P052 Heat shock protein GP96 is essential for maintaining intestinal epithelial architecture by supporting its self-renewal capacity
J. Häfliger1, M. Schwarzfischer1, M. Van de Sande1, R. Manzini1, C. Stanzel1, K. Atrott1, S. Lang1, M. Scharl1, M. Spalinger1
1University Hospital Zurich, University of Zurich, Department of Gastroenterology and Hepatology, Zurich, Switzerland
The intestinal epithelium is characterised by a remarkable self-renewal capacity and a high turnover of intestinal epithelial cells (IEC), which emerge from intestinal stem cells (ISC). Defects in proliferation and differentiation of ISC into mature IEC result in impaired barrier function, which is linked with systemic diseases, such as fatty liver disease and diabetes. Glycoprotein (GP)96 is a master chaperone for cell surface receptors including toll-like receptors (TLRs), integrins and the Wnt co-receptors LRP5/6. Wnt signalling is essential for the maintenance of the ISC niche, thus we here investigated how deletion of GP96 specifically in IEC affects intestinal homeostasis.
To study the role of GP96 in the intestinal epithelium, we used GP96-VillinCre-ERT2 mice which harbour a loxP flanked GP96 gene and Tamoxifen-inducible Cre expression specifically in IEC. To deplete GP96 from IEC, those mice were injected with Tamoxifen at five consecutive days (1 mg/day) and terminated on Day 5, 6, and 7 after the first dose to observe the changes in epithelial integrity over time. As a control, littermates with a loxP flanked GP96 gene without the VillinCre-ERT2 construct were injected with Tamoxifen.
IEC-specific GP96 depletion resulted in rapid weight loss within 6 days after the first Tamoxifen injection. At Day 6, the intestine of GP96-VillinCre-ERT2 mice revealed visible signs of inflammation, characterised by a general shortening of the colon and the small intestine, as well as thickening of the colon wall observed by mouse endoscopy. Colon wall thickening was in sharp contrast to the transparent and fragile appearing small intestine and caecum wall. This was in line with a significant, successive reduction of IEC numbers upon depletion of GP96, as observed when harvesting IEC, and by histological analysis of small intestinal tissue sections. Additionally, the entire intestine was filled with bile-containing intestinal fluid, while solid food or faecal pellets were completely absent. Of interest, apoptosis was not affected upon GP96 depletion in IEC; however, proliferating cells as determined by Ki67 staining, at the crypt base were markedly reduced, indicating that loss of GP96 affects ISC proliferation/function.
Our results underline the importance of GP96 in maintaining homeostasis of the intestinal epithelial architecture. Elucidating molecular mechanisms that are decisive for the fate of the ISC niche will promote the understanding of the pathogenesis of inflammatory diseases associated with barrier defects. Given the pronounced phenotype upon deletion of GP96 in IEC, it might serve as a promising novel therapeutic target in diseases involving intestinal barrier defects.