Muc2-Deficient Mice Spontaneously Develop Colitis, Indicating That MUC2 Is Critical for Colonic Protection

Background & Aims: Expression of mucin MUC2, the structural component of the colonic mucus layer, is lowered in inflammatory bowel disease. Our aim was to obtain insight in the role of Muc2 in epithelial protection. Methods: Muc2 knockout (Muc2−/−) and Muc2 heterozygous (Muc2+/−) mice were characterized and challenged by a colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and differences in intestine-specific protein and messenger RNA levels. Results: The Muc2−/− mice showed clinical signs of colitis (as of 5 weeks), aggravating as the mice aged. Microscopic analysis of the colon of Muc2−/− mice showed mucosal thickening, increased proliferation, and superficial erosions. Colonic goblet cells in the Muc2−/− mice were negative for Muc2, but trefoil factor 3 was still detectable. In Muc2−/− mice, transient de novo expression of Muc6 messenger RNA was observed in the distal colon. On day 2 of DSS treatment, the histologic damage was more severe in Muc2+/− versus wild-type (Muc2+/+) mice, but the disease activity index was not yet different. By day 7, the disease activity index and histologic score were significantly elevated in Muc2+/− versus Muc2+/+ mice. The disease activity index of the Muc2−/− mice was higher (versus both Muc2+/+ and Muc2+/− mice) throughout DSS treatment. The histologic damage in the DSS-treated Muc2−/− mice was different compared with Muc2+/+ and Muc2+/− mice, with many crypt abscesses instead of mucosal ulcerations. Conclusions: This study shows that Muc2 deficiency leads to inflammation of the colon and contributes to the onset and perpetuation of experimental colitis. Background & Aims: Expression of mucin MUC2, the structural component of the colonic mucus layer, is lowered in inflammatory bowel disease. Our aim was to obtain insight in the role of Muc2 in epithelial protection. Methods: Muc2 knockout (Muc2−/−) and Muc2 heterozygous (Muc2+/−) mice were characterized and challenged by a colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and differences in intestine-specific protein and messenger RNA levels. Results: The Muc2−/− mice showed clinical signs of colitis (as of 5 weeks), aggravating as the mice aged. Microscopic analysis of the colon of Muc2−/− mice showed mucosal thickening, increased proliferation, and superficial erosions. Colonic goblet cells in the Muc2−/− mice were negative for Muc2, but trefoil factor 3 was still detectable. In Muc2−/− mice, transient de novo expression of Muc6 messenger RNA was observed in the distal colon. On day 2 of DSS treatment, the histologic damage was more severe in Muc2+/− versus wild-type (Muc2+/+) mice, but the disease activity index was not yet different. By day 7, the disease activity index and histologic score were significantly elevated in Muc2+/− versus Muc2+/+ mice. The disease activity index of the Muc2−/− mice was higher (versus both Muc2+/+ and Muc2+/− mice) throughout DSS treatment. The histologic damage in the DSS-treated Muc2−/− mice was different compared with Muc2+/+ and Muc2+/− mice, with many crypt abscesses instead of mucosal ulcerations. Conclusions: This study shows that Muc2 deficiency leads to inflammation of the colon and contributes to the onset and perpetuation of experimental colitis. Ulcerative colitis and Crohn’s disease are two of the most important inflammatory bowel diseases (IBD), characterized by chronic inflammation and mucosal tissue damage of parts of the gastrointestinal tract. The etiology of these inflammatory disorders remains unknown, but research so far has shown that these diseases are caused by a combination of genetic, environmental, immunoregulatory, and epithelial factors.1Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1501) Google Scholar, 2Einerhand A.W. Renes I.B. Makkink M.K. van der Sluis M. Buller H.A. Dekker J. Role of mucins in inflammatory bowel disease important lessons from experimental models.Eur J Gastroenterol Hepatol. 2002; 14: 757-765Crossref PubMed Scopus (115) Google Scholar, 3Mashimo H. Wu D.C. Podolsky D.K. Fishman M.C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science. 1996; 274: 262-265Crossref PubMed Scopus (623) Google Scholar, 4Hermiston M.L. Gordon J.I. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.Science. 1995; 270: 1203-1207Crossref PubMed Scopus (592) Google Scholar, 5Van Klinken B.J. Dekker J. Buller H.A. Einerhand A.W. Mucin gene structure and expression protection vs. adhesion.Am J Physiol. 1995; 269: G613-G627PubMed Google Scholar, 6Panwala C.M. Jones J.C. Viney J.L. A novel model of inflammatory bowel disease mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis.J Immunol. 1998; 161: 5733-5744PubMed Google Scholar The epithelium and mucus layer in the intestinal tract form a physical barrier between the potential toxic and noxious agents present in the gut lumen and the underlying tissues. Goblet cells exert a vital role since they secrete molecules, which serve protective roles in the gut, like mucins and trefoil factors. Mucins are the building blocks of the mucus layer, and previous studies have shown that human, rat, and mouse colonic epithelium expresses mainly one secretory mucin in high amounts, MUC2.7Tytgat K.M. Buller H.A. Opdam F.J. Kim Y.S. Einerhand A.W. Dekker J. Biosynthesis of human colonic mucin Muc2 is the prominent secretory mucin.Gastroenterology. 1994; 107: 1352-1363Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 8Tytgat K.M. Bovelander F.J. Opdam F.J. Einerhand A.W. Buller H.A. Dekker J. Biosynthesis of rat MUC2 in colon and its analogy with human MUC2.Biochem J. 1995; 309: 221-229Crossref PubMed Scopus (57) Google Scholar, 9Herrmann A. Davies J.R. Lindell G. Martensson S. Packer N.H. Swallow D.M. Carlstedt I. Studies on the “insoluble” glycoprotein complex from the human colon. Identification of reduction-insensitive Muc2 oligomers and C-terminal cleavage.J Biol Chem. 1999; 274: 15828-15836Crossref PubMed Scopus (123) Google Scholar, 10van Klinken B.J. Einerhand A.W. Duits L.A. Makkink M.K. Tytgat K.M. Renes I.B. Verburg M. Buller H.A. Dekker J. Gastrointestinal expression and partial cDNA cloning of murine Muc2.Am J Physiol. 1999; 276: 115-124PubMed Google Scholar, 11Makkink M.K. Schwerbrock N.M. Mahler M. Boshuizen J.A. Renes I.B. Cornberg M. Hedrich H.J. Einerhand A.W. Buller H.A. Wagner S. Enss M.L. Dekker J. Fate of goblet cells in experimental colitis.Dig Dis Sci. 2002; 47: 2286-2297Crossref PubMed Scopus (26) Google Scholar The secretory mucin MUC2 is stored in bulky apical granules of the goblet cells and is the most important factor determining the goblet cell morphology.12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar, 13Yang W. Velcich A. Lozonschi I. Liang J. Nicholas C. Zhuang M. Bancroft L. Augenlicht L.H. Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2−/− mice.Am J Pathol. 2005; 166: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Trefoil factor 3 (Tff3) is a protein also expressed by goblet cells in the intestine and has been shown to play an essential role in the maintenance and repair of the intestinal mucosa.3Mashimo H. Wu D.C. Podolsky D.K. Fishman M.C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science. 1996; 274: 262-265Crossref PubMed Scopus (623) Google Scholar Damage to the epithelium, in particular those events affecting the protective properties as offered by the secretory products of the goblet cells, is a likely cause of the inflammation. Histologic analysis for patients with ulcerative colitis often shows depletion of recognizable goblet cells in the colonic epithelium.14Theodossi A. Spiegelhalter D.J. Jass J. Firth J. Dixon M. Leader M. Levison D.A. Lindley R. Filipe I. Price A. et al.Observer variation and discriminatory value of biopsy features in inflammatory bowel disease.Gut. 1994; 35: 961-968Crossref PubMed Scopus (146) Google Scholar Defects in the intestine, such as loss of E-cadherin expression, multidrug-resistant protein, or Tff3 deficiency, have been shown to lead to higher susceptibility to chronic inflammation that can progressively deteriorate the epithelial barrier function.3Mashimo H. Wu D.C. Podolsky D.K. Fishman M.C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science. 1996; 274: 262-265Crossref PubMed Scopus (623) Google Scholar, 4Hermiston M.L. Gordon J.I. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.Science. 1995; 270: 1203-1207Crossref PubMed Scopus (592) Google Scholar, 6Panwala C.M. Jones J.C. Viney J.L. A novel model of inflammatory bowel disease mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis.J Immunol. 1998; 161: 5733-5744PubMed Google Scholar Furthermore, it has been shown that in patients with ulcerative colitis, the activity of the mucosal inflammation correlates significantly with a decrease in MUC2 synthesis15Tytgat K.M. van der Wal J.W. Einerhand A.W. Buller H.A. Dekker J. Quantitative analysis of MUC2 synthesis in ulcerative colitis.Biochem Biophys Res Commun. 1996; 224: 397-405Crossref PubMed Scopus (128) Google Scholar and secretion,16Van Klinken B.J. Van der Wal J.W. Einerhand A.W. Buller H.A. Dekker J. Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis.Gut. 1999; 44: 387-393Crossref PubMed Scopus (116) Google Scholar again implying that the mucosal barrier plays a key role in the course of the disease. To more specifically address the importance of MUC2 in epithelial protection, Muc2-deficient (Muc2−/−) mice were generated through genetic inactivation of the murine Muc2 gene.12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar In these Muc2−/− mice, the intestinal goblet cells were seemingly absent. Interestingly, mice lacking Muc2 developed adenomas as of 6 months of age, which progressed to invasive adenocarcinoma in the small intestine as well as rectal tumors at an older age. Therefore, Muc2 seems to play a role in the suppression of intestinal cancer,12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar but the exact mechanism whereby Muc2 suppresses tumorigenesis remains unclear. To obtain insight in the role of Muc2 in epithelial barrier function, and thus its possible role in IBD, we characterized the Muc2 knockout (Muc2−/−) and heterozygous (Muc2+/−) mouse from birth to 16 weeks of age, before the development of adenomas, and challenged these mice with the colitis-inducing agent dextran sulfate sodium (DSS). This animal model is the first to provide the possibility to investigate the physiologic function of Muc2 in the intestine of the mouse under unchallenged and luminally challenged conditions. The previously described Muc2−/− mice on mixed genetic background12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar were backcrossed onto a 129SV (Charles River, Maastricht, The Netherlands) genetic background for 9 generations followed by intercrosses to generate mice homozygous for the Muc2 disruption. To obtain experimental groups with a minimal age difference between the different litters, all mice were generated from Muc2+/− breedings. Throughout the backcrossing procedure, the targeted Muc2 gene was monitored via polymerase chain reaction (PCR) assays performed on genomic DNA isolated from tail clips as described by Velcich et al.12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar All the mice were housed in the same specific pathogen–free environment with free access to standard rodent pellets (Special Diets Services, Witham, Essex, England) and acidified tap water in a 12-hour light/dark cycle. All animal care and procedures were conducted according to institutional guidelines (Erasmus MC Animal Ethics Committee, Rotterdam, The Netherlands). Wild-type (Muc2+/+), Muc2+/−, and Muc2−/− littermates were scored weekly until the age of 16 weeks to obtain a disease activity index (DAI) as described by Cooper et al17Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar (Table 1). Briefly, they were scored for the following: weight, softness of the stool, occult fecal blood,18Wahba N. An ortho-tolidine hydrochloride test for the detection of occult blood in faeces without dietary restrictions.J Clin Pathol. 1965; 18: 687-688Crossref PubMed Scopus (5) Google Scholar and general appearance of the mice. One hour before the mice were killed, bromodeoxyuridine (BrdU) 30 mg/kg body wt (Sigma Chemical Co, St Louis, MO) was injected intraperitoneally to be able to study epithelial proliferation.19Boshuizen J.A. Reimerink J.H. Korteland-van Male A.M. van Ham V.J. Koopmans M.P. Buller H.A. Dekker J. Einerhand A.W. Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice.J Virol. 2003; 77: 13005-13016Crossref PubMed Scopus (120) Google Scholar The time curve was started with 16 mice of each genotype: Muc2+/+, Muc2+/−, and Muc2−/−. At the ages of 5, 8, 12, and 16 weeks, groups of 4 male mice per genotype were killed. Small intestine and colon were excised immediately and either fixed in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS), stored in RNAlater (Qiagen, Venlo, The Netherlands) at −20°C, or frozen in liquid nitrogen and stored at −80°C.Table 1DAI ScoreScore% weight lossStool consistencyBlood lossAppearance0None (none)Normal droppingsNoneLively/normal10–17 (0–10)Loose droppingsHemoccult positiveHunched218–35 (10–20)DiarrheaGross bleedingStarey coat3>35 (>20)LethargicNOTE. Criteria were obtained by pooling all data and calculating quartiles. For the initial phenotyping experiment, the percentage weight loss was calculated by comparing the weight of the mice with corresponding wild-type littermates. For the experiment in which the animals were treated with DSS, percentage weight loss was calculated by using the initial body weight of the individual animals. The classification of the weight loss in the DAI as applied in the DSS experiment appears in parentheses. Open table in a new tab NOTE. Criteria were obtained by pooling all data and calculating quartiles. For the initial phenotyping experiment, the percentage weight loss was calculated by comparing the weight of the mice with corresponding wild-type littermates. For the experiment in which the animals were treated with DSS, percentage weight loss was calculated by using the initial body weight of the individual animals. The classification of the weight loss in the DAI as applied in the DSS experiment appears in parentheses. Tissue fixed in 4% (wt/vol) paraformaldehyde in PBS was prepared for light microscopy, and 5-μm-thick sections were stained with H&E to study histologic changes as described previously.20Renes I.B. Verburg M. Van Nispen D.J. Taminiau J.A. Buller H.A. Dekker J. Einerhand A.W. Epithelial proliferation, cell death, and gene expression in experimental colitis alterations in carbonic anhydrase I, mucin MUC2, and trefoil factor 3 expression.Int J Colorectal Dis. 2002; 17: 317-326Crossref PubMed Scopus (72) Google Scholar Grading of intestinal inflammation was determined as described by Rath et al21Rath H.C. Herfarth H.H. Ikeda J.S. Grenther W.B. Hamm Jr, T.E. Balish E. Taurog J.D. Hammer R.E. Wilson K.H. Sartor R.B. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.J Clin Invest. 1996; 98: 945-953Crossref PubMed Scopus (693) Google Scholar with slight modifications (Table 2). In particular, the scoring of both the percentage of the total ulcer area per section and the number of crypt abscesses for each section was modified. To detect differences in mucosal and epithelial thickness in the colon, 10 well-oriented crypts or epithelial cells were chosen per intestinal segment and measured using calibrated Leica Image Manager 500 software (Leica Microsystems BV, Rijswijk, The Netherlands). All scores were obtained in a blinded fashion by 2 independent investigators.Table 2Histologic Score to Quantify the Degree of Gastrointestinal InflammationCriterionScore01234Goblet cells—↓↓↓↓↓↓↓↓↓Mucosa thickening—↑↑↑↑↑↑↑↑↑Inflammatory cells—↑↑↑↑↑↑↑↑↑Submucosa cell infiltration——↑↑↑↑↑↑Destruction of architecture———↑↑↑Ulcers (epithelial cell surface)0%0–25%25%–50%50%–75%75%–100%Crypt abscesses01–34–67–9>10NOTE. Grading of intestinal inflammation was determined as described by Rath et al21Rath H.C. Herfarth H.H. Ikeda J.S. Grenther W.B. Hamm Jr, T.E. Balish E. Taurog J.D. Hammer R.E. Wilson K.H. Sartor R.B. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.J Clin Invest. 1996; 98: 945-953Crossref PubMed Scopus (693) Google Scholar and adapted. In particular, the scoring of both the percentage of the total of ulcer area of the section and the number of crypt abscesses for each section was modified. Open table in a new tab NOTE. Grading of intestinal inflammation was determined as described by Rath et al21Rath H.C. Herfarth H.H. Ikeda J.S. Grenther W.B. Hamm Jr, T.E. Balish E. Taurog J.D. Hammer R.E. Wilson K.H. Sartor R.B. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.J Clin Invest. 1996; 98: 945-953Crossref PubMed Scopus (693) Google Scholar and adapted. In particular, the scoring of both the percentage of the total of ulcer area of the section and the number of crypt abscesses for each section was modified. Five-micrometer-thick sections were cut and prepared for immunohistochemistry as described previously22Verburg M. Renes I.B. Meijer H.P. Taminiau J.A. Buller H.A. Einerhand A.W. Dekker J. Selective sparing of goblet cells and Paneth cells in the intestine of methotrexate-treated rats.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1037-G1047PubMed Google Scholar using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine as staining reagent. Expression of Muc2 was detected using anti-Muc2 antibody H-300 (1:1000 in PBS; SC-15334, Santa Cruz, SanverTech, Heerhugowaard, The Netherlands). To detect goblet cells, anti-Tff3 (1:3000 in PBS; a generous gift from Dr D. K. Podolsky3Mashimo H. Wu D.C. Podolsky D.K. Fishman M.C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science. 1996; 274: 262-265Crossref PubMed Scopus (623) Google Scholar) was used. As a marker for enterocyte differentiation in the colon, villin was used, which was detected with an anti-villin antibody (1:2000 in PBS, generously given by Dr S. Robine23Pringault E. Arpin M. Garcia A. Finidori J. Louvard D. A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture.EMBO J. 1986; 5: 3119-3124Crossref PubMed Scopus (72) Google Scholar). To visualize BrdU incorporation, the sections were incubated with anti-BrdU (Boehringer Mannheim, Mannheim, Germany) as described previously.19Boshuizen J.A. Reimerink J.H. Korteland-van Male A.M. van Ham V.J. Koopmans M.P. Buller H.A. Dekker J. Einerhand A.W. Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice.J Virol. 2003; 77: 13005-13016Crossref PubMed Scopus (120) Google Scholar, 20Renes I.B. Verburg M. Van Nispen D.J. Taminiau J.A. Buller H.A. Dekker J. Einerhand A.W. Epithelial proliferation, cell death, and gene expression in experimental colitis alterations in carbonic anhydrase I, mucin MUC2, and trefoil factor 3 expression.Int J Colorectal Dis. 2002; 17: 317-326Crossref PubMed Scopus (72) Google Scholar CD3+ cells were detected using an anti-human CD3 (DAKO, Heverlee, Belgium; 1:800 diluted in 1% bovine serum albumin, 0.1% Triton X-100 in PBS). Additionally, nonspecific binding was reduced by blocking with TENG-T (10 mmol/L Tris-HCl, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.25% [wt/vol] gelatin, 0.05% [wt/vol] Tween 20). Total RNAs from mouse tissues were prepared using the QIAamp RNA midi-kit (Qiagen) following the manufacturer’s protocol and treated with deoxyribonuclease (Qiagen). Total RNA (1.5 μg) was used to prepare first-strand complementary DNA (Advantage RT-for-PCR kit; BD Biosciences, Clontech, Alphen aan den Rijn, The Netherlands) PCR was performed on 5 μL of complementary DNA using primer pairs specific for Muc5ac (5′-GAGGCCAACAAGGTAGAGCACA-3′ and 5′- TGGGACAGCAGCAGTATTCAGT-3′),24Jonckheere N. Van Der Sluis M. Velghe A. Buisine M.P. Sutmuller M. Ducourouble M.P. Pigny P. Buller H.A. Aubert J.P. Einerhand A.W. Van Seuningen I. Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1.Biochem J. 2004; 377: 797-808Crossref PubMed Google Scholar Muc5b (5′-GAGGTCAACATCACCTTCTGC-3′ and 5′- TCTCATGGTCAGTTGTGCAGG-3′), Muc6 (5′-TGGTCGAAGTACTCATTCTGG-3′ and 5′-GTGGCTTGTGTGGCAACGCC-3′), and β-actin (5′-TCACGCCATCCTGCGTCTGGACT-3′ and 5′- CCGGACTCATCGTACTCCT-3′) as an internal standard.25Escande F. Porchet N. Bernigaud A. Petitprez D. Aubert J.P. Buisine M.P. The mouse secreted gel-forming mucin gene cluster.Biochim Biophys Acta. 2004; 1676: 240-250Crossref PubMed Scopus (39) Google Scholar PCR products were 295, 319, 569, and 582 base pairs, respectively. PCR reactions were performed in 50-μL final solutions as described previously26Mesquita P. Jonckheere N. Almeida R. Ducourouble M.P. Serpa J. Silva E. Pigny P. Silva F.S. Reis C. Silberg D. Van Seuningen I. David L. Human MUC2 mucin gene is transcriptionally regulated by Cdx homeodomain proteins in gastrointestinal carcinoma cell lines.J Biol Chem. 2003; 278: 51549-51556Crossref PubMed Scopus (127) Google Scholar using Invitrogen (Breda, The Netherlands) Taq polymerase. A positive control for each mucin PCR (stomach for Muc5ac and Muc6, larynx for Muc5b) and a negative control without complementary DNA were amplified with each PCR experiment. Annealing temperature was 58°C. PCR products were analyzed on 1.5% ethidium bromide–stained agarose gels run in 1× Tris/borate/EDTA buffer. As a marker, a 100–base pair DNA ladder (Invitrogen) was used. RNA isolation, complementary DNA preparation, and PCR for Muc6 was performed as described above. The resulting 569–base pair PCR product was isolated using the Qiagen gel extraction kit (Qiagen), ligated into the pGEM-Teasy vector (Promega, Madison, WI), and verified for sequence integrity using T7, SP6 primers and ABI Prism 310 and software (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands). Subsequently, the Muc6 fragment was subcloned into the pBluescript KS vector (Stratagene, La Jolla, CA) using the EcoRI restriction enzyme. The pBluescript KS vector contains T3 and T7 RNA polymerase sites. The digoxigenin-11 uridine triphosphate–labeled sense (T3) and anti-sense Muc-6 (T7) RNA probes were prepared according to the manufacturer’s protocol (Roche, Almere, The Netherlands). Nonradioactive in situ hybridization was performed according to the method already described27Lindenbergh-Kortleve D.J. Rosato R.R. van Neck J.W. Nauta J. van Kleffens M. Groffen C. Zwarthoff E.C. Drop S.L. Gene expression of the insulin-like growth factor system during mouse kidney development.Mol Cell Endocrinol. 1997; 132: 81-91Crossref PubMed Scopus (50) Google Scholar with slight modifications.28Renes I.B. Verburg M. Bulsing N.P. Ferdinandusse S. Buller H.A. Dekker J. Einerhand A.W. Protection of the Peyer’s patch-associated crypt and villus epithelium against methotrexate-induced damage is based on its distinct regulation of proliferation.J Pathol. 2002; 198: 60-68Crossref PubMed Scopus (31) Google Scholar, 29van der Sluis M. Melis M.H. Jonckheere N. Ducourouble M.P. Buller H.A. Renes I. Einerhand A.W. Van Seuningen I. The murine Muc2 mucin gene is transcriptionally regulated by the zinc-finger GATA-4 transcription factor in intestinal cells.Biochem Biophys Res Commun. 2004; 325: 952-960Crossref PubMed Scopus (44) Google Scholar The messenger RNA (mRNA) expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 as well as the endogenous housekeeping gene β-actin were quantified using real-time PCR analysis (TaqMan chemistry) based on the intercalation of SYBR Green on an ABI Prism 7700 sequence detection system (PE Applied Biosystems). Primers combinations for β-actin (3′-GGGACCTGACGGACTAC-5′, 3′-TGCCACAGGATTCCATAC-5′), IL-1β (3′-CCCCAACTGGTACATCA-5′, 3′-AGAATGTGCCATGGTTTC-5′), IL-6 (3′-CCCAACAGACCTGTCTAT-5′, 3′-GGCAAATTTCCTGATTAT-5′), and TNF-α (3′-TGGCCTCCCTCTCATC-5′, 3′-GGCTGGCACCACTAGTT-5′) were designed using the OLIGO 6.22 software (Molecular Biology Insights, Cascade, CO) and purchased from Invitrogen. All primers had a melting temperature (Tm; nearest neighbor method) between 65°C and 66.5°C. All PCRs performed with comparable efficiencies of 95% or higher. The PCR was performed using 1× SYBR Green Master mix (Bioké, Leiden, The Netherlands), 1× ROX reference dye, 15 pmol forward and reverse primers, and 5 μL 5× diluted complementary DNA (RNA isolation and complementary DNA synthesis as described above) from each mouse as a template in a total reaction volume of 50 μL in a MicroAmp optical 96-well plate and covered with MicroAmp optical caps (Applied Biosystems). Samples were heated for 10 minutes at 95°C and amplified for 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. To verify the amplification efficiency within each experiment, a serial dilution of complementary DNA derived from an RNA pool of control colon samples in dH2O was amplified in duplicate on each plate. Because all PCRs performed with equal efficiencies, relative mRNA expression levels of TNF-α and IL-1β for each mouse can directly be normalized for input RNA against the β-actin expression of the mice. For this, the relative mRNA expression levels of these target genes were calculated according to the comparative cycle time (Ct) method,30Meijerink J. Mandigers C. van de Locht L. Tonnissen E. Goodsaid F. Raemaekers J. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.J Mol Diagn. 2001; 3: 55-61Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar following this equation: Relative mRNA Expression = 2−(Ct target − Ct β-actin) × 100%. One week before DSS administration, 8-week-old, specified pathogen–free, male Muc2+/+, Muc2+/−, and Muc2−/− littermates were divided into groups (6 mice in each group that received DSS and 4 mice in the control group), given free access to autoclaved tap water, and weighed daily to adjust to the experimental conditions. During the experiment, daily prepared autoclaved tap water supplemented with 2.5% (wt/vol) DSS (37–40 kilodaltons; TdB Consultancy, Uppsala, Sweden) was administered for 5 days, followed by a recovery period during which the DSS was omitted from the drinking water. Scoring of the mice was performed daily, using the DAI as adapted from Cooper et al17Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar as described above. A group of animals was killed on day 2 (onset of disease) and day 8 (severe disease), and a control group was killed at the end of the experiment (no DSS). Tissue segments were obtained, processed, and scored as described above. All data are expressed as mean ± SEM. Statistical significance of the 3 different groups of mice during time was assessed by one-way analysis of variance test and Tukey t test. The Mann–Whitney U test was used to analyze changes in mRNA expression assessed by real-time PCR (Prism, version 4.00; GraphPad software, San Diego, CA). The data were considered statistically significant at P < .05. The Muc2-deficient mouse model12Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (722) Google Scholar was used in this study to explore the in vivo function of Muc2 in the colon. There were no significant differences between the Muc2+/+ and Muc2+/− mice regarding body weight (Figure 1) at any time. Moreover, occult blood