Maria van der Sluis

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.

Methotrexate-induced mucositis in mucin 2-deficient mice

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy‐induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy‐induced mucositis. Mucositis was induced in Muc2 knockout (Muc2−/−) and wild type (Muc2+/+) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2–6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2+/+ and Muc2−/− mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase‐isomaltase and trefoil factor‐3 protein expression levels were comparable between Muc2+/+ and Muc2−/− mice. Up to Day 3 after MTX treatment, percentages of weight‐loss did not differ. Thereafter, Muc2+/+ mice showed a trend towards regaining weight, whereas Muc2−/− mice continued to lose weight. Surprisingly, MTX‐induced intestinal damage of Muc2−/− and Muc2+/+ mice was comparable. Prior to MTX‐injection, tumor necrosis factor‐α and interleukin‐10 mRNAs were upregulated in Muc2−/− mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy‐induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin‐10, an anti‐inflammatory cytokine before MTX‐treatment. J. Cell. Physiol. 210: 144–152, 2007.

Colitis development during the suckling-weaning transition in mucin Muc2-deficient mice

The mucin Muc2 is the structural component of the colonic mucus layer. Adult Muc2 knockout (Muc2(-/-)) mice suffer from severe colitis. We hypothesized that Muc2 deficiency induces inflammation before weaning of mothers milk [postnatal day (P) 14] with aggravation of colitis after weaning (P28). Muc2(-/-) and wild-type mice were killed at embryonic day 18.5 and P1.5, P7.5, P14, P21, and P28. Colonic morphology, influx of T cells, and goblet cell-specific protein expression was investigated by (immuno)histochemistry. Cytokine and Toll-like receptor (TLR) profiles in the colon were analyzed by quantitative RT-PCR. Muc2(-/-) mice showed an increased and persistent influx of Cd3ε-positive T cells in the colonic mucosa as of P1.5. This was accompanied by mucosal damage at P28 in the distal colon but not in the proximal colon. At P14, the proinflammatory immune response [i.e., increased interleukin (IL)-12 p35, IL-12 p40, and tumor necrosis factor-α, expression] in the distal colon of Muc2(-/-) mice presented with an immune suppressive response [i.e., increased Foxp3, transforming growth factor (TGF)-β1, IL-10, and Ebi3 expression]. In contrast, at P28, a proinflammatory response remained in the distal colon, whereas the immune suppressive response (i.e., Foxp3 and TGF-β1 expression) declined. The proximal colon of Muc2(-/-) mice did not show morphological damage and was dominated by an immune suppressive response at P14 and P28. Interestingly, changes in expression of TLRs and TLR-related molecules were observed in the distal colon at P14 and P28 and in the proximal colon only at P28. Colitis in Muc2(-/-) mice is limited before weaning by immune suppressive responses and exacerbates in the distal colon after weaning because of the decline in the immune suppressive response.

Threonine metabolism in the intestine of mice: loss of mucin 2 induces the threonine catabolic pathway.

Objectives: Previous studies have shown that the intestine uses a major part of the dietary threonine intake for the synthesis of the structural component of the protective intestinal mucus layer, the secretory mucin Muc2. In this context, the high intestinal demand for dietary threonine probably results from its incorporation into secretory mucins rich in threonine residues. Therefore, we compared threonine utilization in the colon of Muc2 knockout (Muc2−/−) and wild-type (Muc2+/+) mice to investigate the intestinal dietary threonine metabolism in the absence of Muc2, which results in inflammation of the colon. Materials and Methods: Concentrations and isotopic enrichment of threonine were measured by gas chromatography-isotope ratio mass spectrometry in the serum, colon, and colonic content of mice given a bolus [U-13C]threonine enterally. Results: We retrieved 37.8% and 40.9% of dietary threonine in Muc2+/+ and Muc2−/− mice, respectively, either as free or incorporated threonine. There were no major differences in the availability and concentration of free or incorporated threonine recovered in both serum and colon in both types of mice. However, the Muc2−/− mice did show overall significantly higher threonine oxidation rates compared with Muc2+/+ mice. Conclusions: In the absence of Muc2, dietary threonine is mainly used for constitutive protein synthesis or becomes a substrate for metabolic oxidation. This indicates that inflammation also requires high threonine amounts.