Evidence That Gallbladder Epithelial Mucin Enhances Cholesterol Cholelithogenesis in MUC1 Transgenic Mice  Helen H. Wang, Nezam H. Afdhal, Sandra J. Gendler, David Q.–H. Wang  Gastroenterology  Volume 131, Issue 1, Pages 210-222 (July 2006) DOI: 10.1053/j.gastro.2006.04.011 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Relative mRNA levels for the gallbladder mucin genes as analyzed by quantitative real-time PCR techniques. These measurements are made using the gallbladder RNA of mice (A) on chow (day 0) or (B) fed the lithogenic diet for 4 weeks. For each mRNA expression level, the fold change for MUC1.Tg mice is expressed relative to the level of the corresponding mucin gene transcript in wild-type mice on the same diet, which in each case is arbitrarily set at 1.0. Of note is that gene expression of Muc2 is not detected in the gallbladders of mice. ND, not detectable. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Human MUC1 and mouse Muc1 protein concentrations in gallbladder biles as assayed by Western blot analysis. (A) The human MUC1 mucin is detected in the gallbladder biles of MUC1.Tg mice by HMFG-2, a monoclonal antibody against the tandem repeat region of human MUC1,27,28 which confirms a success in the mice transgenic for the human MUC1 gene. Its concentrations are significantly (P < .01) increased in MUC1.Tg mice in response to the lithogenic diet (right panel) compared with the chow diet (left panel). (B) On chow, total MUC1 mucin concentrations encoded by both the murine Muc1 gene and the human MUC1 gene are similar in 2 groups of mice, as measured by CT2, a monoclonal antibody recognizing the cytoplasmic tail domain of both mouse Muc1 and human MUC1.29 At 28 days on the lithogenic diet, total MUC1 mucin concentrations are 2-fold higher in MUC1.Tg mice than in wild-type mice. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Comparison of gallbladder sizes in mice (A) on chow and (B) at 28 days on the lithogenic diet. Gallbladder volumes are significantly larger in fasted MUC1.Tg mice than in wild-type mice, regardless of whether chow or the lithogenic diet is fed. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Because of gallbladder emptying, bile flow rates and biliary bile salt outputs are increased sharply in response to exogenously administered CCK-8 (as shown by the arrows) in mice (A) on chow or (B) fed the lithogenic diet. We observed that gallbladder contractile function is totally impaired in MUC1.Tg mice and partially in wild-type mice, especially in the lithogenic state. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Gallbladder cholesterol absorption in mice (A) on chow (day 0) and (B) fed the lithogenic diet for 28 days. There are significantly higher cholesterol concentrations in the gallbladder wall tissues of MUC1.Tg mice than in those of wild-type mice, regardless of whether chow or the lithogenic diet is fed. At 24 hours after the perfusion of HDL labeled with [14C]cholesterol, significantly higher radioactivity is found in the gallbladders of MUC1.Tg mice compared with those in wild-type mice, suggesting an increased gallbladder cholesterol absorption in MUC1.Tg mice. Increased cholesterol absorption induces significantly higher expression levels of the gallbladder Acat2 gene in the former compared with the latter. These alterations may promote the conversion of unesterified cholesterol to cholesteryl esters and result in significantly higher cholesteryl ester concentrations in the gallbladder wall tissues of MUC1.Tg mice. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Photomicrographs of mucin gel, liquid crystals, cholesterol monohydrate crystals, and gallstones observed in wild-type and MUC1.Tg mice during 28 days of the lithogenic diet feeding. It should be emphasized that the crystallization, growth, and agglomeration of cholesterol monohydrate crystals, as well as the development of cholesterol gallstones, are significantly accelerated in MUC1.Tg mice compared with wild-type mice. All magnifications are ×800, except for the day 28 panels, which are ×400, by polarizing light microscopy. Gastroenterology 2006 131, 210-222DOI: (10.1053/j.gastro.2006.04.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions