Therapeutic Potential of HNF4α in End-stage Liver Disease

ABSTRACT

The prevalence of end-stage liver disease (ESLD) in the US is increasing at an alarming rate. It can be caused by several factors; however, one of the most common routes begins with nonalcoholic fatty liver disease (NAFLD). ESLD is diagnosed by the presence of irreversible damage to the liver. Currently, the only definitive treatment for ESLD is orthotopic liver transplantation (OLT). Nevertheless, OLT is limited due to a shortage of donor livers. Several promising alternative treatment options are under investigation. Researchers have focused on the effect of liver-enriched transcription factors (LETFs) on disease progression. Specifically, hepatocyte nuclear factor 4-alpha (HNF4α) has been reported to reset the liver transcription network and possibly play a role in the regression of fibrosis and cirrhosis. In this review, we describe the function of HNF4α, along with its regulation at various levels. In addition, we summarize the role of HNF4α in ESLD and its potential as a therapeutic target in the treatment of ESLD.

KEYWORDS:

• ESLD
• HNF4α
• LETFs
• molecular therapy
• hepatocytes

Introduction

End-stage liver disease (ESLD) is a fatal condition with an increasing prevalence in the United States. ESLD is the final stage of chronic liver disease, characterized by irreversible damage to the cells, tissues, and hepatic functions.¹ From 1999 to 2016, the annual liver-associated deaths in the US increased by 65%, resulting in 34,174 deaths.² However, due to a high rate of underreported cases, the true mortality is likely much higher.³

Currently, medical therapy of ESLD involves mitigation of risk factors, nutritional support coupled with the avoidance of harmful medications, and avoidance of high risk, low reward procedures.⁴ Medical interventions can increase life expectancy, but OLT remains the only definitive treatment for ESLD.⁵ However, liver transplantation is extremely limited due to a shortage of donor livers. According to data collected by the Organ Procurement and Transplantation Network, less than half (49.0%) of waiting list registrants listed in 2018 received a deceased donor liver transplant within one year. Additionally, from 2014 to 2016, only 56.0% of adult liver transplant candidates had undergone the necessary transplant.⁶ This critical situation is further exacerbated by a forecasted increase in the cost of liver transplantation in the near future.⁵

Since alternative treatment options for ESLD are urgently needed, different approaches to treat ESLD are currently under investigation.^7,8 Moroni et al.⁸ confirmed the safety of autologous macrophage transplantation for potential treatment of cirrhosis and other fibrotic diseases. One of the most promising methods is the exploration of liver-enriched transcription factors (LETFs). LETFs, along with transactivation factors, have a great influence on the maintenance of liver-specific gene transcription.⁹ Liu et al.¹⁰ reported the initial hypothesis, which described the role of hepatocyte-specific transcription factors in the development of ESLD.^10,11 Interestingly, the expression of hepatocyte nuclear factor 4-alpha (HNF4α) was found to be directly correlated to liver disease in humans.^10,12 HNF4α is a transcription factor that plays an important role in liver morphogenesis and maintenance of proper hepatocyte function in a mature liver.^13–15 Most liver diseases have been associated with altered HNF4α expression, isoform ratios, and localization.¹⁶ Therefore, many studies have indicated that HNF4α may potentially be a target gene for the regression of fibrosis and cirrhosis.^17,18 This review touches upon the functions and regulation of HNF4α in a healthy mature liver, as well as the role of HNF4α in liver disease, particularly in ESLD. Implementation of HNF4α as a key therapeutic target for future ESLD treatment is discussed as well.

ESLD

Patients diagnosed with ESLD often exhibit abnormalities of liver synthetic and excretory functions with the development of ascites, hepatic encephalopathy, and variceal hemorrhage, among other complications.¹⁹ There are many causes of ESLD, including alcohol-related liver disease (ARLD)²⁰ and infection via hepatitis-inducing viruses, hepatitis B virus (HBV) and hepatitis C virus (HCV).^21,22 However, one of the most common maladies that progresses to ESLD in the US is nonalcoholic fatty liver disease (NAFLD).²³ The histological spectrum of NAFLD can range from simple steatosis, characterized by lipid accumulation in the liver, to more severe nonalcoholic steatohepatitis (NASH).^24,25 Patients with NASH are at an increased risk of developing fibrosis, cirrhosis, and potentially hepatocellular carcinoma of the liver.^24,26

It is important to note that cirrhosis exists at two different stages: compensated and decompensated. Compensated cirrhosis is the early asymptomatic stage, while decompensated cirrhosis is synonymous with ESLD.^27,28 Decompensated cirrhosis encompasses serious alterations at different levels, such as fibrosis with severe deposition of rigid extracellular matrix (ECM); disruption of the liver lobular architecture together with disruption of vascular organization, resulting in portal hypertension; and the inability of hepatocytes to perform adequate metabolic functions due to dedifferentiation or apoptosis of cells.^29,30 Nishikawa T et al.³¹ reported an adaptive metabolic shift in early stages of liver injury; hepatocytes start generating energy predominantly from glycolysis rather than oxidative phosphorylation. This adaptive shift results in hepatocytes that are unable to sustain high levels of energy production and subsequently leads to hepatocyte dysfunction.³¹ Hepatocyte dysfunction in individuals with ESLD manifests in the liver with reduced hepatocyte mass, oxidative stress, impaired mitochondrial function, and limited regenerative capacity.¹² It is likely that the dysfunction observed can be attributed to cytokine release or injuries that, by extrinsic mechanisms, alter the expression of the hepatocyte transcription factor network.¹⁸ Unfortunately, the precise molecular mechanisms responsible for the disruption of hepatic functions in liver injury remain enigmatic and require further investigation.

HNF4α function and regulation

There are six families of LETFs responsible for the maintenance of liver-specific gene transcription: HNF-1, HNF-3, HNF-4, HNF-6, CCAAT-enhancer-binding protein (C/EBP), and D-binding protein (DBP). The HNF-4 family is a subset of LETFs that includes HNF4β, HNF4γ, and most importantly HNF4α.⁹ HNF4α is a transcription factor in the superfamily of nuclear receptors^13,32 encoded by the HNF4A gene on chromosome 20 in humans.^33,34 Generally, nuclear receptors exhibit modular structure with six distinct regions, often referred to as regions A-F, which correspond to functional domains (Figure 1).³⁵ As a nuclear receptor, HNF4α utilizes these regions to control the activity of its target genes directly through transactivation mechanisms.³⁶ Full transactivation activity results from the synchronization of HNF4α’s two activation domains, activation function-1 (AF-1) and activation function-2 (AF-2).³⁷ AF-1 and AF-2, located in the A/B and D/E regions, respectively, activate transcription in a cell-type independent manner. Region D in HNF4α acts as a hinge between the DNA binding domain in region C and the ligand-binding domain in region E.^35,38 Additionally, region F has been indicated to express repressor activity.³⁹ Ultimately, region C contains a conserved zinc-finger DNA binding domain, which functions to bind HNF4α as a homodimer to the consensus DNA sequence (Figure 1).^40,41 Consensus DNA binding sequences have been found in the regulatory regions of many genes expressed in the liver and kidney.⁴² Upon binding these sequences, HNF4α recruits transcriptional co-activators and the appropriate accessory proteins to positively regulate the expression of target genes.⁴³

short-legendFigure 1.

As the master regulator of liver homeostasis, HNF4α targets several genes essential in hepatocytes to maintain cellular processes. Traditionally, the target genes of HNF4α have been thought to encode apolipoproteins (apolipoproteins CIII, A-IV), involved in cholesterol homeostasis, transthyretin (TTR), involved in carrying thyroid hormone and Vitamin A in serum, pyruvate kinase, involved in glycolysis, and glutamate synthetase, involved in amino acid biosynthesis.^44,45 Newer target genes are also associated with hepatic lipid homeostasis, xenobiotic detoxification, and drug metabolism.⁴⁶ HNF4α plays a crucial role in the expression of genes within Phase I and Phase II xenobiotic detoxification and drug metabolism.⁴⁶ Phase I is characterized by addition or exposure of a polar functional group to a lipophilic compound.⁴⁶ The most important Phase I gene that HNF4α regulates is CYP3A4, which is crucial to the body’s ability to metabolize drugs.⁴⁶ Phase II is characterized by conjugation reactions that help eliminate Phase I products from the body.⁴⁶ Genes in the SULT and UGT families account for the majority of Phase II metabolism and are regulated by HNF4α.⁴⁶ Bolotin et al., expanded on the classical roles of HNF4α by identifying additional target genes involved in signal transduction pathways (TAOK3, NGEF, FNTB) and in inflammation and immune responses (IL32, BRE, LEAP2).⁴⁷ Additionally, HNF4α has a role in controlling the expression of other LETFs such as HNF1α.⁹

Regulation of HNF4α expression/activity occurs through multiple mechanisms and at different levels.^{37, 48–54} The HNF4α gene is composed of 12 exons and alternative splicing of mRNA is a common way in which HNF4α expression is regulated at the transcriptional level. HNF4α protein variants are encoded by two separate promoters: P1 and P2. Transcription through the P1 or P2 promoters, combined with alternative splicing, produces up to 12 functionally distinct HNF4α isoforms.⁵⁵ Isoforms HNF4α1-6 are derived from the P1 promoter, while isoforms HNF4α7-12 are derived from the P2 promoter.⁴⁸ The P2-derived variants are in higher abundance within the adult pancreas and in the developing liver, whereas the P1-derived variants are in higher abundance within the adult liver.⁴⁹ Among the P1-products, the α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity.⁵⁶ The α4, α5 and α6 isoforms, which use an alternative first exon, display reduced transcriptional potential and inability to recognize the consensus response element of HNF4α.⁵⁶ P2 isoforms encode for a different amino-terminus on the 5ʹ end, which alters the AF-1 domain. This results in differential activation potential and target gene regulation compared to the P1-encoded isoforms.⁵⁷ These structural differences confer different capabilities in interacting with specific cofactors and in the transcriptional regulation of target genes in specific contexts.^56,58The transactivation activity of HNF4α can be potentiated by the binding of specific co-activators at the AF-2 domain, such as steroid receptor co-activator-1 (SRC-1) and glutamate receptor interaction protein-1 (GRIP-1).⁵⁰ Additionally, enhanced activity can occur due to post-translational modifications, such as methylation and acetylation.³⁷ HNF4α methylation at the arginine 91 residue by protein arginine N-methyltransferase 1 (PRMT1) increases the efficiency of HNF4α binding to consensus DNA sequences.⁵¹ Acetylation appears to be the most important post-translational modification in the regulation of HNF4α activity. Regarding this issue, acetylation of residues within the DNA binding domain by CREB-binding protein (CBP) has been shown to increase DNA binding activity as well.⁵² However, acetylation of the lysine 458 residue in the repressor region of the F domain is responsible for normal repression and downregulation of HNF4α activity.⁵³ Conversely, phosphorylation modifications negatively regulate HNF4α activity. More specifically, phosphorylation of serine 304 by AMP-activated protein kinase (AMPK) serves to repress the activity of HNF4α by reducing its ability to form dimers and bind to DNA.⁵⁴

Through its network of transcriptional regulation, HNF4α plays a pivotal role in regulating hepatocyte functions, and it has been documented that HNF4α maintains hepatocyte differentiation and inhibits proliferation. In the adult mouse liver, hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation.⁵⁹ Possible mechanisms by which HNF4α can inhibit cell proliferation involve epigenetic repression of pro-mitogenic genes, crosstalk with other cell cycle regulators including c-Myc and Cyclin D1, regulation of miRNAs, and post-translational modifications of HNF4α.^60,61 These properties are key during initial and final phases of liver regeneration.⁶²

HNF4α in liver disease

The metabolic functions of hepatocytes in the early stages of liver disease are elevated by the upregulation of multiple networks of metabolism-related genes.¹⁰ This elevated demand can only be sustained for a short period of time before hepatocellular failure.¹⁰ Subsequently, it has been demonstrated that the molecular hallmarks in the events that lead to the progression of end-stage cirrhosis and terminal hepatic failure consist of a gradual down-regulation of LETFs, such as HNF4α, FOXA2, CEBPα, PPARα and HNF1α.¹⁸ In an analysis of hepatocytes and liver tissue obtained from rats affected by liver disease, Nishikawa T et al.,¹⁸ reported that the presence of HNF4α in hepatocytes with decompensated function was significantly reduced. Failure of the hepatic transcription system contributing to disease progression within rats, found by Nishikawa T et al.,¹² was later confirmed to occur in humans as well.

In response to acute liver injury, protective mechanisms appear to trigger a decline in HNF4α expression to redistribute transcriptional resources.⁶³ This decline in HNF4α subsequently contributes to a loss of cell-identifying gene expression.⁶³ However, HNF4α loss is temporary, and HNF4α expression can be restored with recovery from the injury.⁶³ On the other hand, prolonged liver injury persistently suppresses HNF4α expression and can contribute to the progression of liver disease.⁶³ In fact, evaluation of patient liver tissue samples at varying stages of decompensated liver function confirmed a correlation between HNF4α expression and degree of liver disease and fibrosis.⁶³

As a transcription factor, expression of HNF4α must be localized to the nucleus of the hepatocyte to properly control gene transcription associated with different hepatocellular processes (Figure 2). In contrast to normal human hepatocytes, Florentino et al.⁶⁴ confirmed that, in addition to low protein expression, the expression of HNF4α in human hepatocytes isolated from livers with decompensated function is increasingly localized to the cytoplasm rather than the nucleus (Figure 2). Markedly, they also showed that reduced HNF4α acetylation at lysine 106 was accompanied by a higher degree of liver dysfunction.⁶⁴ Signaling pathways possibly associated with HNF4α nuclear localization include AKT (protein kinase B) and cMET. AKT plays an important role in numerous cellular processes including development, proliferation, and survival.⁶⁵ Florentino et al. revealed a strong correlation among AKT and HNF4α levels; as AKT levels decreased in human hepatocytes from livers with decompensated function, HNF4α expression also decreased.⁶⁴ In addition, AKT directly influences cyclic adenosine monophosphate response element‐binding protein (CREB), a molecule controlling nuclear retention of transcription factors through acetylation activity.⁵² It is suggested that AKT influences HNF4α nuclear localization through phosphorylation of threonine 308 by controlling the CREB-binding protein. Additionally, cMET, an inducer of AKT, was found to be reduced and directly correlated with decreased HNF4α nuclear localization.⁶⁴

short-legendFigure 2.

Furthermore, Yu et al.⁶⁶ identified that oxidative stress, which is elevated in fatty livers, fosters HNF4α cytoplasmic localization through activation of protein kinase C (PKC). PKC activation leads to phosphorylation of HNF4α and hinders its transfer into the nucleus by importin.⁶⁶ The decreased activity of HNF4α diminishes Apolipoprotein B (ApoB) and lessens very low-density lipoprotein (VLDL) secretion, leading to triglyceride (TAG) buildup in the liver. Consequently, this promotes the development of NAFLD.⁶⁶ Among the causes related to the decreased activity of HNF4α in liver disease, genetic alteration can be relevant. HNF4α mutations are associated with a large spectrum of diseases, including Crohn's disease,⁶⁷ Bowel disease,⁶⁸ and maturity-onset diabetes of the young (MODY).⁶⁹ Mutations in the HNF4α gene linked to maturity-onset diabetes in the young (MODY) are usually associated with a loss of HNF4α function.⁶⁹ The direct relationship between HNF4α mutations and liver disease progression has not been clearly established. Nonetheless, molecular mechanisms have been described. Mutations E276Q and R154X in the HNF4α gene lead to impaired HNF4α transcriptional activity through impaired physical interaction and functional cooperation between HNF4α and p300, a key HNF4α co-activator.⁷⁰ This decrease in HNF4α functional activity results in a decreased expression of its target genes, including HNF1α, which may finally alter the network of transcription factors in liver homeostasis.^41,70,71 These results support clinical findings that liver function can also be impaired in diabetic patients having HNF4α mutations.

Targeting HNF4α in molecular therapy

Liver transplantation remains the only curative treatment for patients with terminal liver failure. Recent advances in the understanding of the pathophysiology of cirrhosis, and the treatment of its complications, have resulted in improved management, leading to an increase in the quality of life and life expectancy of patients with liver disease.72–74 Pharmacological treatments targeting fibrosis and cirrhosis have been investigated with some success, such as the utilization of antifibrotic medications focusing on hepatic stellate cells.^75,76 Unfortunately, these therapeutic approaches fall short in achieving a significant restoration of vital liver function, as the potential side effects (e.g. hepatocarcinogenesis) outweigh the benefits.^{72–74, 76}

In the search for alternative treatments for ESLD, different approaches have revealed HNF4α as a potential target. Previously, HNF4α has been described as a potential therapeutic candidate for other liver diseases. Forced expression of HNF4α in animal models and cell lines of hepatocellular carcinoma (HCC) have shown to reverse invasiveness.⁷⁷ Downregulation of HNF4α promotes hepatic fibrosis and its re-expression alleviates hepatic fibrosis and improves liver function by suppressing the epithelial–mesenchymal transition (EMT). This evidence suggests that HNF4α could be an ideal option for the treatment of HCC or hepatic fibrosis.⁷⁸ Furthermore, it has been shown that the forced re-expression of HNF4α in rat models of end-stage liver disease corrected hepatocyte function at different levels, resetting target genes including liver-enriched transcription factors, serum proteins, coagulation factors and metabolic enzymes, as well as reversing impaired hepatic function.¹⁸

Regarding the role of HNF4α as a possible candidate for transcriptional factor therapy, there are still many problems to be solved. In order to develop therapies based on target genes and bring them to the clinical trial stage, the type of therapy or delivery system must first be determined. The therapeutic potential of HNF4α appears very promising since mRNA-based therapeutics as an exogenous delivery system have been shown to be effective in replacing, reprogramming or modifying protein expression. The development of different anti-cancer therapies⁷⁹ and the generation of vaccines against infectious diseases, such as SARS-CoV-2, have made apparent the power of this technology.^80–82

Interestingly, in a study closely modeling the pathophysiology of cirrhotic human livers, Tafaleng et al. showed that forced re-expression of HNF4α via mRNA treatment is effective in restoring, to some degree, endogenous expression of LETF and hepatocyte functional genes in human hepatocytes isolated from a cirrhotic liver. However, this study showed that this approach was not maximally effective in restoring normal HNF4α function, at least at the level of protein localization (Tafaleng et al., in press). Further investigation is necessary to increase the efficiency of HNF4α as a transcription factor in decompensated human hepatocytes. This may be done by targeting genes related to the function of HNF4α or those associated with its localization.

Currently, the concept of HNF4α as an ideal candidate for the treatment of end-stage liver disease is emerging, but to establish this potential molecular therapy, new challenges must be faced and questions must be answered, paving the way for clinical trials.

Abbreviations

ESLD, end-stage liver disease; NAFLD, nonalcoholic fatty liver disease; OLT, orthotopic liver transplantation; LETFs, liver-enriched transcription factors; HNF4α, hepatocyte nuclear factor 4 alpha; ARLD, alcohol-related liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; NASH, nonalcoholic steatohepatitis; ECM, extracellular matrix; EMT, epithelial–mesenchymal transition; HCC, hepatocellular carcinoma.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the NIH/NIDDK [P30DK120531].