Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro - PubMed (original) (raw)

Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro

Jan A Lammel Lindemann et al. Mol Cell Endocrinol. 2014.

Abstract

Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients.

Keywords: Bile acids; Cholesterol; Cyp7a1; TH; TH receptor.

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Figures

Fig. 1

Adenovirus expression of TRb. (A) Detection of TRβ1 transcripts after adenovirus infection of HepG2 cells. Panel represents qPCR analysis performed on extracts of control and adenovirus TRb infected cells ± T3. Grey panels represent TRβ1 and black panels represent TRα1. Results of _T_-test indicated by * (P < 0.05). (B) Western blot of SDS–polyacrylamide gels used to separate extracts of control adenovirus and adenovirus TRβ1 infected cells. Blots used antibodies against myc epitope tag and β-actin control to ensure equal loading.

Fig. 2

Adenovirus expressed TRβ1 is functional. (A) qPCR analysis of adrenomedullin transcripts in control and adenovirus-TRβ1 infected HepG2 cells treated ± T3. Note enhancement of T3 induction of the endogenous adm gene in the presence of adenovirus TRβ versus induction obtained with endogenous TRβ. (B) Comparative transfection assays performed with a DR-4 responsive luciferase reporter in control (black) and adenovirus TRβ1 infected (dark grey) HepG2 cells and in HepG2 cells that stably express TRβ1 and treated with increasing levels of T3. Note lack of enhancement of luciferase activity with T3 in cells that do not express endogenous TRβ1 and that dose response is similar with Ad-TRβ1 infected HepG2 cells and HepG2-TRβ1 cells. Results of _T_-test indicated by * (P < 0.05).

Fig. 3

Similar T3 regulation of cholesterol metabolic genes in mouse liver, mouse and human liver primaries and a human hepatocyte cell line. (A) Heat map depicting high throughput qPCR analysis of expression levels of indicated genes in livers of hypothyroid mice treated ± T3, mouse and human primary hepatocyte cultures and HepaRG cells infected with control adenovirus or Ad-TRβ1 and treated ± T3. (B) Independent single point qPCR validation of changes in mouse and human Cyp7a1 expression levels ± adenovirus TRβ1 expression and T3.

Fig. 4

T3 induction of Cyp7a11 in HepG2 cells. (A) Results of qPCR analysis of Cyp7a1 transcripts in HepG2 cells that express TRβ1. Cells treated ± T3 and the protein synthesis inhibitor cycloheximide. Results represent mean of 3 replicates ± SEM, P < 0.05 indicated by * (Student's _T_-test). (B) Western blot of SDS-polyacrylamide gels used to separate extracts of HepG2-TRβ1 cells treated ± T3. Antibodies used were anti-Cyp7a1 and anti-tubulin loading control. Individual lanes represent different biological replicates in triplicate. (C) C4 levels detected in culture media for HepG2-TRβ1 cells treated ± T3 using approaches described in Section 2. Results represent mean of 3 replicates ± SEM, P < 0.05 indicated by * (Student's _T_-test).

Fig. 5

TRβ1 is expressed at low levels in HepG2 cells relative to mouse liver. (A) Results of single point T3 binding analysis performed on extracts of HepG2 cells (control, ctl) or HepG2 cells expressing ad-TRβ1 (TRβ) compared to native mouse liver extracts (MM). Results represent mean of 3 replicates ± SEM, P < 0.05 indicated by * (Student's _T_-test). (B) Saturation T3 binding analysis performed with HepG2 cells (HG2), HepG2 infected with Ad-TRβ1 expression vector (HG2-TRβ) and mouse liver extracts (5× dilution; MM). Results of each data point represent mean of 3 replicates ± SEM, P < 0.05 indicated by * (Student's _T_-test). (C) Western analysis of HepG2 cells (HG2), TRβ1 expressing HepG2 cells (stable and adenovirus expression stTRβ1 and adTRβ1) versus mouse liver (5× dilution, MM). Antibodies detect TRβ1 or tubulin as a loading control. Individual lanes represent duplicate biological replicates. (D) Results of gel shift analysis performed with extracts on native HepG2 (HG2), HepG2 infected with ad-TRβ1 (HG2TRβ) and mouse liver (MM). Triplet sets of lanes represent shifting with native DR-4, with unlabeled competitor DR-4 oligonucleotide or mutant oligonucleotide.

Fig. 6

TRs bind to the Cyp7a1 proximal promoter. Upper panel is a schematic of the Cyp7a1 gene locus with positions of regions that were probed with ChIP primers. The lower panel depicts ChIP assays performed upon extracts of cells that express tagged TRα1 or TRβ1, as described in Section 2. Bars represent enrichment over IgG control, which was set to a value of 1. Note that TR binding only exceeds background in the R3 and R5 regions. Results of _T_-test indicated by * (P < 0.05).

Fig. 7

Analysis of function of TREs in TR binding peaks. Upper panel represents approximate positions of defined TREs in TR binding peaks. Lower panel depicts results of luciferase assays performed with reporter genes drive by a standard DR-4 element, the Cyp7a1 promoter or individual predicted TREs in HepG2 cells that stably express TRα or TRβ. Note specific dose-dependent enhancement of luciferase activity with T3. Results of _T_-test indicated by * (P < 0.05).

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Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro - PubMed (original) (raw)