Liver receptor homolog-1 regulates bile acid homeostasis but is not essential for feedback regulation of bile acid synthesis

Abstract

Liver receptor homolog 1 (LRH-1), an orphan nuclear receptor, is highly expressed in liver and intestine, where it is implicated in the regulation of cholesterol, bile acid, and steroid hormone homeostasis. Among the proposed LRH-1 target genes in liver are those encoding cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12alpha-hydroxylase (CYP8B1), which catalyze key steps in bile acid synthesis. In vitro studies suggest that LRH-1 may be involved both in stimulating basal CYP7A1 and CYP8B1 transcription and in repressing their expression as part of the nuclear bile acid receptor [farnesoid X receptor (FXR)]-small heterodimer partner signaling cascade, which culminates in small heterodimer partner binding to LRH-1 to repress gene transcription. However, in vivo analysis of LRH-1 actions has been hampered by the embryonic lethality of Lrh-1 knockout mice. To overcome this obstacle, mice were generated in which Lrh-1 was selectively disrupted in either hepatocytes or intestinal epithelium. LRH-1 deficiency in either tissue changed mRNA levels of genes involved in cholesterol and bile acid homeostasis. Surprisingly, LRH-1 deficiency in hepatocytes had no significant effect on basal Cyp7a1 expression or its repression by FXR. Whereas Cyp8b1 repression by FXR was also intact in mice deficient for LRH-1 in hepatocytes, basal CYP8B1 mRNA levels were significantly decreased, and there were corresponding changes in the composition of the bile acid pool. Taken together, these data reveal a broad role for LRH-1 in regulating bile acid homeostasis but demonstrate that LRH-1 is either not involved in the feedback regulation of bile acid synthesis or is compensated for by other factors.

Figure 1

Generation of Mice Deficient for LRH-1 in Liver or Intestine A, Schematic representation of the targeting strategy to disrupt Lrh-1. The schematic shows exons 4 and 5 of Lrh-1, the targeting vector, and the targeted Lrh-1 allele both before and after successive removal of the MC1-neocassette with FLPe recombinase and exon 5 with cre recombinase. LoxP sites (solid arrowheads), FLPe recombinase target sites (Frt; open boxes), selected restriction enzyme sites and the 5′- and 3′-probes used for Southern blots are indicated. B, Southern blot analysis of DNA derived from wild-type (+/+) or targeted (+/neo-loxP) ES cells digested with NdeI or NheI and probed with 5′- and 3′-probes. Positions of wild-type and targeted alleles and their sizes are shown. C, PCR analysis of Lrh-1 alleles using DNA prepared from tail, liver, and intestine of Lrh-1^+/+, Lrh-1^flox/flox, hepKO, or ieKO mice as indicated. A control PCR reaction performed with no DNA is shown on the left. The positions of the PCR products generated by the wild-type Lrh-1 allele and the Lrh-1^flox/flox (floxed) and deleted exon 5 Lrh-1 (Δexon 5) alleles are indicated by arrows on the left. D and E, RT-qPCR analysis done using a primer set that recognizes Lrh-1 exon 5 and cDNA from liver and ileum of either Lrh-1^flox/flox and hepKO mice (D) or Lrh-1^flox/flox and ieKO mice (E). F, Western blot analysis using in vitro synthesized LRH-1 (IVS) or nuclear extracts prepared from livers of Lrh-1^flox/flox and hepKO mice and antibodies against LRH-1 or TATA-binding protein (TBP). The LRH-1 band migrates at approximately 62 kDa.

Figure 2

LRH-1 Deficiency in Intestinal Epithelium Does Not Affect Proliferation in Duodenum A, mRNA levels of the indicated genes were analyzed by RT-qPCR using intestinal epithelium derived from Lrh-1^flox/flox and ieKO mice (n = 7–8 mice per group). *, P < 0.05. B–D, Villus length (B), crypt depth (C), and BrdU labeling index (D) were measured in duodenum of Lrh-1^flox/flox and ieKO mice.

Figure 3

Effects of LRH-1 Deficiency in Liver or Intestine on Bile Acid Pool Size, Composition, and Excretion A–D, Bile acid pool size and composition were measured in either hepKO (A and B) or ieKO mice (C and D) and corresponding Lrh-1^flox/flox mice. tMCA, Tauromuricholic acid; tCDCA, taurochenodeoxycholic acid; tCA, taurocholic acid; tDCA, taurodeoxycholic acid; α/ωMCA, combined α/ω-muricholic acid; βMCA, β-muricholic acid; CA, cholic acid. All bile acid species shown in supplemental Table 1 were measured but only those at concentrations more than 1 μmol/100 g body weight (bw) are shown. *, P < 0.05. E and F, Fecal bile acid excretion was measured in either hepKO (E) or ieKO mice (F) and corresponding Lrh-1^flox/flox mice. Data are the mean ± sem (n = 4–6 mice per group).

Figure 4

Effect of FXR Activation on Gene Expression in Tissue-Specific LRH-1 Deficient Mice hepKO, ieKO, and control Lrh-1^flox/flox mice were treated with vehicle (Veh) or GW4064 (GW) for 14 h and RNA prepared from liver and ileum. RT-qPCR was used to measure mRNA levels of CYP7A1 (A and D), CYP8B1 (B), and SHP (C) in liver and FGF15 (E) and IBABP (F) in ileum. Data are the mean ± sem (n = 3–4 mice per group).