Frances M. Sladek

Exclusive Homodimerization of the Orphan Receptor Hepatocyte Nuclear Factor 4 Defines a New Subclass of Nuclear Receptors

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor alpha (RXR alpha), in overall amino acid sequence and DNA binding specificity. Since RXR alpha is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR alpha was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR alpha is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.

AMP-Activated Protein Kinase Is Involved in Endothelial NO Synthase Activation in Response to Shear Stress

Objective—The regulation of AMP-activated protein kinase (AMPK) is implicated in vascular biology because AMPK can phosphorylate endothelial NO synthase (eNOS). In this study, we investigate the regulation of the AMPK–eNOS pathway in vascular endothelial cells (ECs) by shear stress and the activation of aortic AMPK in a mouse model with a high level of voluntary running (High-Runner). Methods and Results—By using flow channels with cultured ECs, AMPK Thr172 phosphorylation was increased with changes of flow rate or pulsatility. The activity of LKB1, the upstream kinase of AMPK, and the phosphorylation of eNOS at Ser1179 were concomitant with AMPK activation responding to changes in flow rate or pulsatility. The blockage of AMPK by a dominant-negative mutant of AMPK inhibited shear stress-induced eNOS Ser1179 phosphorylation and NO production. Furthermore, aortic AMPK activity and level of eNOS phosphorylation were significantly elevated in the aortas of High-Runner mice. Conclusions—Our results suggest that shear stress activates AMPK in ECs, which contributes to elevated eNOS activity and subsequent NO production. Hence, AMPK, in addition to serving as an energy sensor, also plays an important role in regulating vascular tone.

International Union of Pharmacology. LXVI. Orphan nuclear receptors.

Half of the members of the nuclear receptors superfamily are so-called “orphan” receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.

MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300.

CBP/p300 recruitment to enhancer‐bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre‐assembled Elk‐1–p300 complexes become activated following Elk‐1 phosphorylation by changes in Elk‐1–p300 interactions rather than recruitment. It is known that Elk‐1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk‐1 by mitogen‐acivated protein kinase (MAPK)‐mediated phosphorylation leads to targeted gene transactivation. We show that Elk‐1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk‐1 C‐terminus and the p300 N‐terminus. Phosphorylation on Ser383 and Ser389 of Elk‐1 by MAPK enhances this basal binding but, most importantly, Elk‐1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk‐1‐associated complex that could play a critical role in chromatin remodeling and gene activation. The pre‐assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate‐early response genes, in particular those involved in stress responses.

Identification of an endogenous ligand bound to a native orphan nuclear receptor.

Orphan nuclear receptors have been instrumental in identifying novel signaling pathways and therapeutic targets. However, identification of ligands for these receptors has often been based on random compound screens or other biased approaches. As a result, it remains unclear in many cases if the reported ligands are the true endogenous ligands, – i.e., the ligand that is bound to the receptor in an unperturbed in vivo setting. Technical limitations have limited our ability to identify ligands based on this rigorous definition. The orphan receptor hepatocyte nuclear factor 4 α (HNF4α) is a key regulator of many metabolic pathways and linked to several diseases including diabetes, atherosclerosis, hemophilia and cancer. Here we utilize an affinity isolation/mass-spectrometry (AIMS) approach to demonstrate that HNF4α is selectively occupied by linoleic acid (LA, C18:2ω6) in mammalian cells and in the liver of fed mice. Receptor occupancy is dramatically reduced in the fasted state and in a receptor carrying a mutation derived from patients with Maturity Onset Diabetes of the Young 1 (MODY1). Interestingly, however, ligand occupancy does not appear to have a significant effect on HNF4α transcriptional activity, as evidenced by genome-wide expression profiling in cells derived from human colon. We also use AIMS to show that LA binding is reversible in intact cells, indicating that HNF4α could be a viable drug target. This study establishes a general method to identify true endogenous ligands for nuclear receptors (and other lipid binding proteins), independent of transcriptional function, and to track in vivo receptor occupancy under physiologically relevant conditions.

Hepatocyte Nuclear Factor-4α Is Essential for Glucose-stimulated Insulin Secretion by Pancreatic β-Cells

Mutations in the hepatocyte nuclear factor (HNF)-4α gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic β-cells. HNF-4α, a transcription factor belonging to the nuclear receptor superfamily, is expressed in pancreatic islets as well as in the liver, kidney, and intestine. However, the role of HNF-4α in pancreatic β-cell is unclear. To clarify the role of HNF-4α in β-cells, we generated β-cell-specific HNF-4α knock-out (βHNF-4αKO) mice using the Cre-LoxP system. The βHNF-4αKO mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1. Pancreatic islet morphology, β-cell mass, and insulin content were normal in the HNF-4α mutant mice. Insulin secretion by βHNF-4αKO islets and the intracellular calcium response were impaired after stimulation by glucose or sulfonylurea but were normal after stimulation with KCl or arginine. Both NAD(P)H generation and ATP content at high glucose concentrations were normal in the βHNF-4αKO mice. Expression levels of Kir6.2 and SUR1 proteins in the βHNF-4αKO mice were unchanged as compared with control mice. Patch clamp experiments revealed that the current density was significantly increased in βHNF-4αKO mice compared with control mice. These results are suggestive of the dysfunction of KATP channel activity in the pancreatic β-cells of HNF-4α-deficient mice. Because the KATP channel is important for proper insulin secretion in β-cells, altered KATP channel activity could be related to the impaired insulin secretion in the βHNF-4αKO mice.

Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4α1

ABSTRACT Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 α1 (HNF4α1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4α2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4α1 and HNF4α2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4α1 and -α2 and that that contact is different in the HNF4α1 and HNF4α2 isoforms. Finally, we propose a model in which the F domain of HNF4α1 acts as a negative regulatory region for transactivation and in which the α2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4α1 and -α2 suggests that this model may be relevant to other nuclear receptors as well.

Integrated approach for the identification of human hepatocyte nuclear factor 4α target genes using protein binding microarrays

Hepatocyte nuclear factor 4 alpha (HNF4α), a member of the nuclear receptor superfamily, is essential for liver function and is linked to several diseases including diabetes, hemophilia, atherosclerosis, and hepatitis. Although many DNA response elements and target genes have been identified for HNF4α, the complete repertoire of binding sites and target genes in the human genome is unknown. Here, we adapt protein binding microarrays (PBMs) to examine the DNA‐binding characteristics of two HNF4α species (rat and human) and isoforms (HNF4α2 and HNF4α8) in a high‐throughput fashion. We identified ∼1400 new binding sequences and used this dataset to successfully train a Support Vector Machine (SVM) model that predicts an additional ∼10,000 unique HNF4α‐binding sequences; we also identify new rules for HNF4α DNA binding. We performed expression profiling of an HNF4α RNA interference knockdown in HepG2 cells and compared the results to a search of the promoters of all human genes with the PBM and SVM models, as well as published genome‐wide location analysis. Using this integrated approach, we identified ∼240 new direct HNF4α human target genes, including new functional categories of genes not typically associated with HNF4α, such as cell cycle, immune function, apoptosis, stress response, and other cancer‐related genes. Conclusion: We report the first use of PBMs with a full‐length liver‐enriched transcription factor and greatly expand the repertoire of HNF4α‐binding sequences and target genes, thereby identifying new functions for HNF4α. We also establish a web‐based tool, HNF4 Motif Finder, that can be used to identify potential HNF4α‐binding sites in any sequence. (HEPATOLOGY 2009.)

What are nuclear receptor ligands

Nuclear receptors (NRs) are a family of highly conserved transcription factors that regulate transcription in response to small lipophilic compounds. They play a role in every aspect of development, physiology and disease in humans. They are also ubiquitous in and unique to the animal kingdom suggesting that they may have played an important role in their evolution. In contrast to the classical endocrine receptors that originally defined the family, recent studies suggest that the first NRs might have been sensors of their environment, binding ligands that were external to the host organism. The purpose of this review is to provide a broad perspective on NR ligands and address the issue of exactly what constitutes a NR ligand from historical, biological and evolutionary perspectives. This discussion will lay the foundation for subsequent reviews in this issue as well as pose new questions for future investigation.

Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4α1: Modulation by the F domain

ABSTRACT 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 4α1 (HNF4α1, 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 HNF4α1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4α-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4α1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4α1 in vitro and repress HNF4α1 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 HNF4α1 and GRIP1, CBP, or p300, SMRT can titer out HNF4α1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4α 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.