Quality Is as Important as the Quantity: Role of Mucin Glycosylation on Intestinal Barrier Function

Abstract

An G, Wei B, Xia B, et al. (University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.) Increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived O-glycans. J Exp Med 2007;204:1417–1429.The intestinal mucus layer covers the gastrointestinal epithelium to form an effective barrier against harmful luminal microflora. Mucin-type glycoproteins, the major constituent of the mucus layer, are glycosylated primarily by O-linked oligosaccharides (O-glycans). In this study, the authors have investigated the biological function of the core 3-derived O-glycans, the predominant O-glycan in the colon, in the pathogenesis of colitis and colorectal carcinoma.To characterize the role of core 3-derived O-glycans in the pathogenesis of intestinal inflammation, An et al developed mice lacking core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT/core 3 synthase), an enzyme vital in the synthesis of core 3-derived O-glycans. The authors confirmed that C3GnT-/- mice did not express core 3-derived O-glycans. C3GnT-/- mice had a substantial reduction of Muc2 protein compared with C3GnT+/+ mice, although mRNA levels of Muc2 as well as total number of goblet cells were not significantly different between the groups. C3GnT-/- mice had increased intestinal permeability to FITC-dextran as well as increased levels of 16S bacterial rDNA in their colon compared to their wild-type (WT) counterparts.The authors then looked at the susceptibility of C3GnT-/- mice to dextran sodium sulfate (DSS)-induced colitis. C3GnT-/- mice had worse clinical scores (as measured by weight loss, diarrhea, fecal bleeding, and shortened colon length) and histologic scores (as measured by ulceration, crypt damage, and inflammatory cell infiltration) compared with WT mice. When exposed to DSS, C3GnT-/- mice had increased infiltration of T cells and monocytes/macrophages in their lamina propria. Splenocytes from C3GnT-/- mice showed increased production of proinflammatory cytokines like tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-17. Interestingly, the authors noted that the expression of TNF-α, IFN-γ, and IL-17 by intraepithelial lymphocytes and lamina propria lymphocytes in C3GnT-/- was high, even in the absence of DSS, suggesting an inflammatory-prone status in the intestines of these mice.The authors next demonstrated that C3GnT-/- had a 5-fold increased incidence of colonic tumors when given azoxymethane/DSS (AOM/DSS) treatment. They hypothesized that the underlying mechanism was increased epithelial cell proliferation and activation of the Wnt signaling pathway, a key signaling pathway in colon cancer development.Thus, An et al concluded that core 3-derived O-glycans play an important role in protecting against colitis and colon cancer by fortifying the barrier defense mechanism and decreasing the interaction between the immune cells and colonic microflora.CommentMucins, expressed by the colonic goblet cells, play a protective role against a variety of colonic insults (Clin Microbiol Rev 2006;19:315–337; Eur J Gastroenterol Hepatol 2002;14:757–765). Mucin depletion and alterations in the quality of mucin is a characteristic histologic feature of ulcerative colitis. Alterations in mucin are also seen in the majority of colon cancers associated with ulcerative colitis (Cancer Metastasis Rev 206:260–276; Front Biosci 2001;6:D1321–D1357). Further, animal studies have shown that certain strains of genetically engineered mice deficient in MUC-2 develop spontaneous colitis. However, it has been difficult to reconcile a causal relationship between MUC-2 deficiency and colitis in humans because decreased MUC-2 levels are not a consistent finding in patients with colitis and MUC-2 single nucleotide polymorphism has not been associated with human disease. This manuscript is among the first to show a causal association between quality of mucin (as determined by glycosylation) and barrier dysfunction, an early epithelial defect that precedes an inflammatory response.C3GnT is the only enzyme responsible for the biosynthesis of core 3 O-glycans (J Biol Chem 277;12802–12809) and thus C3GnT-/- mice serve as an attractive model to study the function of core 3-derived O-glycans. One of the main conclusions of the study is that C3GnT-/- mice are more susceptible to DSS-induced colitis compared with C3GnT+/+ mice. A proposed mechanism for the increased susceptibility of C3GnT-/- mice to DSS is that these mice exhibit increased epithelial permeability at baseline. In support of altered barrier as an inciting event, C3GnT-/- mice exhibit increased 16S bacterial ribosomal DNA in the colonic mucosa as well as increased levels of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in splenic CD4 cells and increased intraepithelial lymphocytes. It should be pointed out that the increased permeability is an indirect effect of mucin depletion in that increased bacterial translocation and induction of cytokines as a result of mucus depletion likely play a role. It is interesting that C3GnT-/- mice did not progress to develop spontaneous colitis despite modest Th1 activation. Mesenteric lymph nodes did not show a significant increase in ribosomal DNA, suggesting that the response was confined to the mucosa and perhaps limited by the mucosal immune system.The second main conclusion of this study was that the C3GnT-/- mice were more at risk to develop colorectal cancer after AOM/DSS treatment. The rationale could be that impaired intestinal barrier may result in activation of mucosal immune cells, which can provoke abnormal epithelial cell growth that contributes to cancer. Decreased Muc2 observed in these mice could play a role as several studies have shown that Muc2 expression is decreased in colorectal adenocarcinoma (Gastroenterology 1994;107:160–172; J Histochem Cytochem 1996;44:1161–1166). Although the observation is correlative at this point, increased cell proliferation and activation of the Wnt pathway in C3GnT-/- mice, even in the absence of AOM, make them more prone to develop colon cancer. Along the same lines, Iwai et al observed that core 3 synthase is down-regulated in human colon cancer cell lines and intensely suppresses the metastatic potential of carcinoma cells (Proc Natl Acad Sci U S A 2005;102:4572–4577). Apart from core 3 O-glycans, several studies have shown that core 1 O-glycans and core 2 O-glycans also play a significant part in the pathogenesis of colitis and colon cancer (Oncogene 2006;25:3267–3276; Glycobiology 1994;4:873–884). Further studies are needed to validate the role of C3GnT deficiency and colon cancer.It is interesting that C3GnT-/- mice did not develop spontaneous inflammation despite a 60% decrease in MUC-2 levels. Several factors such as preserved goblet cell number and intestinal trefoil factor-3 levels may explain this observation. This observation also underscores the importance of mucin quality over quantity in the barrier function. In early studies that examined mucin quality in human inflammatory bowel disease (IBD) and animal models of IBD, altered MUC-2 levels were associated with a change in mucin quality (Digestion 2001;63[Suppl 1]:28–31; Int J Mol Med 2006;18:721–727). It would be important to determine whether C3GnT enzyme levels and/or C3GnT genetic polymorphism in human IBD extend the intriguing observations to human disease.In summary, this study demonstrates a novel role for core 3-derived O-glycans in barrier function. The observations are exciting and highlight an important aspect of mucin, namely, that the quality of the mucin is as important as the quantity for its barrier protective role. Further studies need to be undertaken to fully characterize the role of O-glycans in human colonic disease. An G, Wei B, Xia B, et al. (University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.) Increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived O-glycans. J Exp Med 2007;204:1417–1429. The intestinal mucus layer covers the gastrointestinal epithelium to form an effective barrier against harmful luminal microflora. Mucin-type glycoproteins, the major constituent of the mucus layer, are glycosylated primarily by O-linked oligosaccharides (O-glycans). In this study, the authors have investigated the biological function of the core 3-derived O-glycans, the predominant O-glycan in the colon, in the pathogenesis of colitis and colorectal carcinoma. To characterize the role of core 3-derived O-glycans in the pathogenesis of intestinal inflammation, An et al developed mice lacking core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT/core 3 synthase), an enzyme vital in the synthesis of core 3-derived O-glycans. The authors confirmed that C3GnT-/- mice did not express core 3-derived O-glycans. C3GnT-/- mice had a substantial reduction of Muc2 protein compared with C3GnT+/+ mice, although mRNA levels of Muc2 as well as total number of goblet cells were not significantly different between the groups. C3GnT-/- mice had increased intestinal permeability to FITC-dextran as well as increased levels of 16S bacterial rDNA in their colon compared to their wild-type (WT) counterparts. The authors then looked at the susceptibility of C3GnT-/- mice to dextran sodium sulfate (DSS)-induced colitis. C3GnT-/- mice had worse clinical scores (as measured by weight loss, diarrhea, fecal bleeding, and shortened colon length) and histologic scores (as measured by ulceration, crypt damage, and inflammatory cell infiltration) compared with WT mice. When exposed to DSS, C3GnT-/- mice had increased infiltration of T cells and monocytes/macrophages in their lamina propria. Splenocytes from C3GnT-/- mice showed increased production of proinflammatory cytokines like tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-17. Interestingly, the authors noted that the expression of TNF-α, IFN-γ, and IL-17 by intraepithelial lymphocytes and lamina propria lymphocytes in C3GnT-/- was high, even in the absence of DSS, suggesting an inflammatory-prone status in the intestines of these mice. The authors next demonstrated that C3GnT-/- had a 5-fold increased incidence of colonic tumors when given azoxymethane/DSS (AOM/DSS) treatment. They hypothesized that the underlying mechanism was increased epithelial cell proliferation and activation of the Wnt signaling pathway, a key signaling pathway in colon cancer development. Thus, An et al concluded that core 3-derived O-glycans play an important role in protecting against colitis and colon cancer by fortifying the barrier defense mechanism and decreasing the interaction between the immune cells and colonic microflora. CommentMucins, expressed by the colonic goblet cells, play a protective role against a variety of colonic insults (Clin Microbiol Rev 2006;19:315–337; Eur J Gastroenterol Hepatol 2002;14:757–765). Mucin depletion and alterations in the quality of mucin is a characteristic histologic feature of ulcerative colitis. Alterations in mucin are also seen in the majority of colon cancers associated with ulcerative colitis (Cancer Metastasis Rev 206:260–276; Front Biosci 2001;6:D1321–D1357). Further, animal studies have shown that certain strains of genetically engineered mice deficient in MUC-2 develop spontaneous colitis. However, it has been difficult to reconcile a causal relationship between MUC-2 deficiency and colitis in humans because decreased MUC-2 levels are not a consistent finding in patients with colitis and MUC-2 single nucleotide polymorphism has not been associated with human disease. This manuscript is among the first to show a causal association between quality of mucin (as determined by glycosylation) and barrier dysfunction, an early epithelial defect that precedes an inflammatory response.C3GnT is the only enzyme responsible for the biosynthesis of core 3 O-glycans (J Biol Chem 277;12802–12809) and thus C3GnT-/- mice serve as an attractive model to study the function of core 3-derived O-glycans. One of the main conclusions of the study is that C3GnT-/- mice are more susceptible to DSS-induced colitis compared with C3GnT+/+ mice. A proposed mechanism for the increased susceptibility of C3GnT-/- mice to DSS is that these mice exhibit increased epithelial permeability at baseline. In support of altered barrier as an inciting event, C3GnT-/- mice exhibit increased 16S bacterial ribosomal DNA in the colonic mucosa as well as increased levels of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in splenic CD4 cells and increased intraepithelial lymphocytes. It should be pointed out that the increased permeability is an indirect effect of mucin depletion in that increased bacterial translocation and induction of cytokines as a result of mucus depletion likely play a role. It is interesting that C3GnT-/- mice did not progress to develop spontaneous colitis despite modest Th1 activation. Mesenteric lymph nodes did not show a significant increase in ribosomal DNA, suggesting that the response was confined to the mucosa and perhaps limited by the mucosal immune system.The second main conclusion of this study was that the C3GnT-/- mice were more at risk to develop colorectal cancer after AOM/DSS treatment. The rationale could be that impaired intestinal barrier may result in activation of mucosal immune cells, which can provoke abnormal epithelial cell growth that contributes to cancer. Decreased Muc2 observed in these mice could play a role as several studies have shown that Muc2 expression is decreased in colorectal adenocarcinoma (Gastroenterology 1994;107:160–172; J Histochem Cytochem 1996;44:1161–1166). Although the observation is correlative at this point, increased cell proliferation and activation of the Wnt pathway in C3GnT-/- mice, even in the absence of AOM, make them more prone to develop colon cancer. Along the same lines, Iwai et al observed that core 3 synthase is down-regulated in human colon cancer cell lines and intensely suppresses the metastatic potential of carcinoma cells (Proc Natl Acad Sci U S A 2005;102:4572–4577). Apart from core 3 O-glycans, several studies have shown that core 1 O-glycans and core 2 O-glycans also play a significant part in the pathogenesis of colitis and colon cancer (Oncogene 2006;25:3267–3276; Glycobiology 1994;4:873–884). Further studies are needed to validate the role of C3GnT deficiency and colon cancer.It is interesting that C3GnT-/- mice did not develop spontaneous inflammation despite a 60% decrease in MUC-2 levels. Several factors such as preserved goblet cell number and intestinal trefoil factor-3 levels may explain this observation. This observation also underscores the importance of mucin quality over quantity in the barrier function. In early studies that examined mucin quality in human inflammatory bowel disease (IBD) and animal models of IBD, altered MUC-2 levels were associated with a change in mucin quality (Digestion 2001;63[Suppl 1]:28–31; Int J Mol Med 2006;18:721–727). It would be important to determine whether C3GnT enzyme levels and/or C3GnT genetic polymorphism in human IBD extend the intriguing observations to human disease.In summary, this study demonstrates a novel role for core 3-derived O-glycans in barrier function. The observations are exciting and highlight an important aspect of mucin, namely, that the quality of the mucin is as important as the quantity for its barrier protective role. Further studies need to be undertaken to fully characterize the role of O-glycans in human colonic disease. Mucins, expressed by the colonic goblet cells, play a protective role against a variety of colonic insults (Clin Microbiol Rev 2006;19:315–337; Eur J Gastroenterol Hepatol 2002;14:757–765). Mucin depletion and alterations in the quality of mucin is a characteristic histologic feature of ulcerative colitis. Alterations in mucin are also seen in the majority of colon cancers associated with ulcerative colitis (Cancer Metastasis Rev 206:260–276; Front Biosci 2001;6:D1321–D1357). Further, animal studies have shown that certain strains of genetically engineered mice deficient in MUC-2 develop spontaneous colitis. However, it has been difficult to reconcile a causal relationship between MUC-2 deficiency and colitis in humans because decreased MUC-2 levels are not a consistent finding in patients with colitis and MUC-2 single nucleotide polymorphism has not been associated with human disease. This manuscript is among the first to show a causal association between quality of mucin (as determined by glycosylation) and barrier dysfunction, an early epithelial defect that precedes an inflammatory response. C3GnT is the only enzyme responsible for the biosynthesis of core 3 O-glycans (J Biol Chem 277;12802–12809) and thus C3GnT-/- mice serve as an attractive model to study the function of core 3-derived O-glycans. One of the main conclusions of the study is that C3GnT-/- mice are more susceptible to DSS-induced colitis compared with C3GnT+/+ mice. A proposed mechanism for the increased susceptibility of C3GnT-/- mice to DSS is that these mice exhibit increased epithelial permeability at baseline. In support of altered barrier as an inciting event, C3GnT-/- mice exhibit increased 16S bacterial ribosomal DNA in the colonic mucosa as well as increased levels of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in splenic CD4 cells and increased intraepithelial lymphocytes. It should be pointed out that the increased permeability is an indirect effect of mucin depletion in that increased bacterial translocation and induction of cytokines as a result of mucus depletion likely play a role. It is interesting that C3GnT-/- mice did not progress to develop spontaneous colitis despite modest Th1 activation. Mesenteric lymph nodes did not show a significant increase in ribosomal DNA, suggesting that the response was confined to the mucosa and perhaps limited by the mucosal immune system. The second main conclusion of this study was that the C3GnT-/- mice were more at risk to develop colorectal cancer after AOM/DSS treatment. The rationale could be that impaired intestinal barrier may result in activation of mucosal immune cells, which can provoke abnormal epithelial cell growth that contributes to cancer. Decreased Muc2 observed in these mice could play a role as several studies have shown that Muc2 expression is decreased in colorectal adenocarcinoma (Gastroenterology 1994;107:160–172; J Histochem Cytochem 1996;44:1161–1166). Although the observation is correlative at this point, increased cell proliferation and activation of the Wnt pathway in C3GnT-/- mice, even in the absence of AOM, make them more prone to develop colon cancer. Along the same lines, Iwai et al observed that core 3 synthase is down-regulated in human colon cancer cell lines and intensely suppresses the metastatic potential of carcinoma cells (Proc Natl Acad Sci U S A 2005;102:4572–4577). Apart from core 3 O-glycans, several studies have shown that core 1 O-glycans and core 2 O-glycans also play a significant part in the pathogenesis of colitis and colon cancer (Oncogene 2006;25:3267–3276; Glycobiology 1994;4:873–884). Further studies are needed to validate the role of C3GnT deficiency and colon cancer. It is interesting that C3GnT-/- mice did not develop spontaneous inflammation despite a 60% decrease in MUC-2 levels. Several factors such as preserved goblet cell number and intestinal trefoil factor-3 levels may explain this observation. This observation also underscores the importance of mucin quality over quantity in the barrier function. In early studies that examined mucin quality in human inflammatory bowel disease (IBD) and animal models of IBD, altered MUC-2 levels were associated with a change in mucin quality (Digestion 2001;63[Suppl 1]:28–31; Int J Mol Med 2006;18:721–727). It would be important to determine whether C3GnT enzyme levels and/or C3GnT genetic polymorphism in human IBD extend the intriguing observations to human disease. In summary, this study demonstrates a novel role for core 3-derived O-glycans in barrier function. The observations are exciting and highlight an important aspect of mucin, namely, that the quality of the mucin is as important as the quantity for its barrier protective role. Further studies need to be undertaken to fully characterize the role of O-glycans in human colonic disease.