Ingrid B. Renes

The regulation of intestinal mucin MUC2 expression by short-chain fatty acids: implications for epithelial protection.

SCFAs (short-chain fatty acids), fermentation products of bacteria, influence epithelial-specific gene expression. We hypothesize that SCFAs affect goblet-cell-specific mucin MUC2 expression and thereby alter epithelial protection. In the present study, our aim was to investigate the mechanisms that regulate butyrate-mediated effects on MUC2 synthesis. Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability, and MUC2 protein expression were analysed. SCFA-responsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel-shift assays. The effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. Butyrate (at 1 mM), as well as propionate, induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM propionate, MUC2 mRNA levels remained increased. Promoter-regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 10-15 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. In conclusion, 1 mM butyrate and 1-15 mM propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP-1 and acetylation/methylation of histones at the MUC2 promoter.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon

Objective Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. Design In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1β and tumour necrosis factor α. Results In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. Conclusions Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

The Human Mucin MUC4 Is Transcriptionally Regulated by Caudal-related Homeobox, Hepatocyte Nuclear Factors, Forkhead Box A, and GATA Endodermal Transcription Factors in Epithelial Cancer Cells

The human gene MUC4 encodes a large transmembrane mucin that is developmentally regulated and expressed along the undifferentiated pseudostratified epithelium, as early as 6.5 weeks during fetal development. Immunohistochemical analysis of Muc4 expression in developing mouse lung and gastrointestinal tract showed a different spatio-temporal pattern of expression before and after cytodifferentiation. The molecular mechanisms governing MUC4 expression during development are, however, unknown. Hepatocyte nuclear factors (HNF), forkhead box A (FOXA), GATA, and caudal-related homeobox transcription factors (TFs) are known to control cell differentiation of gut endoderm derived-tissues during embryonic development. They also control the expression of cell- and tissue-specific genes and may thus control MUC4 expression. To test this hypothesis, we studied and deciphered the molecular mechanisms responsible for MUC4 transcriptional regulation by these TFs. Experiments using small interfering RNA, cell co-transfection, and site-directed mutagenesis indicated that MUC4 is regulated at the transcriptional level by CDX-1 and -2, HNF-1α and -1β, FOXA1/A2, HNF-4α and -4γ, and GATA-4, -5, and -6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays. Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair.

Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration

The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (−76%) and SGLT1 (−77%) and between I-FABP (−52%) and L-FABP (-45%). Decreases in GLUT5 (−53%), MUC2 (-43%), and TFF3 (−54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

Mucin Muc2 Deficiency and Weaning Influences the Expression of the Innate Defense Genes Reg3β, Reg3γ and Angiogenin-4

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2−/−) on the expression of regenerating islet-derived protein 3 beta (Reg3β), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2−/− and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3β, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3β and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3β, Reg3γ, and Ang4 differed between WT and Muc2−/− mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3β and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2−/− mice at P28, where and when expression levels of Reg3β, Reg3γ, and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3β and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2−/− mice. The inverse correlation between colitis development and Reg3β, Reg3γ, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

Forkhead box transcription factors Foxa1 and Foxa2 are important regulators of Muc2 mucin expression in intestinal epithelial cells.

The mucin Muc2 is the main component of the intestinal mucus layer and thus plays important roles in intestinal protection. Therefore, it is important to understand its regulation during goblet cell differentiation. Foxa1 and Foxa2 forkhead box transcription factors (TFs) participate in transcriptional programs governing intestinal cell differentiation. Using immunohistochemistry, we showed a spatio-temporal pattern of expression of both TFs in developing and adult mouse intestine and their expression in Muc2-expressing intestinal cells. Down-regulation of Foxa1 and Foxa2 by RNA interference in cultured intestinal cells decreased Muc2 mRNA level by half, and abolished Muc2 protein expression. Chromatin immunoprecipitation and gel shift assays showed that these two TFs directly bind to the Muc2 promoter. Co-transfection experiments indicated that both TFs activate the Muc2 promoter and that mutations of three Foxa cis-elements inhibit Muc2 transactivation. In conclusion, this work identifies Foxa1 and Foxa2 as important regulators of Muc2 expression in the intestine.

Combined defects in epithelial and immunoregulatory factors exacerbate the pathogenesis of inflammation: Mucin 2-interleukin 10-deficient mice

Expression of the mucin MUC2, the structural component of the colonic mucus layer, is lowered in ulcerative colitis. Furthermore, interleukin (IL)-10 knockout (IL-10−/−) mice develop colitis and have reduced Muc2 levels. Our aim was to obtain insight into the role of Muc2 and IL-10 in epithelial protection. Muc2-IL-10 double-knockout (Muc2/IL-10DKO) mice were characterized and compared to Muc2 knockout (Muc2−/−), IL-10−/− and wild-type (WT) mice. Clinical symptoms, intestinal morphology and differences in epithelial-specific protein levels were analyzed. In addition, levels of the pro-inflammatory cytokines in colonic tissue and serum were determined. IL-10−/− mice were indistinguishable from WT mice throughout this experiment and showed no clinical or histological signs of colitis. Muc2/IL-10DKO and Muc2−/− mice showed significant growth retardation and clinical signs of colitis at 4 and 5 weeks, respectively. Muc2/IL-10DKO mice had a high mortality rate (50% survival/5 weeks) compared to the other types of mice (100% survival). Microscopic analysis of the colon of Muc2/IL-10DKO mice showed mucosal thickening, increased proliferation, superficial erosions and a diminished Muc4 expression. Furthermore, pro-inflammatory cytokines were significantly upregulated, both in tissue (mRNA) and systemically in Muc2/IL-10DKO mice. In conclusion, Muc2/IL-10DKO mice develop colitis, which is more severe in every aspect compared to Muc2−/− and IL-10−/− mice. These data indicate that (i) in case of Muc2 deficiency, the anti-inflammatory cytokine IL-10 can control epithelial damage, though to a limited extent and (ii) the mucus layer is most likely a key factor determining colitis.

Protection of the Peyer's patch-associated crypt and villus epithelium against methotrexate-induced damage is based on its distinct regulation of proliferation

The crypt and villus epithelium associated with Peyers patches (PPs) is largely spared from methotrexate (MTX)‐induced damage, compared with the non‐patch (NP) epithelium. To assess the mechanism(s) preventing damage to the PP epithelium after MTX treatment, epithelial proliferation, apoptosis, and cell functions were studied in a rat‐MTX model. Small intestinal segments containing PPs were excised after MTX treatment. Epithelial proliferation and apoptosis were assessed by detection of incorporated BrdU and cleaved caspase‐3, respectively. Epithelial functions were determined by the expression of cell type‐specific gene products at mRNA and protein level. Before and after MTX treatment, the number of BrdU‐positive cells was higher in PP crypts than in NP crypts. BrdU incorporation was diminished in NP crypts, while in PP crypts incorporation was hardly affected. In PP and NP crypts, similar and increased levels of cleaved caspase‐3‐positive cells were observed after MTX. The enterocyte markers, sucrase‐isomaltase, sodium‐glucose co‐transporter 1, glucose transporters 2 and 5, and intestinal and liver fatty acid binding protein, were down‐regulated after MTX in NP epithelium but not in PP epithelium. In contrast, expression of the goblet cell markers, Muc2 and trefoil factor 3, and the Paneth cell marker, lysozyme, was maintained after MTX in both PP and NP epithelium. In conclusion, as MTX‐induced apoptosis was similar in PP and NP crypts, the protection of the PP epithelium seems to be based on differences in the regulation of epithelial proliferation. Enterocyte function in the PP epithelium was unaffected by MTX treatment. Goblet and Paneth cell function was maintained in both NP and PP epithelium. Copyright

Colonic gene expression patterns of mucin muc2 knockout mice reveal various phases in colitis development

Background: Mucin Muc2 knockout (Muc2−/−) mice spontaneously develop colitis. Methods: To identify genes and biological responses which play a pivotal role during colitis development in Muc2−/− mice, gene expression profiles of colonic tissues from 2‐ and 4‐week‐old Muc2−/− and wildtype mice were determined using microarrays. Results: The majority of highly upregulated genes in 2‐week‐old as well as 4‐week‐old Muc2−/− mice were primarily involved in immune responses related to antigen processing/presentation, B‐cell and T‐cell receptor signaling, leukocyte transendothelial migration, and Jak‐STAT signaling. Specifically, Muc2−/− mice expressed high levels of immunoglobulins, murine histocompatibility‐2, proinflammatory cytokines, chemokines, and antimicrobial proteins. Additionally, in 4‐week‐old Muc2−/− mice, expression of genes involved in cell structure related pathways was significantly altered. Particularly, the tight junction‐associated gene claudin‐10 was upregulated, whereas claudin‐1 and claudin‐5 were downregulated. Furthermore, 4‐week‐old Muc2−/− mice showed increased expression of genes regulating cell growth in conjunction with increased crypt length and increased epithelial proliferation. Conclusions: Muc2‐deficiency leads to an active inflammatory response in 2‐ and 4‐week‐old Muc2−/− mice as demonstrated by the altered expression in immune response related genes. In addition, 4‐week‐old Muc2−/− mice also showed a decrease in epithelial barrier function and an increase in epithelial proliferation as indicated by, respectively, the altered expression in tight junction‐related genes and upregulation of genes stimulating cell growth. Remarkably, upregulation of genes stimulating cell growth correlated with increased crypt length and increased epithelial proliferation in 4‐week‐old Muc2−/− mice. Together, these data demonstrate that there are distinct phases in colitis development in 2–4‐week‐old Muc2−/− mice. (Inflamm Bowel Dis 2011;)

Epithelial functions of the residual bowel after surgery for necrotising enterocolitis in human infants.

Objectives: Information on epithelial functions of the residual small or colonic bowel after resection for necrotising enterocolitis (NEC) in human infants is scarce. Our aim is to evaluate epithelial functions in the intestinal resection margins of tissue obtained at bowel resection for acute NEC and consecutive stoma closure. Materials and Methods: Epithelial morphology, proliferation, and protein expression were (immuno)histochemically studied. Results: Acute NEC was associated with severe and mild epithelial damage varying from epithelial loss to fairly unaffected epithelium. Epithelial proliferation was increased both at acute NEC and at stoma closure. In acute NEC, lactase, glucose transporter-2 and -5 expression was down-regulated in severely affected epithelium, whereas sucrase-isomaltase and intestinal fatty acid binding protein expression was maintained. Goblet cells continued to express mucin 2 and trefoil factor 3, however, their numbers were decreased. Moreover, in acute NEC, Paneth cells were weakly lysozyme positive and were reduced in number. At stoma closure, expression of the above cell type–specific markers had completely been re-established. Conclusions: Residual bowel after resection for acute NEC shows a disturbed epithelial proliferation/differentiation balance. Acute NEC was associated with downregulation of distinct enterocyte-specific proteins. Because of goblet cell and Paneth cell loss in acute NEC, mucosal barrier, and defense functions may be impaired.