Regulation of hepatic metabolic pathways by the orphan nuclear receptor SHP

Abstract

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways. Sustained expression of SHP leads to the depletion of hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver. The mechanism responsible for this phenotype includes SHP-mediated direct repression of downstream target genes and the bile acid sensor FXRalpha, and an indirect activation of PPARgamma and SREBP-1c genes. We present evidence for the role of altered chromatin configurations in defining distinct gene-specific mechanisms by which SHP mediates differential transcriptional repression. The multiplicity of genes under its control suggests that SHP is a pleiotropic regulator of diverse metabolic pathways.

Figure 1

SHP expression and histological analysis of SHP-Tg mice. (A) Quantitative RT–PCR analysis of RNAs from the livers of 60-day-old SHP-Tg mouse lines and nontransgenic wild-type littermates (control) was performed using primer sets amplifying both mouse and human SHP cDNA. Numbers at the bottom represent fold difference between GAPDH-normalized values, compared to the first control sample. (B) Western blot analysis of hepatic nuclear extracts from 60-day-old SHP-Tg and wild-type (control) mice was performed by using αSHP antibody, to detect total SHP protein, and αFlag antibody, to detect transgene-derived protein expression. (C) Liver sections from 60-day-old SHP-Tg and wild-type (control) mice were stained with H&E, oil red O and αFlag as indicated.

Figure 2

Analysis of the mRNA levels of genes involved in bile acid (A) and cholesterol (B) homeostasis. RT–PCR assays were performed with liver RNA prepared from 60-days-old SHP-Tg and wild-type (control) mice. The values obtained were normalized to GAPDH values and are expressed as fold change compared to the wild-type data. Bars represent mean values and standard deviations from RNA samples of at least five individual mice. ^*P<0.05; ^**P<0.01. Black bars, control animals. Gray bars, SHP-Tg animals.

Figure 3

Analysis of the mRNA levels of genes involved in lipogenesis (A) and transcription factors (B). The results are presented as in the legend of Figure 2. Black bars, control animals. Gray bars, SHP-Tg animals.

Figure 4

Analysis of transcription factor recruitment (A, C), RNA pol-II occupancy, and chromatin modifications (B). Soluble, formaldehyde-crosslinked chromatin was prepared from the livers of 60-days-old SHP-Tg and wild-type (control) mice and subjected to immunoprecipitations with the indicated antibodies. The DNAs in the precipitates were analyzed by PCR using primer sets amplifying the indicated promoter regions of different genes. Left: Autoradiogram of a representative ChIP experiment from one control and one SHP-Tg mouse. Right: Quantitative analyses of experiments performed on three different control and three different SHP-Tg samples. All data, obtained with liver extracts from individual animals, were first normalized to the corresponding input values (% input) and expressed as fold enrichment over the values obtained with control antibody. Bars represent average fold enrichments calculated from the three experiments. The error bars indicate variations between the signals obtained in samples from three individual mice. White bars, control animals. Black bars, SHP-Tg animals.

Figure 5

Schematic overview of the pathways and genes regulated by SHP. Black flat-headed lines indicate repressive effects while red arrows indicate stimulatory effects. Solid and dashed lines correspond to direct and indirect mechanisms, respectively.