ABSTRACT
The Dmbt1 gene encodes alternatively spliced glycoproteins that are either membrane-associated or secreted epithelial products. Functions proposed for Dmbt1 include it being a tumor suppressor, having roles in innate immune defense and inflammation, and being a Golgi-sorting receptor in the exocrine pancreas. The heavily sulfated membrane glycoprotein mucin-like glycoprotein (Muclin) is a Dmbt1 product that is strongly expressed in organs of the gastrointestinal (GI) system. To explore Muclin's functions in the GI system, the Dmbt1 gene was targeted to produce Muclin-deficient mice. Muclin-deficient mice have normal body weight gain and are fertile. The Muclin-deficient mice did not develop GI tumors, even when crossed with mice lacking the known tumor suppressor p53. When colitis was induced by dextran sulfate sodium, there was no significant difference in disease severity in Muclin-deficient mice. Also, when acute pancreatitis was induced with supraphysiological caerulein, there was no difference in disease severity in the Muclin-deficient mice. Exocrine pancreatic function was impaired, as measured by attenuated neurohormonal-stimulated amylase release from Muclin-deficient acinar cells. Also, by [(35)S]Met/Cys pulse-chase analysis, traffic of newly synthesized protein to the stimulus-releasable pool was significantly retarded in Muclin-deficient cells compared with wild type. Thus Muclin deficiency impairs trafficking of regulated proteins to a stimulus-releasable pool in the exocrine pancreas.
Subject(s)
Gastrointestinal Tract/physiology , Mucins/deficiency , Amylases/metabolism , Animals , Blotting, Western , Calcium-Binding Proteins , Ceruletide , Colitis/chemically induced , Colitis/genetics , Colitis/pathology , DNA-Binding Proteins , Dextran Sulfate , Electrophoresis, Polyacrylamide Gel , Gastrointestinal Neoplasms/epidemiology , Gastrointestinal Neoplasms/genetics , Gastrointestinal Neoplasms/pathology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucins/genetics , Pancreas/cytology , Pancreas/drug effects , Pancreas/metabolism , Pancreatitis/chemically induced , Pancreatitis/pathology , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/physiology , Tumor Suppressor ProteinsABSTRACT
It is not clear how protein cargo is sorted to and retained in forming regulated secretory granules (RSG). Here, the sulfated mucin-type glycoprotein pro-Muclin was tested for its ability to induce RSG in the poorly differentiated rat pancreatic cell line AR42J. AR42J cells express RSG content proteins, but they fail to make granules. Adenovirus-pro-Muclin-infected AR42J cells store amylase, accumulate RSG, and respond to hormonal stimulation by secreting the stored protein. Expression of pro-Muclin combined with the inducing effect of dexamethasone resulted in a significant enhancement of the efficiency of regulated secretion. The effect of pro-Muclin was a strong decrease in constitutive secretion compared with dexamethasone-induction alone. A pro-Muclin construct missing the cytosolic tail domain was less effective at improving the efficiency of regulated secretion compared with the full-length construct. Increased expression of cargo (using adenovirus amylase) also modestly enhanced regulated secretion, indicating that part of pro-Muclin's effect may be due to increased expression of cargo protein. Overall, the data show that pro-Muclin acts as a sorting receptor that can induce RSG, and that its cytosolic tail is important in this process.
Subject(s)
Gene Expression Regulation/physiology , Mucoproteins/biosynthesis , Secretory Vesicles/physiology , Amylases/biosynthesis , Animals , Cell Line , Ceruletide/pharmacology , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Pancreas/cytology , Pancreas/drug effects , Protein Subunits/physiology , RatsABSTRACT
The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.
Subject(s)
Acids/metabolism , Cystic Fibrosis/metabolism , Duodenum/metabolism , Pancreas/metabolism , Amylases/blood , Animals , Chimera , Cystic Fibrosis/blood , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/deficiency , Enzyme Inhibitors/pharmacology , Gastrins/deficiency , Gene Expression/drug effects , Hydrogen-Ion Concentration , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Omeprazole/pharmacology , Pancreas/drug effects , Pancreas/pathology , Pancreatitis/blood , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The CFTR null mouse [cystic fibrosis (CF) mouse] has a severe intestinal phenotype that serves as a model for CF-related growth deficiency, meconium ileus, and distal intestinal obstructive syndrome. DNA microarray analysis was used to investigate gene expression in the CF mouse small intestine. Sixty-one genes exhibited a statistically significant twofold or greater increase in expression, and 98 genes were downregulated twofold or greater. Of the upregulated genes, most were associated with inflammation and included markers for cells of the innate immune system (mast cells and neutrophils) and for acute-phase genes (serum amyloid A and complement factors). The downregulated genes include 10 cytochrome P-450 genes; several are involved in lipid metabolism, and several are involved in various transport processes. Confirmation by quantitative RT-PCR showed gene expression was significantly increased for mast cell protease 2 (27-fold), hematopoietic cell transcript 1 (17-fold), serum amyloid A3 (2.9-fold), suppressor of cytokine signaling 3 (2.0-fold), leucine-rich alpha(2)-glycoprotein (21-fold), resistin-like molecule-beta (49-fold), and Muclin (2.5-fold) and was significantly decreased for cytochrome P-450 4a10 (28-fold) and cubilin (114-fold). Immune cell infiltration was confirmed histologically by staining for mast cells and neutrophils. These data demonstrate that the CF intestine exhibits an inflammatory state with upregulation of components of the innate immune system.
Subject(s)
Cystic Fibrosis/complications , Cystic Fibrosis/pathology , Enteritis/etiology , Intestine, Small/pathology , Animals , Calcium-Binding Proteins , Computer Systems , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/deficiency , DNA-Binding Proteins , Enteritis/pathology , Gene Expression Profiling , Hormones, Ectopic/metabolism , Immune System/pathology , In Situ Hybridization , Intestine, Small/physiopathology , Mice , Mice, Inbred C57BL , Mice, Inbred CFTR , Mucins/metabolism , Oligonucleotide Array Sequence Analysis , Resistin , Reverse Transcriptase Polymerase Chain Reaction , Serum Amyloid A Protein/metabolism , Tumor Suppressor ProteinsABSTRACT
Gastrin regulates gastric acid secretion, believed to be primarily responsible for killing ingested microbes. We examined gastric killing of gavaged E. coli in gastrin-deficient mice, which have decreased gastric acid production. Additionally, the expression of intestinal genes involved in epithelial protection were analyzed: the mucus layer glycoprotein muclin, the polymeric Ig receptor, trefoil factor 3, and small proline-rich protein 2a (sprr2a). Gastric pH was 2.5 pH units greater in gastrin-deficient mice, and E. coli survival was increased greater than 20-fold at 10 min after gavage compared to control. Muclin and sprr2a gene expression were significantly increased (2.0- and 2.6-fold) in the intestine, and antibiotic treatment reversed these effects. In conclusion, reduced gastric acid secretion results in increased survival of ingested microorganisms in gastrin-deficient mice. Bacterial survival is associated with increased expression of muclin and sprr2a in the intestine, indicating that these genes play protective roles in the intestine.
