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. 2024 Feb;28(3):e18110.
doi: 10.1111/jcmm.18110. Epub 2024 Jan 1.

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation

Liwei Pang  1 , Meiying Cui  2 , Shuodong Wu  1 , Jing Kong  1
Affiliations

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation

Liwei Pang et al. J Cell Mol Med. 2024 Feb.

Abstract

Background and aims: The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation.

Methods: Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells.

Results: Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased.

Conclusion: Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

Keywords: BSEP; Cav-1; Hax-1; cell membrane cholesterol; cholesterol cholelithiasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Mouse BSEP expression is influenced by cholesterol. (A) Cholesterol stone in C57BL/6 mice; (B) BSEP mRNA expression levels in mice; (C) Immunohistochemistry analysis of BSEP from mouse liver (2 weeks) (200×) shows strong positive expression of BSEP in experimental group; (D) Immunohistochemistry analysis of BSEP from mouse liver (4 weeks) (200×) show a significant decrease in the expression of BSEP in experimental group; (E) Western blot analysis of BSEP from mouse liver (2 weeks) shows increased expression of BSEP; (F) Western blot analysis of BSEP from mouse liver (4 weeks) shows decreased expression of BSEP.
FIGURE 2
FIGURE 2
BSEP expression and function in cells are influenced by cholesterol. BSEP protein expression first increased, then decreased as the cholesterol concentration increased. (A) Effects of different cholesterol concentrations on the expression of the BSEP protein in HepG2 cells; (B) BSEP mRNA expression levels in HepG2 cells; (C) BSEP protein expression in the cell membrane under different cholesterol concentrations: BSEP protein expression in the cell membrane increased in the low‐cholesterol‐concentration group, but decreased in the high‐ cholesterol‐concentration group; (D) Cell membrane localization of fluorescent BSEP under different cholesterol concentrations: a low‐cholesterol concentration promoted BSEP localization at the cell membrane, while a high‐cholesterol concentration promoted a cytoplasmic localization of BSEP; (E) Effects of cholesterol concentration on BSEP transport activity: BSEP transport activity first increased, then decreased as cholesterol concentration increased; (F) Effect of taurocholic (TC) acid concentration on BSEP transport activity: as the TC substrate concentration increased, BSEP transport activity was higher in the low‐cholesterol‐concentration group than in the control and high‐cholesterol‐concentration groups.
FIGURE 3
FIGURE 3
Mouse Cav‐1 expression is influenced by cholesterol. (A) Immunohistochemistry analysis of Cav‐1 from mouse liver (2 weeks) (200×) shows strong positive expression of Cav‐1 in experimental group; (B) Immunohistochemistry analysis of Cav‐1 from mouse liver (4 weeks) (200×) shows significant decrease in expression of Cav‐1 in experimental group; (C) Western blot of Cav‐1, Hax‐1 and p‐PKCα from mouse liver (2 weeks): p‐PKCα in mouse liver was decreased but Hax‐1 expression showed no obvious changes in vivo; (D) Western Blot of Cav‐1, Hax‐1 and p‐PKCα from mouse liver (4 weeks): p‐PKCα in mouse liver was increased but Hax‐1 expression showed no obvious changes in vivo; (E) Cav‐1 mRNA expression levels in mice: Cav‐1 mRNA expression was higher in the 2‐week experimental group than in the control group, while Cav‐1 mRNA expression decreased in the 4‐week experimental group compared with the control group.
FIGURE 4
FIGURE 4
Cav‐1 expression in cells is influenced by cholesterol. (A) Effects of different cholesterol concentrations on Cav‐1 protein expression in HepG2 cells; (B) Cav‐1 mRNA expression levels in HepG2 cells; (C) BSEP and Hax‐1 co‐immunoprecipitation under low‐cholesterol concentration; (D) BSEP and Hax‐1 co‐immunoprecipitation under high‐cholesterol concentration; (E) BSEP and Hax‐1 double immunofluorescence staining under different cholesterol concentrations.
FIGURE 5
FIGURE 5
Changes in caveolae of cells under different cholesterol concentrations: low‐cholesterol concentration increased both the number of caveolae and invagination in the cell membrane. Further increase in cholesterol, led to a decrease in cell membrane caveolae, but more invagination was observed. (A) Control group (2500×); (B) Control group (5000×); (C) Control group (10,000×); (D) Low‐cholesterol‐concentration group (2000×); (E) Low‐cholesterol‐concentration group (5000×); (F) Low‐cholesterol‐concentration group (10,000×); (G) High‐cholesterol‐concentration group (2000×); (H) High‐cholesterol‐concentration group (5000×); (I) High‐cholesterol‐concentration group (10,000×).
FIGURE 6
FIGURE 6
Effect of Cav‐1 overexpression on BSEP, Hax‐1 and p‐PKCα. (A) Establishment and identification of Cav‐1 overexpression and knockdown cell line; (B) Effects of Cav‐1 overexpression on BSEP, Cav‐1, p‐PKCα and Hax‐1 protein expression; (C) Cav‐1 overexpression promoted BSEP cell membrane localization; (D) After Cav‐1 overexpression, BSEP co‐localizes with Hax‐1 in the cell membrane; (E) Cav‐1 overexpression inhibited BSEP binding to Hax‐1; (F) Effects of Cav‐1 on BSEP transport function.
FIGURE 7
FIGURE 7
Cholesterol regulated BSEP via Cav‐1 rescue experiment. (A) Cav‐1 overexpression could antagonize the inhibitory effect of the high‐cholesterol concentration on BSEP and reverse PKCα phosphorylation; (B) A PKCα inhibitor inhibited PKCα phosphorylation and increased BSEP expression; although the high‐cholesterol concentration still inhibited Cav‐1.
FIGURE 8
FIGURE 8
Mechanism of regulation of BSEP expression and function in HepG2 cells through cholesterol via Cav‐1. (A) Under a low‐cholesterol concentration, Cav‐1 expression was enhanced, PKCα phosphorylation was inhibited, the binding of BSEP to Hax‐1 was attenuated, and BSEP membrane expression and transport function were enhanced; (B) Under a high‐cholesterol concentration, Cav‐1 expression was inhibited, PKCα phosphorylation was enhanced, the binding of BSEP to Hax‐1 was enhanced, BSEP internalization was promoted, and BSEP membrane expression and transport function were weakened, leading to the promoted formation of cholesterol stones.
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References

    1. Portincasa P, van Erpecum KJ, Di Ciaula A, Wang DQ. The physical presence of gallstone modulates ex vivo cholesterol crystallization pathways of human bile. Gastroenterol Rep (Oxf). 2019;7(1):32‐41. doi:10.1093/gastro/goy044 - DOI - PMC - PubMed
    1. vanBerge‐Henegouwen GP, Venneman NG, Portincasa P, Kosters A, van Erpecum KJ, Groen AK. Relevance of hereditary defects in lipid transport proteins for the pathogenesis of cholesterol gallstone disease. Scand J Gastroenterol Suppl. 2004;241:60‐69. doi:10.1080/00855920410011022 - DOI - PubMed
    1. Stieger B, Meier Y, Meier PJ. The bile salt export pump. Pflugers Arch. 2007;453(5):611‐620. doi:10.1007/s00424-006-0152-8 - DOI - PubMed
    1. Dikkers A, Tietge UJ. Biliary cholesterol secretion: more than a simple ABC. World J Gastroenterol. 2010;16(47):5936‐5945. doi:10.3748/wjg.v16.i47.5936 - DOI - PMC - PubMed
    1. Parton RG. Caveolae: structure, function, and relationship to disease. Annu Rev Cell Dev Biol. 2018;34:111‐136. doi:10.1146/annurev-cellbio-100617-062737 - DOI - PubMed

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