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Curr Opin Pharmacol. Author manuscript; available in PMC Dec 1, 2011.
Published in final edited form as:
PMCID: PMC2981672
NIHMSID: NIHMS235543

HNF4α -- role in drug metabolism and potential drug target?

Abstract

Hepatocyte nuclear factor 4α (HNF4α) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors. It is best known as a master regulator of liver-specific gene expression, especially those genes involved in lipid transport and glucose metabolism. However, there is also a growing body of work that indicates the importance of HNF4α in the regulation of genes involved in xenobiotic and drug metabolism. A recent study identifying the essential fatty acid linoleic acid (LA, C18:2) as the endogenous, reversible ligand for HNF4α suggests that HNF4α may also be a potential drug target and that its activity may be regulated by diet. This review will discuss the role of HNF4α in drug metabolism, including the genes it regulates, the factors that regulate its activity, and its potential as a drug target.

Introduction

HNF4α (NR2A1), a highly conserved member of the nuclear receptor superfamily (NR) of ligand-dependent transcription factors (TFs), is known as a master regulator of liver-specific gene expression [1]. It was originally identified as an activity in rat liver that bound the APOC3 gene and characterized as an orphan receptor as its ligand status was unknown [2]. It was subsequently identified as the gene mutated in Maturity Onset Diabetes of the Young 1 (MODY1), an inheritable form of non-insulin-dependent diabetes [3]. Therefore, HNF4α was initially known for its role in carbohydrate and lipid metabolism in the liver and its role in insulin signaling in the pancreas. Subsequently, cytochrome P450 (CYP) genes involved in xenobiotic and drug metabolism were identified as targets of HNF4α [4], although its role in those processes was eclipsed by that of ligand receptors. The recent identification of the endogenous ligand for HNF4α, as well as several genome-wide studies that have greatly expanded the repertoire of HNF4α targets, has renewed interest in the role of HNF4α in drug metabolism. In this review, we will discuss the key findings on this topic and how they relate to the notion of targeting HNF4α for drug discovery.

Background on drug metabolism and HNF4α

The detoxification of xenobiotics and metabolism of drugs occurs primarily in the liver and consists of two phases. Phase I is carried out by hundreds of cytochrome P450 enzymes (CYP450s) and a handful of flavin-containing monooxygenases (FMOs) that add or expose a polar functional group to lipophilic compounds. Phase II, which consists of conjugation reactions that help eliminate Phase I products from the body, is carried out by glutathione-S-transferases (GSTs), UDP-glucuronosyltransferase (UGTs) and steroid-and bile acid-sulfotransferases (SULTs). In addition to acting on exogenous compounds, the Phase I and Phase II enzymes also act on endogenous compounds, such as steroids, bile acids and fatty acids, as well as drugs.

The expression of Phase I and Phase II genes is influenced by a wide variety of factors, including species, gender, age, diet, genetics and the environment [5]. Not surprisingly, many of the proteins responsible for turning on the expression of the Phase I and Phase II genes are TFs that are highly enriched in the liver. Some of those factors were originally identified as hepatocyte nuclear factors (HNFs) (e.g., HNF1, HNF2 (C/EBPα), HNF3 (now FOXA), HNF6) while several other factors belong to the NR superfamily. HNF4α belongs to both groups.

The human HNF4α gene consists of 12+ exons and two promoters and gives rise to many splice variants/isoforms [1]. The proximal P1 promoter is active in the adult liver, kidney and intestine, where most of the xenobiotic/drug metabolism occurs, and gives rise to two major isoforms -- HNF4α1 and HNF4α2. Recent genome-wide location analyses of HNF4α and other liver-enriched TFs (HNF1α, HNF6 and C/EBPα), showed that in hepatocytes HNF4α is much more often found bound to the promoters of 1iver-specific genes than other HNFs [6,7]. Therefore, since one of the main functions of the liver is detoxification of foreign compounds, the notion that HNF4α plays a major role in drug metabolism is a reasonable one.

Phase I and Phase II genes regulated by HNF4α

Phase I and Phase II target genes – identification by classical means

Several CYP450 genes have been identified as HNF4α targets using classical means (e.g., promoter cloning, gel shift analysis, reporter assays, etc.) and have been reviewed previously [8] (Figure 1). Among the Phase I enzymes, CYP450 3A4 (CYP3A4) is arguably one of the most important as it is involved in the metabolism of nearly half of all drugs currently used. Early studies on CYP3A4 gene regulation focused on ligand-activated NRs, pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) [9,10]. Subsequently, it was found that HNF4α is required for both PXR-and CAR-mediated transcriptional activation of CYP3A4 [11]. Tirona et al. examined a variety of NRs, including farnesoid X receptor (FXR, NR1H4), liver receptor homolog-1 (LRH1, NR5A2), liver X receptor α (LXR1, NR1H3), retinoid X receptor α (RXRα, NR2B1), and found HNF4α to have the greatest effect on the CYP3A4 promoter. Furthermore, when an HNF4α binding site in the CYP3A4 enhancer region was mutated, the ability of PXR and CAR to activate the CYP3A4 promoter was eliminated [11]. Thus, HNF4α plays a critical and central role in the regulation of this key Phase I gene.

Figure 1
Central role of HNF4α in the regulation of Phase I and Phase II genes

HNF4α has been shown to activate the expression of several other Phase I genes, including CYP2C8, CYP2C9, CYP2C19, CYP7A1 and CYP8B1 [8,12]. Knockdown of HNF4α by antisense and siRNA in primary human hepatocytes also identified CYP2A6, CYP2B6, CYP2D6, CYP3A4 and CYP3A5 as targets of HNF4α [13,14]. Classical techniques have also shown that HNF4α can bind and activate FMO1 promoter activity in concert with HNF1α [15].

In terms of Phase II genes, HNF4α also plays an important role in regulating the basal SULT2A1 promoter, as well as synergizing with PXR and CAR [16]. The expression of the UGT1A6 and UGT1A9 genes were found to be positively correlated with HNF4α and HNF1α expression in human liver [17] and functional HNF4α and HNF1α binding sites were identified in the promoter regions of both UGT genes using several classical techniques [1720]. Several transporters that are critical for the elimination of drugs and toxicants (ABCB1, ABCB11, ABCC2, SLCO1B1, SLC22A1), have also been found to be decreased when HNF4α is knocked down [14].

Phase I and Phase II target genes – identification by genomic approaches

The sequencing of the human genome has led to the use of genome-wide approaches that are more high throughput and less biased than the classical approach to target gene identification. Chief among those techniques are genome-wide location analysis (ChIP-chip/seq) and expression profiling. Another less well known but highly complementary technique is protein binding microarrays (PBMs) [21]. PBMs are a high throughput in vitro DNA binding assay that allows for the identification of 1000’s of distinct DNA binding sequences in a given experiment. We recently modified the PBMs to characterize the DNA binding specificity of native, full length HNF4α in crude nuclear extracts [22]. This led to the identification of >1400 new binding sequences for HNF4α that we then used to search the regulatory regions of human genes to identify potential new targets of HNF4α. The PBM data were also used to train a Support Vector Machine (SVM) algorithm to predict additional HNF4α binding sites with high accuracy. Finally, cross referencing with expression profiling data from an HNF4α RNAi knockdown in a human liver cancer cell line (HepG2) and published HNF4α ChIP-chip data identified >240 new direct, functional targets of HNF4α [22]. The results for the Phase I and Phase II genes are given in Table 1, along with target genes identified by classical methods. All told there are at least 50 CYP, 7 FMO, 21 GST, 13 SULT and 19 UGT (~110 total) human genes involved in drug metabolism that are predicted or proven targets of HNF4α.

Table 1
Verified and predicted HNF4α Phase I and Phase II human target genes1

Factors that influence HNF4α-mediated regulation of drug metabolism genes

Transcriptional regulation network

Many NRs such as PXR, CAR, LXR, FXR, small heterodimer partner (SHP, NR0B2), Vitamin D receptor (VDR, NR1I1), chicken ovalbumin upstream promoter transcription factor (COUP-TF, NR2F1) and glucocorticoid receptor (GR, NR3C1) interact with HNF4α to regulate the expression of drug metabolism genes in a complex fashion (Figure 1). For example, HNF4α is involved in crosstalk with PXR and CAR on the promoters of the CYP3A4 [23], CYP2C8 [24], CYP2C9 [25], CYP7A1 [26,27] and SULT2A1 [28] genes. While for several of these genes the crosstalk has not been characterized beyond requirements for specific NRs and their respective binding sites, on two genes – CYP3A4 and CYP7A1 -- the mechanisms have been explored. Ligand (rifampacin)-activated PXR recruits HNF4α and co-activator SRC-1 to the CYP3A4 promoter, thereby increasing its expression [23]. HNF4α is one of the few NRs able to activate the human CYP7A1 gene, a key player in bile acid synthesis (see [26]), but its ability to do so is negatively modulated by other NRs. In contrast to CYP3A4, a direct physical interaction between HNF4α and PXR, that is enhanced by rifampacin, displaces the co-activator PGC1α from HNF4α resulting in down regulation of CYP7A1 (and CYP8B1) gene expression [26,29]. Likewise, on CYP7A1, CAR competes with HNF4α for binding both DNA response elements and co-activators GRIP1 and PGC1α, thereby repressing the ability of HNF4α to activate this gene [27]. The VDR also interacts directly with HNF4α to suppress CYP7A1 expression [30]. High levels of SHP, a NR that lacks a DNA binding domain, represses CYP3A4 and CYP7A1 gene expression by interacting with HNF4α and PXR [23,31]. In contrast, rifampacin-activated PXR suppresses SHP, allowing for the crosstalk between HNF4α, PXR and coactivators (PGC1, SRC1) to maximally induce CYP3A4 [23]. FXR also suppresses CYP7A1 through up regulation of SHP. However, down regulation of CYP7A1 reduces bile acid synthesis and subsequently hepatic JNK activity that in turn increases HNF4α expression [32,33]. Interestingly, HNF4α not only co-regulates target genes with PXR, CAR and SHP but it also regulates their expression [14,22,23,3436]. FXR gene is also direct target of HNF4α while FXR protein can indirectly up regulate the expression of the HNF4A gene [22,32] (Figure 2A). Finally, on CYP2C9, HNF4α works with GR and NR coactivator 6 (NCOA6) to mediate maximum induction of the gene [37]. It is anticipated that similar complex networks involving multiple NRs will be found on one or more of the other HNF4α-regulated Phase I and Phase I genes in Table 1.

Figure 2
Factors affecting HNF4α expression and function

In addition to protein-protein and protein-promoter interactions between HNF4α and other NRs, there is also direct competition for binding to common response elements between HNF4α and the COUP-TFs, orphan NRs that tend to repress transcription. Both COUP-TFs and HNF4α bind DNA via direct repeats of a half site AGGTCA with a spacing of 1–2 nucleotides (DR1s and DR2s, respectively) and thus compete for regulation of the rat Cyp2C and human CYP2D6 genes. COUP-TFs also repress the strong transactivation function of HNF4α by binding additional response elements on the rat Cyp3a1 and Cyp3a23 promoters. In humans, COUP-TF2 inhibits HNF4α transactivation activity on the CYP3A4 promoter via both the DNA binding-dependent and –independent mechanisms [8] (Figure 1). Interestingly, HNF4α and COUP-TF2 can transactivate the expression of each others’ expression [38] (Figure 2A). Crosstalk between HNF4α and other TFs, such as C/EBPα and Oct-1, has also been observed on CYP2A6 [8]. More recently, two microRNAs have been identified that down regulate the expression of HNF4α (miR-24 and miR-34a). These miRNAs are up-regulated by reactive oxygen species (ROS) and phorbol ester (PMA), both of which down regulate HNF4α protein levels [39] (Figure 2B).

Genetics

Mutations in HNF4α response elements contribute to diseases such as hemophilia and MODY3 [4], while mutations in the HNF4α P2 promoter result in MODY1 [3]. Likewise an altered HNF4α binding site in a drug metabolism gene promoter could result in dysregulation of that gene and nucleotide changes in the HNF4α P1 promoter could result in altered levels of HNF4α1/2 protein in the liver, kidney and intestine, which would in turn affect Phase I and Phase II gene expression (Figure 2B). Indeed, a single nucleotide polymorphism (SNP) at a putative HNF4α binding site in the CYP2B6 promoter is able to alter the level of expression of that gene [40]. A SNP has also been identified in the HNF4α coding region that affects expression of the CYP2D6 gene that metabolizes a wide variety of drugs including beta blockers for heart disease and Prozacs and other drugs for mental disorders [41]. Numerous genetic variations resulting in altered levels of Cyp2D6 have also been associated with altered metabolism of 4-hydroxy tamoxifen and responsiveness to treatment for breast cancer [42]. While it is not known if HNF4α is directly involved in those effects, it is possible that HNF4α activity in the liver could affect one’s response to chemotherapeutic agents such as tamoxifen via Cyp2D6. Therefore, it will be of great interest to identify SNPs in HNF4α response elements in other Phase I and Phase II promoters, and to further characterize SNPs in the promoter and coding region of the HNF4A gene itself (Figure 2B).

Gender

In humans, it is estimated that there is a 40% difference in pharmacokinetics between men and women [43]. Much of this difference could be due to differential expression of drug metabolism genes between genders. CYP3A4, for example, is expressed at a higher level in women than in men [44], resulting in higher clearance rates of CYP3A4-targeted xenobiotics in women [45]. While these differences in human have not yet been attributed to HNF4α, in rodents HNF4α has been found to be a key player in gender-specific gene expression in liver and to be involved in crosstalk with STAT5b, a key intermediary in the growth hormone-mediated sexual dimorphism seen in hepatic gene expression [46,47]. An additional layer of complexity is achieved by the fact that STAT5b and HNF4α also co-regulate the expression of TFs that regulate gender-specific Cyp genes. For example, in response to growth hormone stimulation, STAT5b and HNF4α bind and activate the promoter of HNF6 (Onecut1) [48], a female-predominant transcription factor [49] that positively regulates the female-specific gene Cyp2c12 [50,51].

Environmental factors

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumor suppressor protein p53. We previously showed that p53 inhibits the transactivation function of HNF4α1 [52] and suppresses the HNF4α P1 promoter in human liver cells exposed to doxorubicin [53]. Other cellular stress conditions known to up regulate p53 expression, such as hypoxia [54] and arsenic trioxide [55], also down regulate the expression of HNF4α, and hence presumably affect Phase I and Phase II gene expression. We have also shown that protein kinase C (PKC) phosphorylates the DNA binding domain (DBD) of HNF4α, thereby inhibiting its ability to bind DNA and increasing its degradation. Importantly, the PKC site in the HNF4α DBD is completely conserved in all non steroid NRs, suggesting that other NRs such as CAR, RXR, FXR and COUP-TF, could also be affected by PKC activators [56]. This suggests that compounds such as phorbol esters that activate PKC could also alter the regulation of drug metabolism enzymes via HNF4α and other NRs. Finally, while HNF4α mRNA levels do not appear to be affected by the circadian clock, as are many other NR mRNAs [57], HNF4α protein does interact with Period (PER), a key component of the circadian clock regulatory pathway [58]. This opens the possibility that HNF4α activity may also be affected by the light/dark cycle.

Diet and the HNF4α Ligand

Diet can influence the levels and hence activity of HNF4α in several ways. HNF4α protein and mRNA levels are up regulated in the liver during fasting [59], as is the potent HNF4α co-activator PGC1α [60]. The effect on HNF4α is due in part to increased levels of insulin after feeding, which up regulates the sterol response element binding proteins SREBPs which in turn down regulate the expression of the HNF4α P1 promoter [59]. The energy/metabolic sensing kinase AMPK also phosphorylates both HNF4α and PGC1α thereby altering their activity [61,62]. Finally, bile acids, as a product of dietary metabolism, alter HNF4α expression in a complex fashion [32,33]. Hence, nutritional status can be expected to affect the transcription of many Phase I and Phase II genes via HNF4α.

One of the most controversial issues regarding HNF4α, and other orphan NRs, has been whether they respond to ligands [63]. HNF4α was known as a constitutively active NR that did not require the addition of exogenous ligand in order to activate transcription [2,64], but the identity of its endogenous ligand remained elusive. The crystallographic structures of bacterially expressed HNF4 showed that it did indeed contain a hydrophobic ligand binding pocket that could accommodate lipophilic compounds [65,66]. However, since those compounds, a mixture of fatty acids, bound HNF4 in an irreversible fashion, the ligand status and potential for HNF4 as a drug target remained in doubt. These issues were resolved once the endogenous HNF4α ligand expressed in the appropriate physiological context – mammalian tissues and cells – was identified. Using an unbiased and highly sensitive approach (affinity isolation followed by mass spectrometry, AIMS), linoleic acid (LA, C18:2\ω6) was found bound to HNF4α in the livers of fed but not fasted mice [36]. This made physiological sense as LA is an essential polyunsaturated fatty acid that must be obtained from the diet. Furthermore, it was shown that LA binds mammalian-expressed HNF4α in a reversible fashion, suggesting that it could be available for drug targeting. While a specific transactivation function of LA on HNF4α, was not observed, a modest but consistent decrease in HNF4α target gene expression in the presence of LA that was associated with a decrease in HNF4α protein levels was noted [36]. These findings raise the intriguing possibility that different diets containing different amounts of LA could affect HNF4α levels, and hence drug metabolism, although this remains to be proven in vivo.

HNF4α as a drug target?

While the function of the endogenous HNF4α ligand remains uncertain, what is clear is that HNF4α contains a ligand binding pocket that is occupied in a reversible fashion dependent upon the feeding state of the host. Hence, it is possible that one could design a drug that could bind in the pocket of HNF4α and alter its ability to activate transcription. However, since HNF4α regulates so many Phase I/II and other genes in the liver, there is a high likelihood of side effects, unless one can develop a drug that is specific to a given HNF4α target. But this is not a new problem, nor is it unique to HNF4α. Any NR or TF with multiple targets faces similar problems. The trick will be to develop drugs that are specific to HNF4α on one or at most a few genes. This specificity could be found in the DNA sequence of the response elements that we now know can vary greatly between different target genes [22].

Perspectives

It is now evident that HNF4α is a key player in the regulation of genes involved in drug metabolism. Indeed, since HNF4α is one of the most ancient of the NRs and since Cyp genes have equally ancient evolutionary origins [63], it is possible that HNF4α was the first NR to control the expression of this critical gene family in animals. The regulation of these genes, however, has evolved, just as the genes themselves. In addition to HNF4α, that regulation now involves many additional NR, and other TFs, in a complex network of synergy, inhibition and mutual regulation that is affected by a variety of internal factors such as SNPs and external factors such as diet and environmental stress. Detailed knowledge of this network and the factors that influence it is essential for understanding individual responses to drug treatment.

Acknowledgements

We apologize to all of our colleagues whose work we were not able to cite due to space limitations. WH-V is funded by a postdoctoral fellowship from Academia Sinica. The Sladek lab is funded by grants from the National Institutes of Health (DK053892 and MH087397).

Footnotes

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