epidermal-growth-factor and gastrin-17

This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.

Topics: Animals; Cell Differentiation; Chick Embryo; Colonic Neoplasms; Endothelial Cells; Epidermal Growth Factor; Gastrins; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Immune Sera; Intercellular Signaling Peptides and Proteins; Isoenzymes; Metalloendopeptidases; Mice; Neovascularization, Physiologic; Omeprazole; Receptor, Cholecystokinin B

The mechanism of regulation of mucus production in the gastric mucosa remains unclear. Recently, we established a gastric surface mucous cell line GSM06, which produces periodic acid-Shiff (PAS)-positive glycoconjugate (mucus) layers on the cell surface, from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. In this study, GSM06 cells were examined for its production of PAS-positive glycoconjugate layers to acid secretagogues and growth factors. The cells were cultured at nonpermissive temperature (39 degrees C) for 3-18 days and stained with PAS. Insulin (1-30 microg/ml; 0.29-8.6 microM) time- and dose-dependently increased production of glycoconjugates on the cell surface. When glycoconjugate layers produced by stimulation of insulin (3-30 microg/ml; 0.86-8.6 microM) were removed from the cell surface of GSM06 cells by a mild trypsin treatment, PAS-positive materials were remarkably decreased (day 18). In addition, morphological findings indicate that a high concentration of insulin (30 microg/ml; 8.6 microM) produced thick PAS-positive glycoconjugate layers just like normal gastric surface mucosa on the cell surface on day 18. In contrast, histamine (0.1-100 microM), carbachol (0.1-100 microM), gastrin-17 (0.1-100 nM), epidermal growth factor (0.01-10 ng/ ml; 1.7-1,700 pM), transforming growth factor-alpha (0.01-10 ng/ml; 1.8-1,800 pM), and fetal bovine serum (1-10%) did not increase glycoconjugate production. These findings suggest that insulin is a stimulator of glycoconjugate production, and stimulates production of glycoconjugate layers on the cell surface in the gastric surface mucous cell line GSM06.

Topics: Animals; Carbachol; Cattle; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gastric Mucosa; Gastrins; Glycoconjugates; Histamine; Hypoglycemic Agents; Insulin; Mice; Mice, Inbred C57BL; Nicotinic Agonists; Serum Albumin, Bovine; Stimulation, Chemical; Transforming Growth Factor alpha

An organ-culture system has been used to investigate the effect of certain gastrointestinal peptides on the morphology and cell proliferation of explants of azoxymethane (AOM)-treated colonic mucosa. Our aim was to ascertain whether such factors play a direct part in the maintenance of hyperplastic changes in the large intestine. Explants of AOM-treated colonic mucosa from 15 animals were maintained in a serum-free medium in the presence of either gastrin-17 (250 pg/ml and 250 ng/ml), peptide YY (80 pmol/l and 160 pmol/l) epidermal growth factor (EGF) (10 ng/ml and 100 ng/ml) or the C-terminal fragment of glucagon-37 (30 pmol/l) for a period of up to 7 days. Other explants (controls) received fresh medium only each day. After 1, 2, 3, 5 and 7 days of culture both experimental and control explants received vincristine (4 micrograms/ml) for 3 h prior to fixation. The proportion of vincristine-arrested metaphases within the explants was determined together with crypt length. Neither gastrin nor peptide YY was found to influence cell division at either concentration. Despite an initial inhibitory effect, both concentrations of EGF exerted a trophic effect which increased with time. The glucagon-37 fragment caused an immediate increase in proliferation which then declined as time progressed. None of these factors, however, were able to maintain the hyperplastic changes seen in the pre-culture samples of AOM-treated mucosae.

Topics: Animals; Azoxymethane; Cell Division; Epidermal Growth Factor; Gastrins; Gastrointestinal Hormones; Gastrointestinal Neoplasms; Glucagon-Like Peptides; Hormones; Intestinal Mucosa; Male; Mitotic Index; Organ Culture Techniques; Peptide YY; Peptides; Rats; Rats, Inbred Strains

Tyrosine phosphorylation seems to be a key event in the control of cellular growth. Several viral transforming proteins, including the src protein of Rous sarcoma virus, the p120 protein of Abelson leukaemia virus and the middle T antigen of polyoma virus, are phosphorylated by associated tyrosine kinases. The levels of kinase activity correlate with the transforming efficiency of the virus. The receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin are also phosphorylated by associated tyrosine kinase activities, which are stimulated by EGF, PDGF and insulin, respectively. The EGF-stimulated kinase and the src protein share similar substrate specificity for tyrosines immediately C-terminal to a sequence of acidic amino acids. Such a sequence is also found adjacent to the phosphotyrosine of middle T antigen, and in the homologous region of the hormone gastrin, adjacent to a tyrosine which is sulphated in approximately half the gastrin isolated from gastric mucosa. Reports that gastrin acts as a growth factor for cells of the gastrointestinal tract suggested that phosphorylation of this tyrosine might be physiologically more relevant than sulphation. We report here that synthetic human gastrin 17 is phosphorylated by the EGF-stimulated tyrosine kinase of A431 cell membranes. The Km values of 53-87 and 223-547 microM obtained in the presence and absence of EGF, respectively, are the lowest reported so far for this enzyme.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Gastrins; Humans; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases