Hepatic Nuclear Factor-4, a Key Transcription Factor at ... - CiteSeerX

Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2007, 1, 166-175

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Hepatic Nuclear Factor-4, a Key Transcription Factor at the Crossroads Between Architecture and Function of Epithelia Agnès Ribeiro1,2,3,*, Amena Archer1,2,3,§ Johanne Le Beyec1,2,3, Anne-Laure Cattin1,2,3, Susan Saint-Just1,2,3, Martine Pinçon-Raymond1,2,3, Jean Chambaz1,2,3, Michel Lacasa1,2,3 and Philippe Cardot1,2,3 1

Centre de Recherche des Cordeliers, Université Pierre et Marie Curie - Paris 6, UMRS 872, Paris, F-75006 France, 2INSERM, U872, Paris, F-75006 France, 3Université Paris Descartes, UMRS 872, Paris, F-75006 France

Received: April 13, 2007; Accepted: April 20, 2007; Revised: May 4, 2007 Abstract: Hepatic nuclear factor-4 (HNF-4) is a transcription factor and a member of the large family of nuclear receptors. It was first cloned from liver but is expressed also in kidney, pancreas and intestine. Three genes encoding three isoforms have been identified, HNF4 and , in mammals, drosophila and xenopus and HNF-4, exclusively in xenopus. HNF-4 is the best studied isoform, especially in liver. Such studies put HNF-4 at the crossroads between architecture and function of epithelia, as it induces expression of cell/cell junction proteins while it also controls glucido-lipidic metabolism and drug metabolizing enzyme genes. Furthermore, mutations in the HNF-4 gene lead to a metabolic disease in humans, Maturity Onset Diabetes of the Young-1 (MODY-1). The existence of a “true ligand” is not clearly established but a “structural” fatty acid is present in the ligand binding pocket of HNF-4 and . Consequently, activity of HNF-4 can be modulated by the interaction with co-regulators or by post-translational modifications. Then, HNF-4 is a potential direct or indirect target for pharmacologic drugs, with a special interest for the intestinal epithelium which is the primary site of metabolic control, due to its roles in nutrient absorption and in sensing energy. The patents related to the HNF-4 gene are also discussed in this article.

Keywords: HNF-4, liver, pancreas, intestine, epithelium, metabolism, differentiation, nuclear receptor, MODY-1, drugs. INTRODUCTION Nuclear receptors represent one of the largest families of transcription factors. These proteins were first recognized as mediators of steroid hormone signalling and provided an important link between transcription regulation and physiology [1]. Today, this superfamily includes 48 members which include members as varied as thyroid hormone receptor (TR), steroid receptors (ER, PR, AR), and glucocorticoid receptor (GR). It also includes retinoic acid receptors (RAR, RXR), vitamin D receptor (VDR) and the more recently discovered receptors for fatty acids, thiazolidindiones and eicosanoids (PPARs), for biliary acids (FXR), and for oxysterols (LXR). In some cases, ligands have not yet been identified and these nuclear receptors are termed “orphan” [2]. Nuclear receptors have a common modular structure: an amino-terminal region A/B which contains the transactivation domain AF-1; the region C which is the DNA binding domain (DBD); the region D which represents a hinge domain; the region E which includes the ligand binding domain (LBD), the dimerisation interface, and a second transactivation domain, and finally the region F, the carboxyterminal domain (Fig. 1). These molecules are targets for numerous physiological, environmental or developmental signals. The importance of nuclear receptors in maintaining the normal physiological state is illustrated by the enormous pharmacopoeia to combat disorders in which nuclear signalling is a pathological determinant [3]. Nuclear receptors can be classified into four groups according to their mechanism of action. The first group corresponds to steroid hormone receptors: a cytoplasmic inactive receptor is complexed with heat-shock proteins. The ligand induces a conformational change and the homodimeric nuclear receptor is translocated in the nucleus, where it transactivates its target genes using a DNA consensus site, termed Hormone Responsive Element (HRE). The steroid hormone receptor HRE is a palindrome of which the half is formed by the sequence AGGTCA. The second group of nuclear *Address correspondence to this author at the UMRS 872, 15 rue de l’école de médecine, 75006 Paris, France; Tel: 33 1 42 34 69 18; Fax: 33 1 43 25 16 15; E-mail: [email protected] § Present address: Karolinska Institute, Department of Biosciences and Nutrition, Novum, SE-141 57 Huddinge, Sweden

1872-2148/07 $100.00+.00

receptors is those which are constitutively present in the nucleus where they are bound to DNA as a heterodimer with RXR. These receptors include TR, RAR, VDR, FXR, LXR, PPARs. For these receptors, HREs are direct AGGTCA repeats separated by 0 to 5 nucleotides, depending on the nature of the receptor. In this case, the receptor represses transcription and binding of the ligand induces a conformational change which allows the receptor to activate transcription. The third group is represented by nuclear receptors which bind DNA as monomers such as NGFI-B or FTZF1. Members of the fourth group are present in the nucleus as homodimers. HNF-4 and COUP-TF belong to this group [4]. Prominent among orphan nuclear receptors is HNF-4 which was cloned first using cDNA from liver by F. Sladek [5]. In the past few years, remarkable inroads have been made into determining the functional importance of HNF-4 isoforms. Numerous studies in vivo, in vitro and by genome-scale location analysis have shown that HNF-4 plays a major role in hepatic function. Recent approaches in vivo and in silico suggest a key role for HNF-4 for intestinal epithelial cell function. The primary functions of the gastrointestinal tract are to process ingested food and to absorb nutrients and water. In addition to digesting and assimilating nutrients, the intestine and associated visceral organs play key sensing and signalling roles in energy homeostasis. These control steps may represent new areas for the development of therapeutic agents for treating metabolic diseases with HNF-4 as the target. GENOMIC STRUCTURE, ISOFORMS AND TISSUE DISTRIBUTION To date, three different genes coding for three different isoforms have been identified, HNF-4 and HNF-4 in mammals, drosophila, and xenopus [6-8], and HNF-4 in xenopus [9]. Isoform HNF-4 is the best studied. The HNF-4 gene contains 13 exons which encode 9 different mRNAs, resulting from alternative splicing and transcription from two different promoters P1 and P2 (Fig. 1). The expression of HNF-4, a 54 kDa protein, is restricted to liver, kidney, small intestine, colon, pancreas and testis [5,6,10]. HNF-4, encoded by a different gene, is expressed in the same tissues, except for liver [6-8]. However, some HNF-4 has been found in a human hepatoma cell line, HepG2 [11,12]. Furthermore,

© 2007 Bentham Science Publishers Ltd.

HNF-4, Master Gene of Epithelial Differentiation

Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2007, Vol. 1, No. 2

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Fig. (1). Schematic representation of the HNF-4 gene and protein isoforms HNF-4 and HNF-4. (A) HNF-4 gene structure. Exons are shown as boxes. Arrows represent the two alternative promoters P1 and P2. In the human genome, P2 promoter is located 45.6 kb upstream P1 promoter. (B) HNF-4 isoforms structure. The P1 promoter initiates transcription from exon 1A and transcribes isoforms HNF-41 to HNF-46, whereas the P2 promoter initiates transcription from exon 1D and transcribes isoforms HNF-47 to HNF-49, with a completely different A/B domain. HNF-44, 5 and 6 are splicing variants of HNF-41, 2 and 3, respectively. The carboxy-terminus of the A/B domain is different. HNF-42, 5 and 8 are splicing variants of HNF-41, 4 and 7, respectively. They have an insertion of 10 amino acids in the carboxy-terminus of domain F. HNF-43, 6 and 9 are characterized by a short and distinct domain F. (C) HNF-4 isoform structure. Murine HNF-4 presents 95% identity with human HNF4. Regions C (DNA binding domain) and D (hinge domain) present 94% homology with the isoform . Region E (ligand binding domain and dimerisation interface) presents 80% homology between HNF-4 and HNF4. More divergent domains are found at the N-terminus (domain A/B) and at the C-terminus (domain F) which are a transactivation domain and a negative regulatory domain, respectively.

HNF-4 and HNF-4 are differentially expressed in intestinal epithelium, HNF-4 being expressed along the entire crypt-villus axis whereas HNF-4 is expressed only in the villus [13]. During mouse development, HNF-4 is detected very early in the visceral endoderm at embryonic day E4,5 and its expression is restricted to yolk sac between E5,5 and 8,5. In embryos, HNF-4 mRNA is expressed in liver and primitive intestine [14, 15]. HNF-4 mRNA is detected at day E 15.5 [8]. There is a differential expression of the different isoforms of HNF-4 depending on the tissue, development stage and cellular differentiation. In mice, HNF-47 is expressed during development until birth, whereas HNF-41 is expressed from E12.5 and increases until the adult age [16, 17]. Hence, HNF-47 is a stronger transcriptional activator of genes expressed during development such as alpha-foetoprotein and transthyretine whereas HNF-41 is

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a stronger activator of genes expressed in differentiated hepatocytes such as apolipoprotein (apo) C-III [18, 19]. In the human carcinoma colic cell line, Caco-2, HNF-41 and 2 isoform expression is correlated with the differentiation in enterocytes [20]. ROLES OF HNF-4 ALPHA Studies on the regulation of liver-specific genes have identified a set of transcription factor families that govern their tissuerestricted expression: the variant homeodomain-containing proteins (HNF-1 and ), the winged helix family proteins HNF-3, and (also called FoxA1, 2, and 3), the basic leucine zipper-containing factor C/EBP, the onecut homeodomain protein HNF-6 and members of the nuclear hormone receptor family, COUP-TFII, LRH-1, FXR, PXR and of course HNF-4 [21]. In all species, HNF-4 expression precedes the expression of HNF-1 and HNF1, implying that HNF-4 is a transcriptional factor higher up in the hierarchy of regulatory proteins. Functional binding site for HNF-4 has been identified in the promoter of the HNF-1 gene [22, 23]. Furthermore, the analysis of embryoid bodies with inactivated HNF-3 or HNF-3 genes revealed that both transcription factors affect HNF-4 and HNF-1 expression [24]. However, an HNF-1 binding site has also been identified in the HNF-4 promoter [25]. More recent data indicate that hepatic transcription factors HNF-1, HNF-3 C/EBP, HNF-6, and HNF-4 act synergistically to control HNF-4 expression in liver [26,27]. All these studies suggest a regulatory network between these transcription factors. The genome-scale location analysis of binding sites of HNF-4 reveals multiple possible regulatory networks in liver and in pancreatic islets, involving HNF-4, HNF-1 and HNF-6 [28]. An in vivo study by the group of I. Talianidis, demonstrates the presence of complex networks in developing and in adult liver, by chromatin immunoprecipitation using HNF-4deficient livers [29]. HNF-4 is a target of C/EBP, HNF-1 and , HNF-6, HNF-3 and HNF-4 itself and is also a regulator of HNF-1 and , HNF-3, HNF-6. Furthermore, HNF-4 controls the expression of other nuclear receptors, LRH-1, PXR and FXR. These authors reveal the complexity and the plasticity of transcription networks in liver. Tissue-specific HNF-4 invalidation, constitutive or conditional, lead to the discovery that HNF-4 plays pleiotropic roles in epithelium. HNF-4 gene invalidation leads to embryo death very early at the gastrulation stage (E6.5), due to a dysfunctional visceral endoderm, unable to properly supply nutrients necessary to foetal development [14, 30]. The rescue of embryonic stem (ES) cells HNF-4-/- by HNF-4+/+ extraembryonic tissues allows the development of the embryo until E12, showing the major role of HNF-4 in early embryogenesis [30]. This rescue model permitted study of the functional loss of HNF-4 during liver development in the embryo. It was shown that HNF-4 is dispensable for hepatic specification since hepatoblasts present normal morphology [31]. On the other hand, HNF-4 is necessary for hepatoblast differentiation into hepatocytes. Loss of HNF-4 has a dramatic effect on hepatocyte gene expression, since mRNA levels of apolipoproteins A-I, A-II, B, C-II, C-III as well as phenylalanine hydroxylase, L-fatty-acid binding protein, transferrin, erythropoietin and aldolase B were almost undetectable [31]. Furthermore, specific invalidation of the HNF-4 gene in fetal liver showed that HNF-4 also controls epithelial morphology. Indeed, at E18.5 hepatocytes present rudimentary cell/cell junctions, associated with mislocalized E-cadherin, ZO-1 and CEACAM1 and absence of micro-villi [32]. Previous studies have shown that the re-expression of HNF-4 in dedifferentiated hepatocytes allowed re-expression of epithelial markers such as Ecadherin [33]. More recently, two different groups showed that the expression of HNF-4 in mesenchymal cells, such as F9 or 3T3 cultured cells, induced the expression and the proper localization of tight junction proteins such as ZO-1, Occludin, Claudin-6 and -9 and adherens junction proteins such as E-cadherin and -catenin

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[32, 34]. Furthermore in F9 cells, HNF-4 expression induced micro-villi formation [35]. Micro-array analysis of HNF-4 gene invalidation in fetal liver showed that genes encoding proteins of all categories of cell junctions (adherens, desmosomes, tight) are targets of HNF-4 [36]. All these studies showed that HNF-4 is an inducer, direct or indirect, of cell junction protein expression. More recently, our group showed that reciprocally, E-cadherin-dependent cell junctions when they are perturbed, are also able to modulate the nuclear HNF-4 level [37]. Some mutations in the HNF-4 gene are associated with a form of type II diabetes in humans, Maturity Onset Diabetes of the Young-1 (MODY-1). The primary effect of these mutations is a defect in insulin secretion by pancreatic cells. These mutations also induce pleiotropic effects, especially in liver [38]. In the same way, specific invalidation in pancreatic cells of HNF-4 gene inhibits insulin secretion in response to glucose, reproducing the main characteristic of MODY-1 [39]. Interestingly, genome-scale location analysis revealed that HNF-4 is bound to 12% of hepatic genes and 11% of -pancreatic cell genes present on the DNA micro-array, suggesting that HNF-4 is a master regulating factor of hepatocyte and islet transcription [28]. In vivo, conditional invalidation of the HNF-4 gene in adult mouse liver, underlined the key role of HNF-4 in hepatic function since it controls genes of the glucido-lipidic metabolism. The mutant mice present hepatomegaly, associated with glycogen and lipid accumulation, dyslipoproteinemia and a drastic reduction of mRNA for various proteins such as apos A-I, A-II, A-IV, B, microsomal triglyceride transfer protein (MTP), and cholesterol 7-hydroxylase CYP7A1 [40]. HNF-4 is also critical for urea homeostasis by direct regulation of the Ornithine Transcarbamylase (OTC) gene [41]. HNF-4 is a key transcription factor for the expression of cytochrome P450 proteins involved in biliary acid synthesis and for drug metabolizing enzymes. Indeed, the CYP2D6 activity is decreased more than 50% in mice with a conditional invalidation of the HNF-4 gene [42]. CYP3A4, thought to be involved in the metabolism of nearly 50% of all drugs currently prescribed, is controlled by HNF-4, in vivo in fetal mice with conditional deletion of hnf-4 [43]. CYP2C9, CYP1A1, CYP1A2 are also targets for HNF-4 [44, 45]. Furthermore, the expression of HNF4 is induced in response to phenobarbital [46]. Bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are expressed in this organ. In vivo studies with mice lacking the hepatic HNF-4 gene showed that HNF-4 directly controls bile acid transporters [40], enzymes which conjugate bile acids with taurine such as bile acid-CoA ligase and bile acid-CoA:amino acid N-acyl transferase (BAT) [47] and enzymes which are involved in hydroxylation and side chain oxidation of cholesterol, such as CYP8B1 [48]. HNF-4 is also implicated in the porto-central gradient of gene expression in liver [49]. It is involved in the periportal inhibition of enzymes such as glutamine synthetase and ornithine aminotransferase. All these studies reveal HNF-4 as a key transcription factor for hepatic function (Fig. 2). In developing colon, HNF-4 gene invalidation inhibits the expression of a panel of genes necessary for colonic epithelium functions: transport such as aquaporine 4, absorption such as aldolase B and adhesion such as mucine 3 [50]. Furthermore, crypts are abnormal, with a defect in secreting cell lineage maturation and fewer proliferative cells [50]. This defect in proliferation is contradictory with an in vitro study which showed that HNF-4 expression in F9 cells activates p21CIP/WAF1 and inhibits proliferation [51]. Recent approaches using mouse small intestinal villi, crypts and fetal intestinal epithelium, based on transcriptome,

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Fig. (2). Summary of pleiotropic effects of HNF-4 in liver. Numerous studies in vivo and in vitro have shown that HNF-4 plays a major role in hepatic function (see text for details and references).

metabolome and bioinformatic analyses, hypothesized that HNF-4 is involved in the regulation of lipid metabolism in enterocytes [5254]. Furthermore, with intra-duodenal injections of a dominant form of HNF-4, inhibiting HNF-4 and activities, our group showed that apo A-IV, a marker gene for differentiated enterocyte expression and involved in lipid metabolism, is controlled by both isoforms of HNF-4 [55]. In vivo and in vitro, the induction of apo A-IV transcription by intestinal lipids is also controlled by HNF-4 [56]. From these numerous functional in vitro, in vivo and in silico studies, mainly in liver, and recently in intestine, HNF-4 appears as “an orchestra conductor” at a crossroads between epithelial morphogenesis and epithelial functions. Given the fundamental role of HNF-4 as a master transcription factor, its activity must be tightly regulated to allow HNF-4 to play its pleiotropic functions. HOW HNF-4 ACTIVITY CAN BE MODULATED? It is accepted that ligand binding to a nuclear receptor induces a conformational change in the DNA bound receptor which leads to co-repressor dissociation and co-activator recruitment to activate transcription. In the case of HNF-4 and HNF-4, the existence of a “true” ligand is not clearly established, but the activity of HNF-4 and can be modulated either by the interaction with co-regulators modulated by domain F, or by post-translational modifications. Furthermore, the presence of two promoters and various isoforms (1 to 9 and ) adds supplementary levels of regulation. Although the HNF-4 gene is organized like that of HNF-4, which supposes that splicing isoforms could be generated, only one HNF-4 isoform has been described.



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Fig. (3). Schematic representation of the HNF-4 protein. (A) Functional domains of HNF-4. DBD: DNA binding domain. LBD: ligand binding domain. NRD: negative regulatory domain. RD: repressive domain. Interactions with different co-regulators are represented (B) Direct post-translationnal modifications of HNF-4 (see text for details and references).

1- Differential Expression from Two Promoters In liver, the transcription of HNF-4 from the P1 promoter, is dependent on transcription factors HNF-1 and HNF-1, GATA-6, HNF-6, RXR/RAR, and COUP-TF, the later being a repressor whereas the others are activators [26]. The enhancer, 6 kb upstream from the P1 promoter binds transcription factors HNF-3, HNF-1 and , HNF-4, GR and C/EBP [27]. During enterocytic differentiation, it has been proposed that there is a communication between the enhancer with bound HNF-3, C/EBP and HNF-1 and the P1 promoter with bound HNF-6 and HNF-1, leading to the formation of a stable complex of transcription initiation which activates

transcription of HNF-4 [57]. The regulation of HNF-4 by the P2 promoter is dependent on transcription factors HNF-1 and , and HNF-6 and leads to the expression of HNF-47 to 9 isoforms in the liver [17]. Interestingly, these authors have shown that HNF41 is able to inhibit P2 promoter activity and suggest that there is a “progressive switch” between the P2 promoter and the P1 promoter during development, leading to exclusive transcription from the P1 promoter in adult liver. In the embryo, although the P2 promoter is activated by HNF-1 and HNF-6, increase of HNF-41 during development leads to inhibition of P2 promoter activity and the activation of the P1 promoter, then decreasing the expression of HNF-47 and increasing the expression of HNF-41 [17]. Some


studies reported that in pancreatic islets, HNF-4 transcription is driven almost exclusively by the P2 promoter, placing hnf-4 downstream of hnf-1, on the contrary to what has been reported to occur in liver [58-60]. A contradictory study showed that the P1transcribed isoforms, 1 to 6, are expressed in pancreatic islets. As they have stronger transactivation properties than isoforms 7 to 9, due to the presence of the AF-1 domain, the authors have highlighted the potential role of these isoforms in the control of pancreatic -cells [61]. Most of these transcription factors have been identified first in the liver and their roles have been extensively studied in this tissue, underlying their importance in hepatocyte differentiation. Some of these transcription factors are also present in intestine and play a role in the development of this epithelium, suggesting than in enterocytes, mechanisms controlling promotor election of the gene HNF-4 could be similar. 2- Differential Activity Due to Functional Differences in the HNF-4 Structure Dimerisation A possible way to regulate nuclear receptor activity is to modulate its structure by dimerisation or ligand binding. HNF-4 presents a similar structure than other nuclear receptors. The DNA binding domain (DBD) or region C is well defined (position 50 to 116 aa) (Fig. 3A) and contains two zinc fingers. It can be extended to position 142 aa by inclusion of the T-box (from 117 to 125 aa) and the A-box (from 126 to 142 aa) which are respectively necessary for dimerisation and high affinity DNA binding [62]. Mutant analyses and crystallographic studies have shown that the interface of dimerisation is contained in region E or the ligand binding domain (LBD). This region E (from 175 to 370 aa) contains 12 -helices (H1 to H12) and the dimerisation interface is formed by 3 helices, H9 to H11 (Fig. 3A) [63-65]. The crystal structure of HNF-4 is similar to that of HNF-4 [66]. Bogan et coll. suggest that the charge compatibility in helix H9 and H10 is responsible for the strict homodimerisation of HNF-4 [67]. Since sequence analyses revealed a charge compatibility with HNF-4, these authors also suggest a possibility of heterodimerisation between HNF-4 and HNF-4, which would be a possible way to modulate the HNF-4 activity in intestine. However, in our laboratory, by coimmunoprecipitation of over-expressed HNF-4 and , we have never been able to demonstrate hetero-dimerisation between HNF4 and (personal communication). Furthermore, we showed that the relative DNA binding affinities and transactivation properties are similar between these two isoforms [55]. Transactivation Domains HNF-4 has two transactivation domains, AF-1 and AF-2. AF-1 corresponds to aa 1 to 24 of the amino-terminal part (Fig. 3A) [63]. Isoforms 7 to 9, transcribed from the P2 promoter, have a region A/B (encoded by the 1D exon) without the AF-1 domain. An in vivo study, with mice invalidated either for HNF-41 or for HNF47, showed that there is a redundancy of these two isoforms and that AF-1 is dispensable [68]. However, phenotypes of the two mouse mutants are not completely similar. Mice with only the HNF-41 isoform present a mild dyslipidemia with genes encoding apos C-III and A-IV, and CAR (constitutive androstane receptor) being completely repressed whereas mice with only HNF-47 isoform present a moderate glucose intolerance. The AF-2, localized in the LDB, has been restricted to the H12 helix (360 to 366 aa) (Fig. 3A) [63]. For non-orphan receptors, this helix H12 is responsible for the conformational change of the receptor after ligand binding, closing the ligand binding pocket, stabilizing the complex and then generating an active conformation of the receptor which is able to interact with co-activators. Crystallography studies with the LBD of HNF-4 showed that a homo-dimer presents two conformational states, one with an opened helix H12 and the other with helix H12 closed on a constitutive ligand, which is a fatty acid [65]. On the contrary with other nuclear receptors, it is the

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interaction with a co-activator, and not with the ligand, which allows an active stable state by closing the two helices H12 of the homodimer [69]. Essential residues in the ligand binding pocket have been identified and involved helices H3, H5 and H10/H11 [70, 71]. Ligand Binding Domain HNF-4 is constitutively active and has been considered for a long time as an orphan receptor. Some controversial studies showed that HNF-4 activity could be modulated by long chain fatty acids [72-74]. For example, saturated fatty acids such as palmitate (C16:0) increase the activating and DNA binding properties of HNF-4 whereas poly-unsaturated fatty acids such as linolenate (C18:3) or eicosapentaenoate (C20:5) inhibit them. Furthermore, FRET (Fluorescence Resonance Energy Transfer) analyses showed an interaction between acyl-CoA and HNF-4, which induces a conformational change [75]. However, Bogan et al. showed that acyl-CoA did not induce this conformational change and did not modify interaction with co-activators [67]. Crystallography data with the LBD of HNF-4 or HNF-4 confirmed this hypothesis. The LBD crytallises with a fatty acid within the ligand binding pocket. This fatty acid is not exchangeable and it seems to be a structural ligand [65, 66, 69]. Divergence between studies showing exchangeable fatty acids and those showing a structural fatty acid could be due to the presence or not of domain F. Indeed, HNF-4 is the only nuclear receptor with a large domain F (85 residues), known to negatively modulate HNF-4 activity (see text below) [63, 76]. Recently, it has been shown by FRET analysis that full-length HNF-4 (with the F domain) is necessary to obtain a high affinity for fatty acids [77]. Furthermore, the same group showed that domain F has a thio-esterase activity and is a binding site for acylCoA thioesters [78]. Their hypothesis is that HNF-4 would be synthesized with a free fatty acid in its ligand binding pocket, as a structural ligand. Acyl-CoA thio-esters would bind to domain F and would be hydrolysed by the thio-esterase activity of domain F, then allowing an exchange with the free fatty acid in the ligand binding pocket. Depending on the nature of the fatty acid, the activity of HNF-4 would be modulated. Domain F Domain F has a negative effect on transactivation by HNF-4, inhibiting AF-2 activity and the interaction with co-activators [64]. A repressive domain has also been identified, from 429 to 441 aa (Fig. 3A) [76]. The repressive effect of domain F is variable depending on the isoform. For example, domain F of HNF-42 is more repressive than that of HNF-41. The inhibitory effect is not completely due to the repressive domain since the domain F of isoform HNF-43 does not contain this sequence and is repressive [79]. It seems rather that the tri-dimensional structure of domain F modulates the interactions with co-activators and co-repressors [80]. As domain F is a region with little homology between HNF4 and HNF-4 (only 32 %), one can hypothesize that these isoforms would recruit different co-regulators, especially in the intestinal epithelium where they are differentially expressed [13]. Indeed, it has been shown that different HDACs (Histone DeAcetylase) are expressed during intestinal development [81] and that different co-activators can be recruited by different HNF-4 isoforms [18, 19]. 3- Modulation by Interactions with Co-Regulators Co-activators and co-repressors contain histone acetyl transferase (HAT) and HDAC activities. HATs and HDACs ultimately control the balance between chromatin decondensation, a transcriptionally permissive state, and chromatin condensation, a transcriptionally silent state [82]. Co-activators which interact with HNF-4 are represented by three large families, the p160 family (with at SRC-1/NcoA-1, TIF-2/GRIP-1/NcoA-2 and RAC3/TRAM1/ACTR/pCIP), the CBP/p300 family and p/CAF family (Fig. 3A). Members of these families contain intrinsic HAT activity. Members



of the co-repressor family SMRT/NcoR contain intrinsic HDAC activity [1]. Another co-activator family has also been identified: PGC-1 and which do not contain HAT activity. These coactivators then recruit members of CBP/p300 and SRC-1 families [83]. SHP (Small heterodimer partner), a particular orphan nuclear receptor without a DNA binding domain interacts with numerous nuclear receptors such as CAR, LXR, PPAR, RAR, RXR and also with HNF-4. SHP inhibits the transactivation properties of HNF-4 by competition for DNA binding and for co-activator interactions by recruitment of co-repressors [84]. More and more studies show that interactions between HNF-4 and its co-regulators depend on gene and physiological conditions studied [43, 85-89]. For example GRIP-1 and PGC-1 are involved in the cross-talk between HNF4 and CAR to regulate transcription of CYP7A1 and PEPCK genes [89]. A second example is transcription of CYP2C9, CYP1A1, and CYP1A2 genes which requires the recruitment of PGC-1 and SRC-1 by HNF-4 [43]. And as a third example, the proper time-specific transcription of the glucose-6 phosphatase gene is dependent on HNF-6, PGC-1 and HNF-4 [88]. 4- Regulation by Post-Translational Modifications HNF-4 contains 21 serine residues, 6 threonine residues and 7 tyrosine residues which are potentially phosphorylable [90]. Global HNF-4 phosphorylation on Ser/Thr residues and Tyr residues is necessary for the full transactivation and high DNA binding and specificity [90, 91]. HNF-4 also contains acetylable Lys residues in the DNA binding domain, from 91 to 142 aa. This region contains a part of the nuclar localization sequence which overlaps the second Zn finger. Acetylation of HNF-4, mediated by CBP, is

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crucial for its proper retention in the nucleus (Fig. 3B) [92]. The phosphorylation of Ser 78, located in the DBD, by protein kinase C, also impairs the nuclear localization of HNF-4 (Fig. 3B) [93]. Furthermore, HNF-4 can also be methylated by PRMT1 on the Arg residue 91 (Fig. 3B) [94]. This methylation regulates, first, DNA binding activity and then allows a synergistic effect, involving methylation of histones. These numerous possibilities for post-translational modifycations suggest that HNF-4 is a good candidate to be a target for various signalling pathways. For example, at the time of oxidative stress, HNF-4 exhibits a specific phosphorylation pattern associated with transcriptional activity, suggesting a pathway involving redox-sensitive kinases [95]. HNF-4 is also a direct target for protein kinase A (PKA) which inhibits its DNA binding activity in response to fasting or inducers of intracellular cyclic AMP (Fig. 3B) [96]. AMP-activated protein kinase (AMPK), which is a central component of signalling pathways that regulate intracellular energy levels, regulates HNF-4 activity by decreasing its protein level [97] (Fig. 4A). AMPK directly phosphorylates HNF4 at residue Ser 304 in the LBD, inhibiting dimerisation and DNA binding, then decreasing protein stability (Fig. 3B) [98]. Cytokines such as IL-1, IL-6 or TNF induce signal pathways which modulate negatively HNF-4 activity and decrease DNA binding through JAK-2 kinase [99] or NF-B pathway [100]. TGF antagonizes TNF effects on the apo C-III promoter through interactions between HNF-4 and SMAD factors [101, 102] (Fig. 5). HNF-4 is also an indirect target of signalling pathways (Fig. 4B). For example, hypoxic stress modulates interaction between

Fig. (4). Schema of direct (A) and indirect (B) phosphorylations which modulate HNF-4 activity. Metabolic stress (1) and fasting (2) induce AMPK and PKA, respectively. These kinases phosphorylate HNF-4 decreasing its stability [98] and its DNA binding [96]. In response, transcription of key glycolytic enzymes, GK and PK are repressed. On the other hand, insulin (4) induces the phosphorylation of FKHR by Akt/PKB kinase. FKHR, which interacts with HNF-4 in nucleus, is translocated from nucleus to cytoplasm, releasing HNF-4. HNF-4 ultimately transactivates glycolytic genes GK and PK [106]. HNF4 is also a direct target of the signalling pathway induces by oxidative stress (3) although the involved kinase remains unknown [95]. Venous pO2, in synergy with insulin (5) and hypoxic stress (6) induce interaction between HIF-1 and HNF-4 and ultimately increases the transcriptional rate of EPO and GK genes [103-105]. Dashed lines represent uncharacterized steps.


Ribeiro et al.

Fig. (5). Schematic view of signaling pathways induced in response of bile acid, TNF and TGF, which modulate HNF-4 activity. 1- Bile acids induce transcription of the SHP gene by FXR. Increased level of SHP inhibits HNF-4 activity by protein/protein interactions and ultimately decreases the OAT2 transcription. Bile acids may also inhibit the transcription of HNF-4 itself [110]. 2- MAP kinase pathway, by MEKK1 and ERK2, inhibits the expression of C/EBP. The decrease of C/EBP level inhibits the HNF-4 transcription which is C/EBP dependent [111]. 3- Bile acids and TNF induce MAP kinase pathway by MAKK1, SEK1 and JNK. This signaling pathway decreases the transactivation potential of HNF-4 and ultimately the transcriptional rate of CYP7A1 [108, 109]. 4- TNF also triggers NK-kB pathway which induces an inhibition of HNF-4 DNA binding and then the transcription of apo C-III [100]. This pro-inflammatory effect is antagonized by TGF signaling through interactions between SMAD proteins and HNF-4 [101, 102]. Dashed lines represent uncharacterized steps.

HNF-4 and the transcription factor HIF-1 and then transcription of the erythropoietin (EPO) gene [103]. Venous pO2 in synergy with insulin, via Akt/PKB kinase, induce transcription of the glucokinase (GK) gene in the liver perivenous zone, by recruitment of HNF-4, HIF-1 and CBP/p300 [104, 105]. In nucleus, HNF-4 is also able to interact with FKHR, a member of the FoxO family. This interaction inhibits HNF-4 activity. Insulin, by activating Akt/PKB kinase, induces the phosphorylation of FKHR and its translocation to cytoplasm, releasing an active form of HNF-4 in the nucleus. Then HNF-4 activates glycolysis genes such as glucokinase (GK) and pyruvate kinase (PK) [106] (Fig. 4B). The MAP kinase pathway can also modulate indirectly HNF-4 activity. Extra-cellular signals such as biliary acids or TNF activate MEKK1 and downstream JNK or ERK2 to inhibit transcription of HNF-4-dependent genes such as apo C-III [107], Cyp7A1 [108, 109] or organic anion transporter 2 (OAT2) [110], or to inhibit HNF-4 transcription itself [111] (Fig. 5). CURRENT & FUTURE DEVELOPMENTS HNF-4 plays key functions in physiology as an effective regulator of glucido-lipidic metabolism, by affecting the synthesis of enzymes and transporters that control numerous metabolic steps. Despite recent therapeutic advances, the growing epidemics of obesity, type 2 diabetes and coronary artery disease in industrial

societies necessitates the identification and development of new therapy targets. Orphan receptors have raised several questions concerning the existence of a specific ligand and have become the subject of intensive investigations. Although, HNF-4 contains a “structural” ligand in the ligand binding pocket, it can still be a valid pharmaceutical target, potentially regulated by drugs. Modulation of the activity by inhibition of phosphorylation (patent WO 06043701A1) or by inhibition of the degradation (patent EP1669088A1; patent US 20070004636A1) has already been proposed (Table 1) [112-114]. Moreover, regulation of energy balance presents numerous potential targets for intervention, among them the intestine which is an innovative field for new therapy targets. Significant progress is being made in deciphering the precise role of HNF-4 in intestinal epithelium function using murine models. ABBREVIATIONS AMPK Apo AR BAT

= = = =

C/EBP

=

AMP-activated protein kinase Apolipoprotein Androgen receptor Bile acid-CoA amino acid N-acyl transferase CAAT/enhancer binding protein


Table 1.


173

List of Patents Involving Directly or Indirectly in the Control of HNF-4 Activity

Patent number

Patent Title

Country/Organization

WO06043701A1

Method of inhibiting phosphorylation of transcriptional factor for gluconeogenesis-associated gene and phosphorylation inhibitor

World Intellectual Property Organization (WIPO)

EP1669088A1

Method of treating Diabetes by inhibiting degradation of at least one of CREBL1, ATF6 and HNF-4A by HtrA2

European Patent Office (EPO)

US20070004636A1

Method of degrading transcriptional factors of saccharometabolism-associated gene, method for inhibiting the degradation and degradation inhibitor

United States of America

CAR COUP-TF

= =

CYP DBD ER ES FoxO

= = = = =

FRET

=

FTZ-F1 FXR GK GR HAT HDAC HIF HNF HRE JAK JNK LBD LXR MAP MODY

= = = = = = = = = = = = = = =

MTP

=

NGFI-B OAT OTC PEPCK PK PKA PKB PPAR

= = = = = = = =

PR PXR RAR RXR

= = = =

constitutive androstane receptor Chicken ovalbumin upstream promoter -transcription factor Cytochrome P450 DNA binding protein Estrogen receptor Embryonic stem cell Forkhead box-containing protein, O sub-family Fluorescence resonance energy transfer Fushi tarazu-factor 1 Farnesoid X receptor Glucokinase Glucocorticoid receptor Histone acetyl transferase Histone de acetylase Hypoxia -inducible factor Hepatic nuclear factor Hormone responsive element Janus kinase Jun kinase Ligand binding domain Liver-X-Receptor Mitogen activated protein Maturity Onset Diabetes of the Young Microsomal triglyceride transfer Protein NGF-induced clone B Organic anion transporter Ornithine transcarbamylase Phosphoenolpyruvate carboxykinase Pyruvate kinase Protein kinase A Protein kinase B Peroxisome Proliferator Activated Receptor Progesterone receptor Pregnane X receptor Retinoid Acid Receptor Retinoid-X-Receptor

SHP TGF TNF TR VDR ZO

= = = = = =

Small heterodimer partner Transforming growth factor Tumor necrosis factor Thyroid hormone receptor Vitamin D receptor Zona occludens

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