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P008. The amount of keratins in colonic epithelial cells matter for stress protection

M.N. Asghar1, J.S.G. Silvander1, L.E. Fortelius1, J.O. Misiorek1, I. Lähdeniemi1, T. Helenius1, D.M. Toivola1, 1Åbo Akademi University, Department of Biology, Turku, Finland

Background

Colonic epithelial cells are composed of keratins (K) of type II (K7, K8) and type I (K18, K19, K20). Keratins have an important role in cellular stress protection and absence of K8 (K8−/−) leads to e.g. liver damage and colitis in mice. This colonic phenotype includes hyperproliferation of crypts, decreased apoptosis and membrane protein mistargeting leading to diarrhoea, due to faulty ion transporters. K8−/− mice develop Th2-type colitis, resembling human ulcerative colitis (UC) which can be reversed by antibiotic treatment. It is still, however, unclear if patients with K8 mutations are predisposed to IBD. The K8 heterozygote (K8+/−) mouse colon has mild mistargeting of ion transporters, but the colon appears histologically normal. This gave rise to the hypothesis that loss of one K8 allele leads to less keratins and altered stress protection.

Methods

K8 wild-type (K8+/+), K8+/− and K8−/− mouse colonic tissues were analysed. Keratin protein and mRNA amounts were determined by western blotting and qPCR. Keratin crypt distribution was visualised by immunofluorescence (IF) staining and crypt lengths were measured by microscopy of histological samples from H&E-stained colon sections. Whole colon samples were cultured in order to determine the rate of apoptosis. Baseline inflammation was visualised by in vivo animal imaging and myeloperoxidase IF staining. Experimental colitis was induced by 2–5% dextran sodium sulphate (DSS).

Results

K8+/− colon has 50% less keratin mRNA and protein compared to K8+/+. The lengths of K8+/− colonic crypts are slightly longer than K8+/+, but shorter than K8−/−, indicating a mild but significant hyperproliferation. No resistance to apoptosis is, however, seen in K8+/−. The distribution of keratins in K8+/− crypts is similar to K8+/+, except for K7 that in K8+/− is expressed exceptionally high up in crypts in cells closest to the lumen. Compared to K8−/−, no baseline inflammation is observed in K8+/− colon. DSS treatment shows that K8+/− mice are more susceptible to induced colitis compared to K8+/+.

Conclusion

K8+/− mice express ∼50% less keratins which leads to a baseline colonic phenotype which is milder than in K8−/− mice. This indicates that amount of keratin matters for homeostasis in colonocytes. K8+/− mice are also more sensitive to induced colitis compared to K8+/+, suggesting that keratins are important in stress protection. K8+/− mice can serve as a suitable model when studying molecular mechanisms of keratins in the lack of inflammation.