Gastroenterology

Gastroenterology

Volume 146, Issue 4, April 2014, Pages 1048-1059.e7
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Vitamin D Receptor Activation Down-regulates the Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol

https://doi.org/10.1053/j.gastro.2013.12.027Get rights and content

Background & Aims

Little is known about the effects of the vitamin D receptor (VDR) on hepatic activity of human cholesterol 7α-hydroxylase (CYP7A1) and cholesterol metabolism. We studied these processes in mice in vivo and mouse and human hepatocytes.

Methods

Farnesoid X receptor (Fxr)−/−, small heterodimer partner (Shp)−/−, and C57BL/6 (wild-type control) mice were fed normal or Western diets for 3 weeks and were then given intraperitoneal injections of vehicle (corn oil) or 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3; 4 doses, 2.5 μg/kg, every other day). Plasma and tissue samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and rodent fibroblast growth factor (Fgf) 15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter and mobility shift assays in transfected human embryonic kidney 293 cells, quantitative polymerase chain reaction with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation assays with mouse liver.

Results

We first confirmed the presence of Vdr mRNA and protein expression in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and decreased in unison. Changes in hepatic Cyp7a1 messenger RNA (mRNA) correlated with those of Cyp24a1 (a Vdr target gene) and inversely with Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation with 1,25(OH)2D3 increased levels of Cyp24a1/CYP24A1 and Cyp7a1/CYP7A1 mRNA in mouse and human hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr−/− and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp−/− mice given 1,25(OH)2D3 and fed Western diets. Truncation of the human small heterodimer partner (SHP) promoter and deletion analyses revealed VDR-dependent inhibition of SHP, and mobility shift assays showed direct binding of VDR to enhancer regions of SHP. In addition, chromatin immunoprecipitation analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr and rodent retinoid X receptor to the Shp promoter.

Conclusions

Activation of the VDR represses hepatic SHP to increase levels of mouse and human CYP7A1 and reduce cholesterol.

Section snippets

Materials and Methods

In the Supplementary Material, we provide information on materials, mouse strains, antibodies, plasmids, and procedures for real-time polymerase chain reaction, Western blotting, microsomal preparation for Cyp7a1 activity, and assay procedures for bile acid pool size, cholesterol, and 1,25(OH)2D3.

Vdr Protein Tissue Distribution and Liver Immunostaining

Nuclear Vdr protein was present at similar levels in the ileum and kidney of wild-type and Fxr−/− mice, although levels were considerably lower in liver and brain, as found previously.24, 25 Vdr protein was found in the lysate of primary hepatocytes prepared from wild-type mice (data not shown) and Vdr+/+ but not Vdr−/− kidney (Figure 1A). Vdr protein was identified in mouse hepatocytes by immunostaining (Figure 1B), and specificity of the antibody was further confirmed by staining liver

Discussion

We observed cholesterol lowering in response to 1,25(OH)2D3 treatment, an effect associated with elevated Cyp7a1 mRNA and protein expression and microsomal activity, with correspondingly larger bile acid pool sizes and/or greater fecal bile acid excretion in hypercholesterolemic wild-type and Fxr−/− mice. We showed that increased Cyp7a1 expression and activity was achieved via Vdr-repression of Shp after steady-state treatment of 1,25(OH)2D3. The inhibition of Shp and induction of Cyp7a1 by Vdr

Acknowledgments

The authors thank Drs Nan Wu and Martin Wagner, Baylor College of Medicine, Texas Medical Center, Houston, for assistance with studies on the bile acid pool sizes of Shp−/− mice.

Han-Joo Maeng's current affiliation is College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, South Korea.

Sayeepriyadarshini Anakk's current affiliation is Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, Urbana, IL.

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    Conflicts of interest The authors disclose no conflicts.

    Funding The authors (KSP, CC, ECYC, LM, HPQ, and MRD) gratefully acknowledge support from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the Ontario Graduate Scholarship Program. SA and DDM were supported by CPRIT grant RP120138, and R. P. Doherty, Jr. – Welch Chair in Science Q-0022.

    Author names in bold designate shared co-first authorship.

    Authors share co-third authorship.

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    Authors share co-senior authorship.

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