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1.
Exp Physiol ; 93(11): 1174-89, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18567601

RESUMO

The gastric acid-secreting parietal cell exhibits profound morphological changes on stimulation. Studies in gastrin null (Gas-KO) mice indicate that maturation of parietal cell function depends on the hormone gastrin acting at the G-protein-coupled cholecystokinin 2 receptor. The relevant cellular mechanisms are unknown. The application of differential mRNA display to samples of the gastric corpus of wild-type (C57BL/6) and Gas-KO mice identified the cytoskeletal linker protein, ezrin, as a previously unsuspected target of gastrin. Gastrin administered in vivo or added to gastric glands in vitro increased ezrin abundance in Gas-KO parietal cells. In parietal cells of cultured gastric glands from wild-type mice treated with gastrin, histamine or carbachol, ezrin was localized to vesicular structures resembling secretory canaliculi. In contrast, in cultured parietal cells from Gas-KO mice, ezrin was typically distributed in the cytosol, and this did not change after incubation with gastrin, histamine or carbachol. However, priming with gastrin for approximately 24 h, either in vivo prior to cell culture or by addition to cultured gastric glands, induced the capacity for secretagogue-stimulated localization of ezrin to large vesicular structures in Gas-KO mice. Similarly, in a functional assay based on measurement of intracellular pH, cultured parietal cells from Gas-KO mice were refractory to gastrin unless primed. The priming effect of gastrin was not attributable to the paracrine mediator histamine, but was prevented by inhibitors of protein kinase C and transactivation of the epidermal growth factor receptor. We conclude that in gastrin null mice there is reduced ezrin expression and a defect in ezrin subcellular distribution in gastric parietal cells, and that both can be reversed by priming with gastrin.


Assuntos
Diferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Gastrinas/metabolismo , Células Parietais Gástricas/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Receptores ErbB/metabolismo , Ácido Gástrico/metabolismo , Gastrinas/deficiência , Gastrinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histamina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Parietais Gástricas/enzimologia , Proteína Quinase C/metabolismo , Transporte Proteico , Vesículas Secretórias/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo
2.
Proc Natl Acad Sci U S A ; 105(3): 961-6, 2008 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-18195351

RESUMO

The thymus is essential for a functional immune system, because the thymic stroma uniquely supports T lymphocyte development. We have previously identified the epithelial progenitor population from which the thymus arises and demonstrated its ability to generate an organized functional thymus upon transplantation. These thymic epithelial progenitor cells (TEPC) are defined by surface determinants recognized by the mAbs MTS20 and MTS24, which were also recently shown to identify keratinocyte progenitor cells in the skin. However, the biochemical nature of the MTS20 and MTS24 determinants has remained unknown. Here we show, via expression profiling of fetal mouse TEPC and their differentiated progeny and subsequent analyses, that both MTS20 and MTS24 specifically bind an orphan protein of unknown function, Placenta-expressed transcript (Plet)-1. In the postgastrulation embryo, Plet-1 expression is highly restricted to the developing pharyngeal endoderm and mesonephros until day 11.5 of embryogenesis, consistent with the MTS20 and MTS24 staining pattern; both MTS20 and MTS24 specifically bind cell lines transfected with Plet-1; and antibodies to Plet-1 recapitulate MTS20/24 staining. In adult tissues, we demonstrate expression in a number of sites, including mammary and prostate epithelia and in the pancreas, where Plet-1 is specifically expressed by the major duct epithelium, providing a specific cell surface marker for this putative reservoir of pancreatic progenitor/stem cells. Plet-1 will thus provide an invaluable tool for genetic analysis of the lineage relationships and molecular mechanisms operating in the development, homeostasis, and injury in several organ/tissue systems.


Assuntos
Células Epiteliais/metabolismo , Proteínas da Gravidez/metabolismo , Células-Tronco/imunologia , Células-Tronco/metabolismo , Timo/embriologia , Timo/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Biomarcadores , Linhagem Celular , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/imunologia , Epitélio/metabolismo , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Ductos Pancreáticos/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/imunologia , RNA Mensageiro/genética , Timo/imunologia , Fatores de Tempo
3.
J Cell Sci ; 116(Pt 14): 3017-26, 2003 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-12808021

RESUMO

Epithelial cell responses to bacterial infection include induction of matrix metalloproteinase 7 (MMP-7). Here, we identify increased MMP-7 expression in the gastric epithelium in response to the oncogenic bacterium Helicobacter pylori, and report on the mechanisms and consequences for gastric epithelial cell migration. In patients infected with H. pylori, there was increased MMP-7 in gastric biopsies detected by western blot. MMP-7 was localized to the advancing edge of migrating gastric epithelial cell colonies, including lamellipodia. Rates of spreading of gastric gland cells were higher in H. pylori-infected cultures compared with control, and this was inhibited by antisense oligonucleotides to MMP-7. Complementary data were obtained in a gastric cancer cell line (AGS cells). In the latter, H. pylori induced expression of an MMP-7-luciferase promoter/reporter vector through mechanisms that involved activation of Rho and Rac. RhoA acted through activation of both NF-kappaB and AP-1, whereas Rac activated NF-kappaB but not AP-1. MMP-7 is commonly upregulated in gastric cancer; since H. pylori is a recognized gastric carcinogen, the data suggest a new mechanism by which the bacterium might predispose towards gastric neoplasia.


Assuntos
Células Epiteliais/citologia , Mucosa Gástrica/metabolismo , Helicobacter pylori/metabolismo , Metaloproteinase 7 da Matriz/fisiologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Células Epiteliais/enzimologia , Genes Reporter , Vetores Genéticos , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica , Oligonucleotídeos Antissenso/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Radioimunoensaio , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Regulação para Cima , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/fisiologia
4.
Gastroenterology ; 123(1): 271-80, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12105855

RESUMO

BACKGROUND & AIMS: The gastric hormone gastrin regulates acid secretion, gene expression, and the functional development and cellular composition of the gastric mucosa. Using a gene array, we sought to identify major, novel, gastrin-regulated genes. METHODS: A cancer gene array was probed with samples from the gastric cancer cell line AGS, expressing the gastrin-cholecystokinin(B) receptor and stimulated with gastrin. The expression of gastrin-regulated genes was further characterized by Western blots and enzyme-linked immunosorbent assay in tissue and blood of hypergastrinemic patients. Gene expression was studied using promoter-luciferase reporter constructs. RESULTS: Plasminogen activator inhibitor 2 (PAI-2) was identified as a major, previously unknown target of gastrin in the gastric cancer cell line AGS. The relevance was confirmed by showing elevated tissue and plasma PAI-2 in hypergastrinemic patients (pernicious anemia and multiple endocrine neoplasia type 1). PAI-2 promoter-luciferase constructs showed that gastrin stimulated expression via pathways involving Galpha and Gbetagamma subunits, protein kinase C, RhoA, and the transcription factors CREB and AP1. The tumor suppressor menin inhibited transcription. In addition, gastrin stimulated expression in adjacent cells via a paracrine mechanism involving protein kinase C and RhoA but not CREB. CONCLUSIONS: A gene array showed PAI-2 to be a novel gastrin-regulated gene, stimulated in part through CREB and AP-1 and inhibited by the tumor suppressor menin.


Assuntos
Gastrinas/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Neoplasias/fisiologia , Inibidor 2 de Ativador de Plasminogênio/genética , Proteínas Proto-Oncogênicas , Proteínas rho de Ligação ao GTP/fisiologia , Idoso , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Feminino , Mucosa Gástrica/metabolismo , Gastrinas/sangue , Gastrinas/farmacologia , Genes Reporter , Humanos , Luciferases/genética , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Comunicação Parácrina , Inibidor 2 de Ativador de Plasminogênio/sangue , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteína Quinase C/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/fisiologia , Células Tumorais Cultivadas
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