and intraperitoneal administration, in both sexes, in multiple strains, and does not depend on estrogen, though acid secretion inhibition is partially protective. Thus, substantial gastric toxicity is a heretofore unappreciated tamoxifen side effect.
Assuntos
Celulas Principais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/toxicidade , Tamoxifeno/toxicidade , Administração Oral , Animais , Atrofia , Celulas Principais Gástricas/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Integrases/genética , Óperon Lac , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Parietais Gástricas/patologia , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Especificidade da Espécie , Tamoxifeno/administração & dosagem , Fatores de TempoRESUMO
The regulation and stem cell origin of normal and neoplastic gastric glands are uncertain. Here, we show that Mist1 expression marks quiescent stem cells in the gastric corpus isthmus. Mist1(+) stem cells serve as a cell-of-origin for intestinal-type cancer with the combination of Kras and Apc mutation and for diffuse-type cancer with the loss of E-cadherin. Diffuse-type cancer development is dependent on inflammation mediated by Cxcl12(+) endothelial cells and Cxcr4(+) gastric innate lymphoid cells (ILCs). These cells form the perivascular gastric stem cell niche, and Wnt5a produced from ILCs activates RhoA to inhibit anoikis in the E-cadherin-depleted cells. Targeting Cxcr4, ILCs, or Wnt5a inhibits diffuse-type gastric carcinogenesis, providing targets within the neoplastic gastric stem cell niche.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nicho de Células-Tronco , Neoplasias Gástricas/metabolismo , Microambiente Tumoral , Animais , Anoikis , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transplante de Medula Óssea , Caderinas/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Senescência Celular , Quimiocina CXCL12/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Receptores CXCR4/metabolismo , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Tempo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5a , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Chronic inflammation is a major risk factor for cancer, including gastric cancers and other gastrointestinal cancers. For example, chronic inflammation caused by autoimmune gastritis (AIG) is associated with an increased risk of gastric polyps, gastric carcinoid tumors, and possibly adenocarcinomas. In this study, we characterized the progression of gastric cancer in a novel mouse model of AIG. In this model, disease was caused by CD4(+) T cells expressing a transgenic T-cell receptor specific for a peptide from the H(+)/K(+) ATPase proton pump, a protein expressed by parietal cells in the stomach. AIG caused epithelial cell aberrations that mimicked most of those seen in progression of human gastric cancers, including chronic gastritis followed by oxyntic atrophy, mucous neck cell hyperplasia, spasmolytic polypeptide-expressing metaplasia, dysplasia, and ultimately gastric intraepithelial neoplasias. Our work provides the first direct evidence that AIG supports the development of gastric neoplasia and provides a useful model to study how inflammation drives gastric cancer.
Assuntos
Doenças Autoimunes/etiologia , Linfócitos T CD4-Positivos/imunologia , Gastrite/complicações , ATPase Trocadora de Hidrogênio-Potássio/imunologia , Inflamação/complicações , Receptores de Antígenos de Linfócitos T/fisiologia , Neoplasias Gástricas/etiologia , Adenocarcinoma/etiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Citometria de Fluxo , Imunofluorescência , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/patologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/imunologia , Inflamação/patologia , Interferon gama/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Metaplasia/complicações , Metaplasia/imunologia , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag(+) H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag(+) H. pylori strain PMSS1 or a PMSS1 cagE(-) isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5(+) cells also stained positively for the stem cell marker, Lrig1, and KLF5(+)/Lrig1(+) cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.