Gastroenterology

Gastroenterology

Volume 140, Issue 5, May 2011, Pages 1664-1674
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
NPC2 Regulates Biliary Cholesterol Secretion via Stimulation of ABCG5/G8-Mediated Cholesterol Transport

https://doi.org/10.1053/j.gastro.2011.01.050Get rights and content

Background & Aims

Biliary cholesterol secretion helps maintain cholesterol homeostasis; it is regulated by the cholesterol exporter adenosine triphosphate–binding cassettes G5 and G8 (ABCG5/G8) and the cholesterol importer Niemann–Pick C1-like 1 (NPC1L1). We studied another putative regulator of cholesterol secretion into bile, Niemann–Pick C2 (NPC2)—a cholesterol-binding protein secreted by the biliary system—and determined its effects on transporter-mediated biliary secretion of cholesterol.

Methods

Mice with hepatic knockdown of Npc2 or that overexpressed NPC2 were created using adenovirus-mediated gene transfer; biliary lipids were characterized. The effects of secreted NPC2 on cholesterol transporter activity were examined in vitro using cells that overexpressed ABCG5/G8 or NPC1L1.

Results

Studies of mice with altered hepatic expression of NPC2 revealed that this expression positively regulates the biliary secretion of cholesterol, supported by the correlation between levels of NPC2 protein and cholesterol in human bile. In vitro analysis showed that secreted NPC2 stimulated ABCG5/G8-mediated cholesterol efflux but not NPC1L1-mediated cholesterol uptake. Consistent with these observations, no significant changes in biliary cholesterol secretion were observed on hepatic overexpression of NPC2 in ABCG5/G8-null mice, indicating that NPC2 requires ABCG5/G8 to stimulate cholesterol secretion. Analyses of NPC2 mutants showed that the stimulatory effect of biliary NPC2 was independent of the function of lysosomal NPC2 as a regulator of intracellular cholesterol trafficking.

Conclusions

NPC2 is a positive regulator of biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport.

Section snippets

Adenoviruses

Adenoviruses expressing the short hairpin RNA sequence for knockdown of the mouse Npc2 gene (accession no. BC007190) (Ad-shNpc2 #1 and #2) and adenoviruses expressing human NPC2 complementary DNA (cDNA) (accession no. BC002532) attached to a Myc tag and 6×His tag sequences (Ad-NPC2-Myc-His), human ABCG5 cDNA (accession no. NM022436) attached to a Myc tag sequence (Ad-Myc-ABCG5), and human ABCG8 cDNA (accession no. NM022437) attached to an HA tag sequence (Ad-HA-ABCG8) were constructed and

Biliary Cholesterol Secretion Is Reduced in Hepatic Npc2-Knockdown Mice

To test the hypothesis that biliary NPC2 affects cholesterol secretion into bile, Npc2-knockdown mice were created by intravenous administration of adenoviruses (Ad-shNpc2 #1 and Ad-shNpc2 #2) expressing short hairpin RNA against Npc2. Following infection with Ad-shNpc2 #1 and #2, the hepatic expression of Npc2 mRNA was significantly reduced to 53% and 19% of basal levels, respectively, whereas expression of Npc2 mRNA in the intestine and brain was not affected (Figure 1A). Corresponding to the

Discussion

The major contribution of the present study was to identify the physiologic function of biliary NPC2 as a positive regulator of cholesterol secretion into bile, and this novel function of NPC2 is accounted for by the stimulation of ABCG5/G8-mediated cholesterol efflux. It is known that the 2 half ABC transporters, ABCG5 and ABCG8, which act on the bile canalicular membrane,23 are essential for the biliary secretion of cholesterol; Abcg5/g8-deficient mice have a significantly reduced biliary

Acknowledgments

Yoshihide Yamanashi and Tappei Takada contributed equally to this study.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Grant-in-Aid for Scientific Research on Innovative Areas HD-physiology (22136015).

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