Expression and regulation of scavenger receptor class B type I (SR-BI) in gall bladder epithelium

Abstract

Background and aims: Biliary lipid absorption by the gall bladder mucosa and the cholesterol content of the gall bladder wall appear to play a role in cholesterol gall stone formation. As the scavenger receptor class B type I (SR- BI) regulates cellular cholesterol uptake, we studied its expression in human and murine gall bladders, its regulation by increased biliary lipid content, and its role in gall stone formation.

Methods and results: Using immunohistochemistry, SR-BI was found in the apical domain of human gall bladder epithelial cells. Immunoblotting of isolated membranes from gall bladder epithelial cells showed a specific signal for the 82 kDa SR-BI protein. In C57BL/6 mice, SR-BI was also found in the gall bladder epithelium. Using western blot analysis, an inverse relationship was observed between biliary cholesterol concentration and SR-BI expression in murine gall bladder mucosa. By comparing lithogenic diet fed wild-type and SR-BI deficient mice, gall bladder wall cholesterol content and gall stone formation were not found to be dependent on SR-BI expression.

Conclusions: (i) SR-BI is expressed in both human and murine gall bladder epithelium; (ii) biliary cholesterol hypersecretion is associated with decreased gall bladder SR-BI expression in mice; and (iii) murine SR-BI is not essential in controlling gall bladder wall cholesterol content and gall stone formation during diet induced cholelithiasis.

Figure 1

Haematoxylin-eosin (H&E) staining (A, B) and immunohistochemical reaction for scavenger receptor class B type I (SR-BI) (C, D) in human gall bladder (GB) epithelium. Immunohistochemistry (performed as described in materials and methods) is based on a polyclonal anti-SR-BI as the primary antibody. (A, B) H&E staining revealed a well preserved monolayer of typical GB epithelial cells with an apical domain (arrows), and subepithelial capillary vessels (open arrowhead). (C, D) Strong immunoperoxidase reaction for SR-BI protein was restricted to the plasma membrane of the apical domain of the GB epithelial cells (arrows). No staining was observed in the basolateral domain or in the tissues of the lamina propria. Original magnification: A and C, 200×; B and D, 400×.

Figure 2

Immunoblot analysis of scavenger receptor class B type I (SR-BI) in crude membrane fractions from isolated human gall bladder epithelial cells (GBEC) and from male murine liver (chow diet). GBECs were harvested from a fresh human GB without cholesterolosis, and crude plasma membranes were isolated as described in materials and methods. Liver from a control C57BL/6 mouse was used for isolation of crude plasma membrane. Crude membranes (20 μg protein each) were subjected to sodium dodecyl sulphate-polyarylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose filter. Immunoblot analysis was carried out using anti-human SR-BI polyclonal antibody. Bound antibodies were visualised as described in materials and methods.

Figure 3

Haematoxylin-eosin staining (A, B) and immunohistochemical localisation of scavenger receptor class B type I (SR-BI) (C, D) in gall bladder (GB) epithelium of mice on a chow diet. Immunohistochemistry is based on a polyclonal anti-SR-BI as the primary antibody. (A, B) The section shows GB epithelial cells and their apical domain (arrows), together with tissues of the lamina propria and tunica serosa (open arrowhead). (C, D) A clear signal for SR-BI protein is restricted to the plasma membrane in the apical domain of the GB epithelium (arrows); no staining is observed in the basolateral domain, or in the subepithelial tissues. Original magnification: A and C, 200×; B and D, 400×.

Figure 4

Immunoblot analysis of scavenger receptor class B type I (SR-BI) in crude membrane fractions from gall bladder (GB) and proximal jejunum epithelia of C57BL/6 male mice fed a diet of chow alone, chow supplemented with 2% cholesterol (Chol), or a lithogenic diet for four weeks or 10 days. Epithelium from GBs and jejuna from each group of mice were pooled (n=4 each), crude membranes were prepared, and aliquots (20 μg protein) were subjected to sodium dodecyl sulphate-polyarylamide gel electrophoresis analysis and transferred electrophoretically to a nitrocellulose filter, as described in the experimental procedures. Immunoblot analysis was carried out with rabbit anti-SR-BI as the primary antibody and bound antibodies were visualised as described in materials and methods. The degree of changes in SR-BI protein levels was compared with those of mice on the chow diet and are representative of two independent experiments. Quantitation of bands was determined after correction for loading differences with APN, as described in materials and methods.

Figure 5

Correlation between gall bladder (GB) bile cholesterol content, GB wall total cholesterol content, and scavenger receptor class B type I (SR-BI) expression on administration of either a chow or lithogenic diet. C57BL/6 mice were fed with a control or lithogenic diet for 3, 6, 12, or 30 days. Total cholesterol content of GB bile (A), protein expression of SR-BI in the GB wall, and total cholesterol content of GB tissue (B) were assessed as described in materials and methods over 30 days of diet administration. Total cholesterol content in bile increased progressively over time and correlated inversely with expression of SR-BI in the GB wall. Total cholesterol content of GB tissue correlated directly with SR-BI expression only during the first 12 days of administration of a lithogenic diet.

Figure 6

Total cholesterol content of gall bladder (GB) wall in wild-type and scavenger receptor class B type I (SR-BI) knockout mice on administration of either chow or a lithogenic (lith) diet for 6, 12, and 30 days. No differences were observed in total cholesterol content of the GB wall between wild-type and SR-BI null mice fed with either type of diet for the indicated periods. In both experimental groups, feeding a lithogenic diet elicited a biphasic response with a marked decrease in total cholesterol content of the GB wall during the first 12 days of diet administration which returned to control values after 30 days, similar to the response seen in C57BL/6 mice (see fig 5 ▶).