Jan Dekker

Gastrointestinal expression and partial cDNA cloning of murine Muc2

To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM resembled other mucins in physicochemical properties. Anti-MCM recognized MCM as well as rat and human MUC2 on Western blots, interacting primarily with peptide epitopes, indicating that MCM was identical to murine Muc2. Using anti-MCM and previously characterized anti-human and anti-rat MUC2 antibodies, we identified a murine Muc2 precursor in the colon of approximately 600 kDa, which appeared similar in size to rat and human MUC2 precursors. Western blotting, immunoprecipitation of metabolically labeled mucins, and immunohistochemistry showed that murine Muc2 was expressed in the colon and the small intestine but was absent in the stomach. To independently identify murine Muc2, we cloned a cDNA fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine Muc2. The NH2 terminus of murine Muc2 showed 86 and 75% identity to the corresponding rat and human MUC2 peptide sequences, respectively. Northern blotting with a murine Muc2 cDNA probe showed hybridization to a very large mRNA, which was expressed highly in the colon and to some extend in the small intestine but was absent in the stomach. In situ hybridization showed that the murine Muc2 mRNA was confined to intestinal goblet cells. In conclusion, by two independent sets of experiments we identified murine Muc2, which appears homologous to rat and human MUC2. Because Muc2 is prominently expressed in the colon, it is most likely to be the predominant mucin in the colonic mucus layer.To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM resembled other mucins in physicochemical properties. Anti-MCM recognized MCM as well as rat and human MUC2 on Western blots, interacting primarily with peptide epitopes, indicating that MCM was identical to murine Muc2. Using anti-MCM and previously characterized anti-human and anti-rat MUC2 antibodies, we identified a murine Muc2 precursor in the colon of ∼600 kDa, which appeared similar in size to rat and human MUC2 precursors. Western blotting, immunoprecipitation of metabolically labeled mucins, and immunohistochemistry showed that murine Muc2 was expressed in the colon and the small intestine but was absent in the stomach. To independently identify murine Muc2, we cloned a cDNA fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine Muc2. The NH2 terminus of murine Muc2 showed 86 and 75% identity to the corresponding rat and human MUC2 peptide sequences, respectively. Northern blotting with a murine Muc2 cDNA probe showed hybridization to a very large mRNA, which was expressed highly in the colon and to some extend in the small intestine but was absent in the stomach. In situ hybridization showed that the murine Muc2 mRNA was confined to intestinal goblet cells. In conclusion, by two independent sets of experiments we identified murine Muc2, which appears homologous to rat and human MUC2. Because Muc2 is prominently expressed in the colon, it is most likely to be the predominant mucin in the colonic mucus layer.

Epithelial proliferation, cell death, and gene expression in experimental colitis: alterations in carbonic anhydrase I, mucin MUC2, and trefoil factor 3 expression

AbstractAbstractn Background and aims. To gain insight in intestinal epithelial proliferation, cell death, and gene expression during experimental colitis rats were treated with dextran sulfate sodium (DSS) for 7xa0days.n Materials and methods. Proximal and distal colonic segments were excised on days 2, 5, 7, and 28. Epithelial proliferation, cell death, enterocyte gene expression (carbonic anhydrase I (CAxa0I) and goblet cell gene expression (mucin, MUC2; trefoil factor 3, TFF3) were studied immunohistochemically and biochemically.n Results. Proliferative activity was decreased in the proximal and distal colon at the onset of disease (day 2). However, during active disease (days 5–7) epithelial proliferation was increased in the entire proximal colon and in the proximity of ulcerations in the distal colon. During DSS treatment the number of apoptotic cells in the epithelium of both colonic segments was increased. In the entire colon surface enterocytes became flattened and CAxa0I negative during active disease (day 5–7). Additionally, CAxa0I levels in the distal colon significantly decreased during this phase. In contrast, during the regenerative phase (day 28) CAxa0I levels were restored in the distal colon and up-regulated in the proximal colon. During all disease phases increased numbers of goblet cells were observed in the surface epithelium of the entire colon. In the distal colon TFF3 expression extended to the bottom of the crypts during active disease. Finally, MUC2 and TFF3 expression was increased in the proximal colon during disease.n Conclusion. DSS affected the epithelium by inhibiting proliferation and inducing apoptosis. DSS-induced inhibition of CAxa0I expression indicates down-regulation of specific enterocyte functions. Accumulation of goblet cells in the surface epithelium and up-regulation of MUC2 and TFF3 expression in the proximal colon underline the importance of goblet cells in epithelial protection and repair, respectively.

Interleukin 10-Deficient Mice Exhibit Defective Colonic Muc2 Synthesis Before and After Induction of Colitis by Commensal Bacteria

Germ-free (GF) interleukin 10-deficient (IL-10−/−) mice develop chronic colitis after colonization by normal enteric bacteria. Muc2 is the major structural component of the protective colonic mucus. Our aim was to determine whether primary or induced aberrations in Muc2 synthesis occur in GF IL-10−/− mice that develop colitis after bacterial colonization. GF IL-10−/− and wild-type mice were colonized with commensal bacteria for various intervals up to 6 weeks. Colitis was quantified by histologic score and IL-12 secretion. Muc2 synthesis, total level of Muc2, and Muc2 sulfation were measured quantitatively. GF IL-10−/− mice showed 10-fold lower Muc2 synthesis and Muc2 levels compared with GF wild-type mice, but Muc2 sulfation was not different. When bacteria were introduced, IL-10−/− mice developed colitis, whereas wild-type mice remained healthy. Muc2 synthesis was unchanged in wild-type mice, but IL-10−/− mice showed a peak increase in Muc2 synthesis 1 week after bacterial introduction, returning to baseline levels after 2 weeks. Total Muc2 levels decreased 2-fold in wild-type mice but remained at stable low levels in IL-10−/− mice. Upon introducing bacteria, Muc2 sulfation increased 2-fold in wild-type mice, whereas in IL-10−/− mice Muc2 sulfation decreased 10-fold. In conclusion, a primary defect in colonic Muc2 synthesis is present in IL-10−/− mice, whereas bacterial colonization and colitis in these mice led to reduced Muc2 sulfation. These quantitative and structural aberrations in Muc2 in IL-10−/− mice likely reduce the ability of their mucosa to cope with nonpathogenic commensal bacteria and may contribute to their susceptibility to develop colitis.

Quantitative em study of projection terminals in the rat's AV thalamic nucleus. Autoradiographic and degeneration techniques compared

An attempt was made to determine whether the labeled amino acid tracing technique combined with electron microscope (EM) autoradiography can be used to identify reliably the terminals of different to a cell group in the brain, using the rats antero-ventral thalamic (AVTh) nucleus as a model. For this purpose in one group of rats [3H]leucine was injected in cingulate cortex, hippocampus and mammilary bodies, respectively, and in another group lesions were made in these structures. With one month autoradiographic exposure time it could be demonstrated that after cortical and hippocampal injections the radioactivity was transported to small terminals on distal dendrites, while after mammillary body injections it was transported to large terminals on proximal dendrites. After 4 months exposure time altogether 12% of the small terminals and 80% of the large ones had been labelled; A comparison with the degeneration findings showed that the EM autoradiography is in many respects more efficient in identifying the various terminals than the EM degeneration technique.

Morphology of rat's AV thalamic nucleus in light and electron microscopy

Abstract The structure of the rats antero-ventral thalamic (AV Th) nucleus has been investigated in order to provide background information for the accompanying study in which an attempt was made to identify the synaptic terminals of the different afferent fiber systems to this nucleus by means of both EM autoradiography and the EM degeneration techniques. Nissl stained sections showed that the rats AV Th nucleus contains mainly relatively light staining neurons which in Golgi material were found to possess tufted dendrites. In EM material three types of synaptic terminals were found which showed a topical distribution over the neuronal surface. Soma and stem dendrites carry a limited number of terminals with symmetrical synapses and flattened vesicles. Proximal dendrites carry mainly large asymmetrical synaptic terminals while distal dendrites are crowded with small asymmetrical synaptic terminals.

Fate of goblet cells in experimental colitis

We sought to correlate the characteristic changes in goblet cell morphology in the chronically inflamed large intestine of IL10−/− mice to specific changes in goblet cell gene expression. In healthy as well as IL10−/− mice, marked differences were found among the large intestinal regions in goblet cell morphology and gene expression. The mucin Muc2, which is a major determinant of goblet cell morphology, was expressed in most goblet cells, yet only in cells staining positive for both Alcian blue and high iron diamine. TFF3 was expressed in only a small subset of goblet cells. Inflamed colon of IL10−/− mice still contained high numbers of small, hypotrophic goblet cells with similar histochemical staining and Muc2 and TFF3 expression patterns, contradicting the often reported “goblet cell depletion” in colitis. Quantitatively, the Muc2 and TFF3 levels remained relatively stabile in IL10−/− mice. Muc2 in distal IL10−/− colon contained significantly less sulfate residues than in controls, which may compromise its protective properties.

The human mucus-glycoprotein MUC2 may be an innocent bystander in Helicobacter pylori infection

In the paper by Van de Bovenkamp et al. [1] recently published in Helicobacter, the authors claim that MUC5AC (MUC5AC mucin) and the carbohydrate structure Leb carried by MUC5AC are primary receptor structures for Helicobacter pylori in the gastric mucosa. We recently published results, partially concurrently with those of Van de Bovenkamp et al., in a series where we evaluated the expression profiles of mucins (MUC1, MUC2, MUC5AC and MUC6) in cases with and without H. pylori colonization in gastric mucosa with gastritis and intestinal metaplasia (IM) [2]. Our study showed adherence of H. pylori to MUC5AC-expressing foveolar cells and absence of colonization of the MUC6-expressing cells in the glands of nonmetaplastic gastric mucosa. These observations [2] are in agreement with those reported by Van de Bovenkamp et al. and support the conclusion that MUC5AC has a major role in H. pylori adhesion to the gastric mucosa [1]. We and other groups have also reported that most areas of IM do not show H. pylori colonization [2,3]. In our series, only four of the 22 biopsies displaying IM showed H. pylori colonization [2]. Curiously, in these four biopsies, IM was of the incomplete type with high levels of expression of MUC1 and MUC5AC and no expression of MUC2. All the other cases with IM (either complete or incomplete type) and without H. pylori adhesion showed expression of the intestinal mucin MUC2. The absence of MUC2 expression in the foci of incomplete IM with adherent microorganisms suggests that the colonization of H. pylori in incomplete IM (25% of the cases of our series) [2] may depend both on the expression of MUC1 and MUC5AC, and on the absence of MUC2. Taking into consideration our results and the observations by Van de Bovenkamp et al. [1], we suggest that H. pylori requires gastric mucins, particularly MUC5AC, as well as absence of the intestinal mucin MUC2, for the colonization of normal and metaplastic gastric tissues.