RESUMO
Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function, and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of PthlhLacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild-type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and posttranscriptional mechanisms.NEW & NOTEWORTHY We show that the growth factor parathyroid hormone-like hormone (PTHLH) is expressed in acid-secreting parietal cells of the mouse stomach. We define the specific PTHLH mRNA isoforms expressed in human stomach and in human gastric cancer cell lines and show that gastrin induces PTHLH expression via transcription activation and mRNA stabilization. Our findings suggest that PTHLH is a gastrin-regulated growth factor that might contribute to gastric epithelial cell homeostasis.
Assuntos
Gastrinas/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Células Parietais Gástricas/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Gastrinas/deficiência , Gastrinas/genética , Gastrinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Genótipo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Relacionada ao Hormônio Paratireóideo/genética , Células Parietais Gástricas/metabolismo , Fenótipo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Fatores de Tempo , Ativação Transcricional , Regulação para CimaRESUMO
Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSC(vect)) or short hairpin RNA (shRNA) targeting the Shh gene (stMSC(ShhKO)). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSC(Shh)), stMSC(vect), or stMSC(ShhKO) cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSC(Shh) and stMSC(vect) cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSC(ShhKO) cells. Compared with stMSC(ShhKO)-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSC(vect)- and wtMSC(Shh)-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation.
Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Gastrite/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/citologia , Mucosa Gástrica/patologia , Gastrinas/deficiência , Gastrite/patologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND & AIMS: Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice. METHODS: We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin. RESULTS: Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib. CONCLUSIONS: Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.
Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Proteínas Hedgehog/metabolismo , Gastropatias/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Células Epiteliais/patologia , Mucosa Gástrica/patologia , Gastrinas/administração & dosagem , Gastrinas/deficiência , Gastrinas/genética , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Infusões Parenterais , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Gastropatias/genética , Gastropatias/patologia , Fatores de Tempo , Via de Sinalização Wnt , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) promote gastric cancer in response to gastritis. In culture, BM-MSCs are prone to mutation with continued passage but it is unknown whether a similar process occurs in vivo in response to gastritis. AIM: The purpose of this study was to identify the role of chronic gastritis in the transformation of BM-MSCs leading to an activated cancer-promoting phenotype. METHODS: Age matched C57BL/6 (BL/6) and gastrin deficient (GKO) mice were used for isolation of stomach, serum and mesenchymal stem cells (MSCs) at 3 and 6 months of age. MSC activation was assessed by growth curve analysis, fluorescence-activated cell sorting and xenograft assays. To allow for the isolation of bone marrow-derived stromal cells and assay in response to chronic gastritis, IRG/Vav-1(Cre) mice that expressed both enhanced green fluorescent protein-expressing hematopoietic cells and red fluorescent protein-expressing stromal cells were generated. In a parabiosis experiment, IRG/Vav-1(Cre) mice were paired to either an uninfected Vav-1(Cre) littermate or a BL/6 mouse inoculated with Helicobacter pylori. RESULTS: GKO mice displayed severe atrophic gastritis accompanied by elevated gastric tissue and circulating transforming growth factor beta (TGFß) by 3 months of age. Compared to BM-MSCs isolated from uninflamed BL/6 mice, BM-MSCs isolated from GKO mice displayed an increased proliferative rate and elevated phosphorylated-Smad3 suggesting active TGFß signaling. In xenograft assays, mice injected with BM-MSCs from 6-month-old GKO animals displayed tumor growth. RFP+ stromal cells were rapidly recruited to the gastric mucosa of H. pylori parabionts and exhibited changes in gene expression. CONCLUSIONS: Gastritis promotes the in vivo activation of BM-MSCs to a phenotype reminiscent of a cancer-promoting cell.
Assuntos
Transformação Celular Neoplásica , Mucosa Gástrica/patologia , Gastrite Atrófica/patologia , Células-Tronco Mesenquimais/patologia , Fenótipo , Animais , Biomarcadores/metabolismo , Proliferação de Células , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Gastrinas/deficiência , Gastrite Atrófica/metabolismo , Gastrite Atrófica/microbiologia , Proteínas Hedgehog/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori , Immunoblotting , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parabiose , Reação em Cadeia da Polimerase em Tempo Real , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Progastrin (PG) is processed into a number of smaller peptides including amidated gastrin (Gamide), non-amidated glycine-extended gastrin (Ggly) and the C-terminal flanking peptide (CTFP). Several groups have reported that PG, Gamide and Ggly are biologically active in vitro and in vivo, and are involved in the development of gastrointestinal cancers. CTFP is bioactive in vitro but little is known of its effects in vivo. This study investigated the bioactivity of CTFP in vivo in normal tissues using gastrin deficient (GASKO) mice and in two mouse models of cancer (SCID mice bearing xenograft tumors expressing normal or knocked-down levels of gastrin and a mouse model of hepatic metastasis). As with Ggly, CTFP treatment stimulated colonic proliferation in GASKO mice compared to control. CTFP also significantly increased apoptosis in the gastric mucosa of male GASKO mice. CTFP did not appear to effect xenograft growth or the incidence of liver metastases. This is the first demonstration that CTFP has specific biological activity in vivo in the colon and stomach.
Assuntos
Apoptose , Divisão Celular/fisiologia , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Precursores de Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Gastrinas/deficiência , Gastrinas/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Fragmentos de Peptídeos/metabolismoRESUMO
Gastrins are peptide hormones important for gastric acid secretion and growth of the gastrointestinal mucosa. We have previously demonstrated that ferric ions bind to gastrins, that the gastrin-ferric ion complex interacts with the iron transport protein transferrin in vitro, and that circulating gastrin concentrations positively correlate with transferrin saturation in vivo. Here we report the effect of long-term dietary iron modification on gastrin-deficient (Gas(-/-)) and hypergastrinemic cholecystokinin receptor 2-deficient (Cck2r(-/-)) mice, both of which have reduced basal gastric acid secretion. Iron homeostasis in both strains appeared normal unless the animals were challenged by iron deficiency. When fed an iron-deficient diet, Gas(-/-) mice, but not Cck2r(-/-) mice, developed severe anemia. In iron-deficient Gas(-/-) mice, massive splenomegaly was also apparent with an increased number of splenic megakaryocytes accompanied by thrombocytosis. The expression of the mRNA encoding the iron-regulatory peptide hepcidin, Hamp, was down-regulated in both Cck2r(-/-) and Gas(-/-) mice on a low-iron diet, but, interestingly, the reduction was greater in Cck2r(-/-) mice and smaller in Gas(-/-) mice than in the corresponding wild-type strains. These data suggest that gastrins play an important direct role, unrelated to their ability to stimulate acid secretion, in hematopoiesis under conditions of iron deficiency.
Assuntos
Gastrinas/deficiência , Hematopoese , Deficiências de Ferro , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Transporte de Cátions/genética , Eritropoetina/sangue , Gastrinas/sangue , Hepcidinas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Colecistocinina B/fisiologia , Esplenomegalia/etiologia , Trombopoetina/sangueRESUMO
BACKGROUND: Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets. RESULTS: Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues. CONCLUSIONS: In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its direct targets. Furthermore we show that miR-449 induces senescence and apoptosis by activating the p53 pathway.
Assuntos
Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adenoma/patologia , Animais , Sequência de Bases , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Gastrinas/deficiência , Gastrinas/metabolismo , Infecções por Helicobacter/complicações , Infecções por Helicobacter/genética , Helicobacter pylori/fisiologia , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Dados de Sequência Molecular , Antro Pilórico/metabolismo , Antro Pilórico/patologia , Transdução de Sinais/genética , Neoplasias Gástricas/complicações , Proteína Supressora de Tumor p53/metabolismoRESUMO
Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Células Secretoras de Gastrina/metabolismo , Gastrinas/genética , Proteínas de Fluorescência Verde/genética , Helicobacter felis/genética , Envelhecimento/metabolismo , Animais , Azoximetano , Carcinógenos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sulfato de Dextrana , Regulação para Baixo , Jejum , Feto/metabolismo , Ácido Gástrico/metabolismo , Gastrinas/deficiência , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Camundongos , Camundongos Transgênicos , Antro Pilórico/metabolismo , Antro Pilórico/patologia , Somatostatina/administração & dosagem , Distribuição Tecidual , Transcrição Gênica , Transgenes , Regulação para CimaRESUMO
BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.
Assuntos
Transformação Celular Neoplásica/genética , Gastrinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Peptídeos/genética , Neoplasias Gástricas/prevenção & controle , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Modelos Animais de Doenças , Feminino , Gastrinas/deficiência , Gastrinas/genética , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter felis/patogenicidade , Histonas/metabolismo , Humanos , Masculino , Metilnitrosoureia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeos/deficiência , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Fatores de Tempo , Transfecção , Fator Trefoil-1 , Proteínas Supressoras de Tumor/metabolismoRESUMO
RegI (Regenerating islet derived-1) was originally characterized as a growth factor involved in pancreatic islet cell regeneration. It is also considered a gastrointestinal mitogen as its expression is increased during pathologies involving aberrant cell proliferation that can lead to neoplasia. However, the absolute requirement for RegI to directly stimulate gastric mucosal cell proliferation in vivo requires further investigation. We used RegI-deficient mice to determine the requirement for RegI in normal gastric mucosal development, wound healing, hyperplasia and tumourigenesis. We found that epithelial repair of acetic acid ulcers in compound mutant RegI/gastrin-deficient mice was significantly reduced compared to wild type, RegI-deficient or gastrin-deficient mice. In contrast, RegI was dispensable for normal gastric mucosal development, hyperplasia in HKbeta-deficient mice and tumourigenesis in gp130(F/F) mice. Although RegI was not required for proliferation in these pathological models, expression of multiple Reg family members were increased during gp130(F/F) tumourigenesis. Interestingly, loss of RegI in gp130(F/F) mice resulted in decreased expression of other Reg family members. Our results indicate that RegI and gastrin may synergistically regulate gastric mucosal proliferation during certain pathological settings like wound healing while gastric epithelial proliferation in other pathologies may require coordinated expression of multiple Reg genes.
Assuntos
Gastrinas/deficiência , Hiperplasia/fisiopatologia , Litostatina/genética , Neoplasias Gástricas/fisiopatologia , Úlcera Gástrica/patologia , Cicatrização , Animais , Sequência de Bases , Proliferação de Células , Mucosa Gástrica/citologia , Gastrinas/genética , Immunoblotting , Litostatina/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Alinhamento de Sequência , Cicatrização/genéticaRESUMO
PURPOSE OF REVIEW: Update on the role of gastrointestinal peptides in regulating gastric acid secretion. RECENT FINDINGS: A novel transgenic mouse that expresses the entire human gastrin gene locus in G-cells of gastrin-null mice will facilitate investigation of gastrin gene regulatory elements. Isolation of a highly homogeneous population of G-cells permits the elucidation of stimulatory and inhibitory ligands without the confounding presence of other neuroendocrine cells. The use of somatostatin receptor knockout mice demonstrated the plasticity of gastric acid regulatory mechanisms and compensation by upregulation of the galanin pathway which inhibits secretion by enterochromaffin-like cells. The importance of adenosine in regulating somatostatin release was shown using adenosine receptor knockout mice. SUMMARY: The importance of gastrointestinal peptides for regulating gastric acid is evident. Ongoing investigations will characterize the mechanisms underlying actions of these agents on gastric acid secretion, particularly with regard to their combinatorial effects and interplay with other acid-regulating pathways.
Assuntos
Ácido Gástrico/metabolismo , Hormônios Gastrointestinais/fisiologia , Animais , Células Secretoras de Gastrina/metabolismo , Células Secretoras de Gastrina/fisiologia , Gastrinas/deficiência , Gastrinas/genética , Gastrinas/fisiologia , Grelina/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeos Natriuréticos/fisiologia , Neuropeptídeos/fisiologia , Orexinas , Peptídeos/fisiologia , Receptores de Somatostatina/deficiência , Receptores de Somatostatina/genética , Somatostatina/fisiologiaRESUMO
The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant progastrin sequentially activated the transcription of Wnt and Notch target genes in progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of progastrin targeting for the clinical management of CRC.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Colorretais/metabolismo , Gastrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Gastrinas/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/biossíntese , Precursores de Proteínas/deficiência , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Notch/biossíntese , Receptores Notch/genética , Proteínas Serrate-Jagged , Transdução de Sinais , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima , beta Catenina/biossíntese , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The unprocessed gastrin precursor, progastrin (PG), is often overexpressed in colon cancer and other malignancies where it appears to stimulate colonic growth. Overexpression of progastrin also stimulates proliferation of normal colonic mucosa, but the receptors mediating these effects have not been identified. Here we report the development of a non-radioactive assay for assessment of PG binding to normal and transformed cells. Progastrin was labeled using biotinylation, and binding of biotinylated PG to cells was assessed using flow cytometry. Using this approach, we show strong and specific binding of PG to some cell lines (IEC-6, IEC-18, HT-29, COLO320) and minimal binding to others (HeLa, DC2.4, Jurkat). We also found PG binding to several non-gut epithelial lines, such as CHO-K1, COS-6 and HEK293 cells. The specificity of binding was confirmed by competition with cold, unlabeled PG but not with glycine-extended gastrin or amidated gastrin-17. Binding was not influenced by the presence of the classical CCK-2 receptor, but was partially dependent on the charged glycosaminoglycans (GAG). The analysis of primary colonic tissues isolated from wild type C57BL/6 mouse, revealed a small epithelial subpopulation of non-hematopoietic (CD45-negative) cells that strongly interacted with PG. Surprisingly, this population was greatly expanded in gastrin knockout mice. This non-radioactive, FACS-based assay should prove useful for further characterization of cells expressing the progastrin receptor.
Assuntos
Gastrinas/metabolismo , Trato Gastrointestinal/metabolismo , Precursores de Proteínas/metabolismo , Animais , Anexina A2/metabolismo , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Colo/citologia , Colo/metabolismo , Cricetinae , Cricetulus , Células Epiteliais/metabolismo , Citometria de Fluxo , Gastrinas/deficiência , Gastrinas/genética , Trato Gastrointestinal/citologia , Glicosaminoglicanos/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Colecistocinina B/metabolismoRESUMO
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 TempoRESUMO
Gastrin is secreted from neuroendocrine cells residing in the adult antrum called G cells, but constitutively low levels are also expressed in the duodenum and fetal pancreas. Gastrin normally regulates gastric acid secretion by stimulating the proliferation of enterochromaffin-like cells and the release of histamine. Gastrin and progastrin forms are expressed in a number of pathological conditions and malignancies. However, the DNA regulatory elements in the human versus the mouse gastrin promoters differ suggesting differences in their transcriptional control. Thus, we describe here the expression of the human gastrin gene using a bacterial artificial chromosome (BAC) in the antral and duodenal cells of gastrin null mice. All 5 founder lines expressed the 253 kb human gastrin BAC. hGasBAC transgenic mice were bred onto a gastrin null background so that the levels of human gastrin peptide could be analyzed by immunohistochemistry and radioimmunoassay without detecting endogenous mouse gastrin. We have shown previously that chronically elevated gastrin levels suppress somatostatin. Indeed, infusion of amidated rat gastrin depressed somatostatin levels, stimulated gastric acid secretion and an increase in the numbers of G cells in the antrum and duodenum. In conclusion, human gastrin was expressed in mouse enteroendocrine cells and was regulated by somatostatin. This mouse model provides a unique opportunity to study regulation of the human gastrin promoter in vivo by somatostatin and possibly other extracellular regulators contributing to our understanding of the mechanisms involved in transcriptional control of the human gene.
Assuntos
Gastrinas/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/genética , Duodeno/citologia , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Células Secretoras de Gastrina/efeitos dos fármacos , Células Secretoras de Gastrina/metabolismo , Gastrinas/deficiência , Gastrinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antro Pilórico/citologia , Antro Pilórico/efeitos dos fármacos , Antro Pilórico/metabolismo , Somatostatina/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Gastrin and gastrin receptor-deficient mice have been used for genetic dissection of the role of gastrins in maintaining gastric homeostasis and control of acid secretion. The gastrin knockout mice are achlorhydric due to inactivation of the ECL and parietal cells. Moreover, this achlorhydria is associated with intestinal metaplasia and bacterial overgrowth, which ultimately leads to the development of gastric tumours. The association between progastrin, progastrin-derived processing intermediates and colorectal carcinogenesis has also been examined through genetic or chemical cancer induction in several mouse models, although the clinical relevance of these studies remains unproven. While others have focused on models of increased gastrin production, the present review describes the lessons learned from gastrin-deficient mice. Study of these mice helps our understanding of how dysregulation of gastrin secretion may be implicated in human disease.
Assuntos
Acloridria/microbiologia , Gastrinas/genética , Neoplasias Gástricas/etiologia , Acloridria/complicações , Acloridria/patologia , Animais , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrinas/deficiência , Regulação da Expressão Gênica , Enteropatias/patologia , Metaplasia/microbiologia , Metaplasia/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Receptor de Colecistocinina B/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologiaRESUMO
BACKGROUND & AIMS: Gastrin deficiency and proton pump inhibitor treatment cause achlorhydria, which predisposes to disease. To elucidate the underlying molecular biology, we examined the changes in gastric gene expression in both types of achlorhydria. We also explored the associated changes in the gastric microflora and the long-term consequences of gastrin-deficient achlorhydria. METHODS: Expression profiles were generated from gastric RNA from wild-type mice, gastrin knockout (KO) mice, gastrin KO mice after 1 week of gastrin infusion, and wild-type mice treated for 1 month with a proton pump inhibitor. The results were confirmed using real-time polymerase chain reaction and immunohistochemistry. Selective media were used to characterize the gastric microflora. RESULTS: The number of gastric bacteria was increased in both gastrin KO and PPI-treated mice. The expression profiles revealed activation of immune defense genes, interferon-regulated response genes, and intestinal metaplasia of the gastric mucosa. In young gastrin-deficient mice, gastrin infusions reversed the changes. Over time, the changes accumulated, became irreversible, and progressed into metaplasia and polyp development. Finally, the study showed that gastrin regulated the expression of genes encoding extracellular matrix proteins. CONCLUSIONS: Independently of gastrin, achlorhydria is associated with gastric bacterial overgrowth and intestinal gene expression patterns and is associated with predisposition to disease. Gastrin is therefore essential for prevention of gastric disease, mainly through control of acid secretion but to a lesser extent also through control of gastric gene expression. The gastrin-deficient mouse serves as a useful new model for gastric metaplasia and neoplasia.
Assuntos
Mucosa Gástrica/patologia , Gastrinas/deficiência , Gastrite/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Fator de Transcrição CDX2 , DNA de Neoplasias/genética , Modelos Animais de Doenças , Feminino , Mucosa Gástrica/metabolismo , Gastrinas/uso terapêutico , Gastrite/genética , Gastrite/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Hormônios/deficiência , Hormônios/uso terapêutico , Imuno-Histoquímica , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Metaplasia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Neoplasias Gástricas/genética , Neoplasias Gástricas/prevenção & controle , Fatores de Transcrição/genéticaRESUMO
Previous studies demonstrated that mice with a null mutation in the gene encoding the hormone gastrin have impaired gastric acid secretion. Hence, the aim of this study was to evaluate changes in the acid-secreting parietal cell in gastrin-deficient (GAS-KO) mice. Analysis of several transcripts encoding parietal cell proteins involved in gastric acid secretion showed reduced abundance in the GAS-KO stomach, including H+,K+-ATPase alpha- and beta-subunits, KCNQ1 potassium channel, aquaporin-4 water channel, and creatine kinase B, which were reversed by gastrin infusion for 1 wk. Although mRNA and protein levels of LIM and SH3 domain-containing protein-1 (LASP-1) were not greatly changed in the mutant, there was a marked reduction in phosphorylation, consistent with its proposed role as a cAMP signal adaptor protein associated with acid secretion. A more comprehensive analysis of parietal cell gene expression in GAS-KO mice was performed using the Affymetrix U74AV2 chip with RNA from parietal cells purified by flow cytometry to >90%. Comparison of gene expression in GAS-KO and wild-type mice identified 47 transcripts that differed by greater than or equal to twofold, suggesting that gastrin affects parietal cell gene expression in a specific manner. The differentially expressed genes included several genes in signaling pathways, with a substantial number (20%) known to be target genes for Wnt and Myc.
Assuntos
Gastrinas/metabolismo , Perfilação da Expressão Gênica , Células Parietais Gástricas/metabolismo , Animais , Proteínas do Citoesqueleto , Citometria de Fluxo , Ácido Gástrico/metabolismo , Fundo Gástrico/citologia , Gastrinas/deficiência , Gastrinas/genética , ATPase Trocadora de Hidrogênio-Potássio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas de Neoplasias/metabolismo , Células Parietais Gástricas/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The antral hormone gastrin and its intestinal relative, cholecystokinin (CCK), are pivotal in the regulation of gastric functions. Other gastric hormones like ghrelin, peptide YY (PYY), and islet amyloid polypeptide (IAPP), however, also contribute to the regulation of acid secretion, motility, and feeding. Because gastrin and CCK are crucial for gastric homeostasis, we examined how loss of gastrin alone and gastrin plus CCK affected the expression of ghrelin, IAPP, and PYY and ghrelin secretion. The expression of ghrelin, IAPP, and PYY and the CCK-A receptor genes were examined in both gastrin and gastrin-CCK double-knockout (KO) mice using immunocytochemistry and quantitative RT-PCR. Ghrelin concentrations in plasma were measured using RIA. Gastrin and CCK were infused in gastrin-CCK KO mice using osmotic minipumps. The number of ghrelin cells and ghrelin gene expression were unaffected, albeit the ghrelin cells were located closer to the base of the glands in both KO mouse strains when freely fed. However, lack of both gastrin and CCK attenuated fasting-induced ghrelin expression and secretion. Fundic ghrelin cells expressed the CCK-A receptor, and ghrelin expression increased after CCK infusion. Furthermore, gastric IAPP and PYY expression as well as the number of IAPP- and PYY-containing cells were reduced in both gastrin and gastrin-CCK KO mice. Gastrin infusion increased gastric IAPP but not PYY expression. In conclusion, lack of gastrin plus CCK but not gastrin alone reduced ghrelin secretion in response to fasting through both direct and indirect mechanisms. Both gastrin and combined gastrin-CCK deficiency reduced the gastric IAPP and PYY expression.
Assuntos
Amiloide/genética , Colecistocinina/deficiência , Gastrinas/deficiência , Regulação da Expressão Gênica , Hormônios Peptídicos/genética , Peptídeo YY/genética , Amiloide/sangue , Animais , Colecistocinina/administração & dosagem , Colecistocinina/genética , Colecistocinina/farmacologia , Jejum , Fundo Gástrico/fisiologia , Gastrinas/administração & dosagem , Gastrinas/genética , Gastrinas/farmacologia , Grelina , Infusões Parenterais , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Camundongos Knockout , Hormônios Peptídicos/sangue , Reação em Cadeia da Polimerase , Antro Pilórico/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
The present study was aimed at investigating the role of gastrin in startle, startle habituation and prepulse inhibition (PPI). There were no significant differences between gastrin knockout mice and their wildtype controls in any of these baseline parameters. The disruption of PPI by treatment with 5 mg/kg of amphetamine was absent in gastrin knockout mice. However, a higher dose of amphetamine disrupted PPI in both genotypes. Similarly, treatment with the dopamine receptor agonist, apomorphine, the N-methyl-D-aspartate receptor antagonist, MK-801, and the serotonin-1A receptor agonist, 8-hydroxy-di-propylaminotetralin (8-OH-DPAT) modulated PPI similarly in gastrin knockout mice and wildtype controls. These data suggest a role of gastrin in the brain in modulating dopamine release in areas involved in PPI.