Synergistic regulation of the mouse orphan nuclear receptor SHP gene promoter by CLOCK-BMAL1 and LRH-1

Small heterodimer partner (SHP; NR0B2) is an orphan nuclear receptor and acts as a repressor for wide variety of nuclear hormone receptors. We demonstrated here that mouse SHP mRNA showed a circadian expression pattern in the liver. Transient transfection of the mSHP promoter demonstrated that CLOCK-BMAL1, core circadian clock components, bound to E-box (CACGTG), and stimulated the promoter activity by 4-fold. Liver receptor homologue-1 (LRH-1; NR5A2) stimulated the mSHP promoter, and CLOCK-BMAL1 synergistically enhanced the LRH-1-mediated transactivation. Interestingly, SHP did not affect the CLOCK-BMAL1-mediated promoter activity, but strongly repressed the synergistic activation of CLOCK-BMAL1 and LRH-1. Furthermore, in vitro pull-down assays revealed the existence of direct protein-protein interaction between LRH-1 and CLOCK. In summary, this study shows that CLOCK-BMAL1, LRH-1 and SHP coordinately regulate the mSHP gene to generate the circadian oscillation. The cyclic expression of mSHP may affect daily activity of other nuclear receptors and contribute to circadian liver functions.     

A key role for orphan nuclear receptor liver receptor homologue-1 in activation of fatty acid synthase promoter by liver X receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.     

The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity

SHP (small heterodimer partner, NR1I0) is an atypical orphan member of the nuclear receptor subfamily in that it lacks a DNA-binding domain. It is mostly expressed in the liver, where it binds to and inhibits the function of nuclear receptors. SHP is up-regulated by primary bile acids, through the activation of their receptor farnesoid X receptor, leading to the repression of cholesterol 7alpha-hydroxylase (CYP7alpha) expression, the rate-limiting enzyme in bile acid production from cholesterol. PXR (pregnane X receptor, NR1I2) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs as well as endogenous compounds such as bile acid precursors. Upon activation, PXR induces CYP3A and inhibits CYP7alpha, suggesting that PXR can act on both bile acid synthesis and elimination. Indeed, CYP7alpha and CYP3A are involved in biochemical pathways leading to cholesterol conversion into primary bile acids, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. Here, we show that PXR is a target for SHP. Using pull-down assays, we show that SHP interacts with both murine and human PXR in a ligand-dependent manner. From transient transfection assays, SHP is shown to be a potent repressor of PXR transactivation. Furthermore, we report that chenodeoxycholic acid and cholic acid, two farnesoid X receptor ligands, induce up-regulation of SHP and provoke a repression of PXR-mediated CYP3A induction in human hepatocytes as well as in vivo in mice. These results reveal an elaborate regulatory cascade, tightly controlled by SHP, for both the maintenance of bile acid production and detoxification in the liver.     

Redundant pathways for negative feedback regulation of bile acid production

The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.     

Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain

For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4alpha1 (HNF4alpha1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4alpha1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4alpha-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4alpha1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4alpha1 in vitro and repress HNF4alpha1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4alpha1 and GRIP1, CBP, or p300, SMRT can titer out HNF4alpha1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4alpha can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.     

Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.     

Bile acids reduce SR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.     

The orphan nuclear receptor SHP regulates PGC-1alpha expression and energy production in brown adipocytes

Brown adipocytes increase energy production in response to induction of PGC-1alpha, a dominant regulator of energy metabolism. We have found that the orphan nuclear receptor SHP (NR0B2) is a negative regulator of PGC-1alpha expression in brown adipocytes. Mice lacking SHP show increased basal expression of PGC-1alpha, increased energy expenditure, and resistance to diet-induced obesity. Increased PGC-1alpha expression in SHP null brown adipose tissue is not due to beta-adrenergic activation, since it is also observed in primary cultures of SHP(-/-) brown adipocytes that are not exposed to such stimuli. In addition, acute inhibition of SHP expression in cultured wild-type brown adipocytes increases basal PGC-1alpha expression, and SHP overexpression in SHP null brown adipocytes decreases it. The orphan nuclear receptor ERRgamma is expressed in BAT and its transactivation of the PGC-1alpha promoter is potently inhibited by SHP. We conclude that SHP functions as a negative regulator of energy production in BAT.