Goblet cell-specific expression mediated by the MUC2 mucin gene promoter in the intestine of transgenic mice.
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The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5'-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases -2864 and +17 of the 5'-flanking region. (+info)
Epidermal growth factor system regulates mucin production in airways.
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Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor alpha (TGFalpha), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor alpha (TNFalpha). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNFalpha. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFalpha induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue-periodic acid-Schiff staining (reflecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNFalpha, EGF, or TGFalpha alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFalpha-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways. (+info)
Weaning anorexia may contribute to local inflammation in the piglet small intestine.
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Compromising alterations in villus-crypt structure are common in pigs postweaning. Possible contributions of local inflammatory reactions to villus-crypt alterations during the weaning transition have not been described. This study evaluated local inflammatory responses and their relationship with morphological changes in the intestine in 21-d-old pigs (n = 112) killed either at weaning (Day 0) or 0.5, 1, 2, 4 or 7 d after weaning to either milk- or soy-based pelleted diets. Cumulative intake averaged <100 g during the first 2 d postweaning, regardless of diet. During this period of weaning anorexia, inflammatory T-cell numbers and local expression of the matrix metalloproteinase stromelysin increased while jejunal villus height, crypt depth and major histocompatibility complex (MHC) class I RNA expression decreased. Upon resumption of feed intake by the fourth d postweaning, villus height and crypt depth, CD8(+) T cell numbers, MHC class I RNA expression and local expression of stromelysin returned to Day 0 values. Together the results indicate that inadequate feed intake during the immediate postweaning period may contribute to intestinal inflammation and thereby compromise villus-crypt structure and function. (+info)
Cloning of the gene gob-4, which is expressed in intestinal goblet cells in mice.
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We isolated the novel cDNA gob-4, which was shown to be expressed in intestinal goblet cells. The deduced amino acid sequence is similar to the gene coding for the Xenopus laevis cement gland-specific XAG-2. These sequence and expression data suggest this gene may be involved in the secretory function. (+info)
Nitric oxide synthase inhibitor attenuates intestinal damage induced by zinc deficiency in rats.
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A nitric oxide synthase (NOS) inhibitor, NG-nitro-L -arginine methyl ester (L-NAME), was given to zinc-deficient (ZD) rats to determine whether it prevents the intestinal damage usually observed under these conditions. Weanling male rats were given free access to a ZD diet (2 mg zinc/kg), whereas control rats including pair-fed (PF) and ad libitum consumption (AL) groups were given a zinc-supplemented (50.8 mg zinc/kg) diet for 4 wk. Half of the ZD rats received L-NAME (0.3 g/L in drinking water) for 3 wk starting at the wk 2 of the deficient period. Plasma zinc concentration in ZD rats was significantly lower (P < 0.05) than that of AL and PF rats. Administration of L-NAME did not alter this concentration. Intestinal zinc concentration did not differ among groups. However, metallothionein-1 (MT-1) mRNA level was significantly lower in the intestine of ZD rats than in AL or PF rats. Treatment of ZD rats with L-NAME did not affect this level. Intestinal microvascular permeability evaluated by Evans blue showed significantly higher extravasation in ZD rats than in AL rats, whereas L-NAME administration inhibited the extravasation. Expression of inducible NOS mRNA was observed in intestine of ZD but not of AL or PF rats, and there was no significant difference between ZD rats, regardless of L-NAME treatment. The activity ratio of inducible NOS to total NOS in ZD rats not receiving L-NAME was significantly higher than that in AL rats or ZD rats treated with L-NAME (P < 0.05). The number of apoptotic-positive and goblet cells in intestinal villi was significantly higher in ZD rats compared with AL or PF rats. L-NAME administration in ZD rats reversed this effect. These results indicate that inhibition of NOS ameliorates zinc deficiency-induced intestinal damage in rats. (+info)
Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation.
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Gastrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Gly's contribute to colonic mucosal proliferation in vivo. (+info)
IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo.
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Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells. (+info)
Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion.
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PURPOSE: To determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctival goblet cells and whether cholinergic agonists and VIP stimulate goblet cell secretion. METHODS: Immunofluorescence studies were performed using antibodies against the m1, m2, and m3 muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva were placed on filter paper and incubated for 15 to 120 minutes, with or without increasing concentrations of the cholinergic agonist carbachol or VIP. The muscarinic antagonist atropine and the muscarinic receptor-subtype-selective antagonists pirenzepine (M1), gallamine (M2), and 4-4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M3) were incubated with carbachol to determine specificity of receptor activation. RESULTS: Immunoreactivity to M2 and M3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M1 receptor was not on goblet cells but was on the stratitfied squamous cells. Immunoreactivity to VIPR2 was found on goblet cells with a localization similar to that of the M2 and M3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol, at 10(-4) M, induced a threefold increase in glycoconjugate secretion, which was completely inhibited by atropine (10(-5) M). Carbachol-induced secretion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by gallamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS: Cholinergic agonists, through M2 and/or M3 muscarinic receptors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, suggesting that goblet cell secretion in vivo is under the control of parasympathetic nerves. (+info)