Defining a Role for MUC2 Mucin‐Associated Proteins in Colitis and Restitution

Goblet cells produce MUC2 mucin that forms the mucus bilayers in the colon that protect the single layer of epithelial cells lining the gut. The mucus layer serves not only as a viscous physical barrier to protect the epithelial cell surface, but also provides a substrate and nutrient source for colonic microbiota. MUC2 mucin together with microbiota plays a major role in maintaining colonic health and is severely altered in infectious colitis and inflammatory bowel diseases. MUC2 mucin also contains numerous mucus‐associated proteins (MAPs) that are uncharacterized and there is much interest in what role they play in mucosal health and disease. These proteins include calcium‐activated chloride channel regulator 1 (CLCA1), F c fragment of IgG binding protein (FCGBP) and kallikrein 1 (KLK1). In this study we functionally characterized the expression of MAPs in colonic goblet cells and interrogated their role in colitis and restitution. To quantify MAPs expression we used human LS174T colonic goblet cells and generated CRISPR/Cas9 MUC2 KO cells to determine if MUC2 production regulated the expression of MAPs. Changes in mRNA and protein levels were determined by QPCR and Western blotting, respectively. To model epithelial injury in vitro , LS174T and CRISPR/Cas9 MUC2 KO cells were mechanically wounded. For in vivo studies we used Muc2 +/+ and Muc2 −/− littermates to basally characterize the expression of MAPs and to quantify what role they play in DSS‐induced colitis. In LS174T cells the temporal expression MUC2 , FCGBP , CLCA1 and KLK1 peaked at day 5 and remained high up to day 9, whereas CLCA1 was drastically increased in CRISPR/Cas9 MUC2 KO cells. In LS174T wounded cells, all MAPs increased as early as 2 days post wound that was more pronounced in CRISPR/Cas9 MUC2 KO cells as compared to the LS174T controls. In Muc2 +/+ and Muc2 −/− littermates and organoids derived from Muc2 +/+ and Muc2 −/− colons, MAPs were expressed at similar levels regardless of if Muc2 was present or not. In DSS colitis, MAPs mRNA transcripts and protein levels steadily decreased up to day 15 in Muc2 +/+ but not in Muc2 −/− littermates. Clca1 expression disappeared during peak of colitis (days 8–12) and Fcgbp, Clca1 and Klk1 were not restored by day 15 during restitution. In active ulcerative colitis (UC), colonic biopsies showed a marked increase in MUC2, FCGBP and KLK1 and a decrease in CLCA1 mRNA expression that returned to basal levels as healthy controls and UC in remission. This study demonstrates that MUC2 mucin and MAPs are not mucus‐associated and are expressed and developmentally regulated independently. In DSS colitis and active UC, MAPs are dysregulated and their reappearance coincides with restitution. Support or Funding Information Supported by CIHR This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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