Ann Båvner

The orphan nuclear receptor SHP utilizes conserved LXXLL-related motifs for interactions with ligand-activated estrogen receptors.

ABSTRACT SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor.

Transcriptional corepression by SHP: molecular mechanisms and physiological consequences.

Small heterodimer partner (SHP; NR0B2), an exceptional member of the mammalian nuclear receptor family, directly modulates the activities of conventional nuclear receptors by acting as an inducible and tissue-specific corepressor. Recent progress in dissecting underlying molecular mechanisms, identifying target factors and target genes, and uncovering physiological functions points to the regulatory involvement of SHP in diverse metabolic and intracellular pathways that awaits future clarification. In this review, we carry out a comprehensive survey of all published data and discuss our current understanding of molecular mechanisms and physiological consequences governing SHP action.

Involvement of corepressor complex subunit GPS2 in transcriptional pathways governing human bile acid biosynthesis.

Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4α (HNF4α), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1- and HNF4α-dependent expression of cholesterol 7α hydroxylase (CYP7A1) and sterol 12α hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4α, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand‐binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator‐binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A‐like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression‐defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300‐dependent coactivator functions.

Functional conservation of interactions between a homeodomain cofactor and a mammalian FTZ‐F1 homologue

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ‐F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand‐independent manner. Thus, developmental and tissue‐specific cofactors may be particularly important in modulating the transcriptional activities of FTZ‐F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ‐F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor–nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co‐repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver.

Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo

Abstract.  Shafaati M, Olin M, Båvner A, Pettersson H, Rozell B, Meaney S, Parini P, Björkhem I (Karolinska University Hospital Huddinge, Huddinge, Sweden; Dublin Institute of Technology, Dublin, Ireland). Enhanced production of 24S‐hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo. J Intern Med 2011; 270: 377–387.

EID3 is a novel EID family member and an inhibitor of CBP-dependent co-activation

EID1 (E1A-like inhibitor of differentiation 1) functions as an inhibitor of nuclear receptor-dependent gene transcription by directly binding to co-regulators. Alternative targets include the co-repressor small heterodimer partner (SHP, NR0B2) and the co-activators CBP/p300, indicating that EID1 utilizes different inhibitory strategies. Recently, EID2 was characterized as an inhibitor of muscle differentiation and as an antagonist of both CBP/p300 and HDACs. Here, we describe a third family member designated EID3 that is highly expressed in testis and shows homology to a region of EID1 implicated in binding to CBP/p300. We demonstrate that EID3 acts as a potent inhibitor of nuclear receptor transcriptional activity by a mechanism that is independent of direct interactions with nuclear receptors, including SHP. Furthermore, EID3 directly binds to and blocks the SRC-1 interacting domain of CBP, which has been implicated to act as the interaction surface for nuclear receptor co-activators. Consistent with this idea, EID3 prevents recruitment of CBP to a natural nuclear receptor-regulated promoter. Our study suggests that EID-family members EID3 and EID1 act as inhibitors of CBP/p300-dependent transcription in a tissue-specific manner.

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase.

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1−/− mouse with 2H7-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of 2H7-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.

On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.

Is it possible to improve memory function by upregulation of the cholesterol 24S-hydroxylase (CYP46A1) in the brain?

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30–60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions.