HNF4, hepatocyte nuclear factor 4

Outcomes of HBV infection are affected by both virus (such as: virus variation, virus protein, virus genotype, etc.) and host factors (such as: cellular immunity, cytokines, apoptosis, etc.) [4-8], and evidence have showed that viral replication is mainly correlated to the development or pro- gression of diseases [2,4]. In fact, viral replication is not only regulated by host factors [9], but also can cause changes in the level and activity of host factors, which would result to severe hepatic damage due to cell dysfunc- tion. Hepatic nuclear factors (HNFs) are a group of impor- tant host transcription factors that mainly reside in the liver and regulate numerous liver-expressed genes [10]. One of our previous work suggested that HNF4a and HNF3b likely participated in HBV replication in patients with HBV infection, or that HBV replication may some- how influence the expression of hepatocyte nuclear factor 4alpha (HNF4 a ) and 3 beta (HNF3 b ) in the liver [11]. Using cell culture and animal models, we also found that HNF4 a supports HBV replication in non-hepatic cells and HNF3 b inhibits HBV replication [12]. And in recent years,

Nuclear receptor hepatocyte nuclear factor 4-α (HNF4α) activates the promoters of multiple genes expressed in he- patocytes that play a role in lipoprotein metabolism [24]. The proximal promoter of the SHBG gene contains an HNF4α binding site, and overexpression of transcription factor HNF4α in HepG2 cells can stimulate transcription of the SHBG promoter [25]. Several studies found that monosaccharide and lipid, as well as other factors, could regulate the SHBG expression via changes in HNF4 α gene expression [26–28]. Among them, liver lipids were consid- ered to be a crucial factor in regulating HNF4α-SHBG. Adiponectin treatment of HepG2 cells activated AMPK, which decreased the hepatic lipid content, then increased HNF4α levels and upregulated SHBG expression [28]. SHBG mRNA is not naturally expressed in rodent liver; therefore, to date, research on SHBG gene expression using in vivo models is limited, and studies concerning SHBG expression in humans are rarely found.

Initiation of gene expression is executed by specific bind- ing of transcription factors to gene regulatory elements, and molecules affecting this process can regulate corre- sponding gene expression. The concrete structure and regulation mechanisms of APOC3 and APOA5 gene ex- pression have been reviewed elsewhere [9], and we will focus here on regulators that are shared by APOC3 and APOA5. Indeed, several molecules have been implicated in the same direction regulation of APOC3 and APOA5 ex- pression, including upregulation with hepatocyte nuclear factor 4-α (HNF4-α) [15, 16] and glucose [17, 18], and downregulation with AMP-activated protein kinase [15, 19], insulin [20–22] and tumor necrosis factor-α (TNF-α) [23, 24]. Noticeably, these substances, except for TNF-α, are all important components directly involved in glucose metabolism, suggesting APOC3 and APOA5 dysregula- tion may contribute to diabetic dyslipidemia. Opposite direction regulation was also found in that peroxi- some proliferator-activated receptor-α (PPAR-α) and farnesoid X-activated receptor (FXR) promoted APOA5 [13, 14] while inhibited APOC3 expression [25, 26]. In contrast to APOA5, the human APOC3 gene promoter doesn’t contain PPAR-α and FXR posi- tive response elements. Actually, these two nuclear receptors acted indirectly by interfering the binding of other transcriptional factors, like HNF4-α, to specific elements of APOC3, thereby further inhibiting APOC3 gene transcription [26, 27]. Thus, the plasma TG low- ering effect of fibrates, one type of PPAR-α agonists, may be partly mediated by increasing the circulating concentration of apoA5 and/or decreasing apoC3 levels. Indeed, recent studies showed that both fenofi- brates and omega-3 polyunsaturated fatty acids ther- apy significantly decreased plasma apoC3 levels in humans [28, 29].

replication by truncated HNF3␤ polypeptides. Mouse NIH 3T3 fibro- blasts were transiently transfected with the HBV DNA (4.1-kbp) con- struct (lanes 1 to 10) plus the HNF4 expression vector (lanes 2 to 10) and the HNF3␤ expression vectors (lanes 3 to 10). (A) RNA (North- ern) filter hybridization analysis of HBV transcripts. The glyceralde- hyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single- stranded DNA. The amino acids of the HNF3␤ polypeptides for panels A and B are shown below the gel in panel B. (C) Quantitative analysis of the 3.5-kb HBV RNA and DNA replication intermediates. The levels of the 3.5-kb HBV RNA and HBV DNA replication interme- diates (HBV DNA RI) are reported relative to the levels of the HBV DNA (4.1-kbp) construct in the presence of HNF4 expression (lane 2), which are set at 1.0. The mean RNA and DNA levels plus standard deviations (indicated by the error bars) from two independent analyses are shown. (D) The effects of truncated HNF3␤ polypeptides on tran- scription from the nucleocapsid and large surface antigen promoter constructs CpLUC and PS1pLUC, respectively, were examined. Rel- ative activities of the constructs in mouse fibroblast in the absence or

Abstract: Hepatitis B virus (HBV) infection is a major factor in development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play key role in development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting ERK signaling pathway. Our data showed that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provided several proofs for the effect of HBV infection in manipulating HNF4α regulatory pathway in HCC development.

nuclear factor/forkhead homologue (HFH)-4 is a 421–amino acid member of the winged helix family (16, 17). Expression of HFH-4 in mice and humans has been localized to the lung, spermatids, oviduct, choroid plexus, and fetal kidney (17–19). In the developing mouse and human lung, HFH-4 expression is restricted to the proximal pulmonary epithelium and is asso- ciated with the differentiation of the proximal from the distal respiratory epithelium during the late pseudoglandular stage of lung development (17, 19). Expression of HFH-4 is also de- velopmentally regulated in spermatids, choroid plexus, and re- nal epithelium (17–19). These tissues are all sites of ciliated cells consistent with a role for HFH-4 in the differentiation of ciliated epithelium. We report here that targeted disruption of the mouse hfh-4 gene demonstrates an essential role for HFH-4 in the development of cilia and, in addition, the nonrandom determination of left-right asymmetry.

The transcription factors that regulated HBV RNA synthe- sis have been extensively examined (10, 12, 13, 20, 24). The roles of specific liver-enriched transcription factors in deter- mining the level of HBV pregenomic RNA expression and viral replication have also been characterized in cell culture (21). These studies have demonstrated that the nuclear hor- mone receptors, HNF4 and RXR␣ plus PPAR␣, are a major determinant restricting HBV viral replication to cells of he- patic origin (21). These analyses also demonstrated that the liver-enriched transcription factor HNF3 negatively regulated nuclear hormone receptor-mediated HBV pregenomic RNA synthesis and viral replication in cell culture (21, 22). In this study, the possibility that HNF3␤ might also negatively regu- late viral transcription and replication in vivo in a manner similar to that observed in cell culture was investigated by using an HBV transgenic mouse model (8). This was achieved by characterizing the viral transcripts and replication intermedi- ates in HBV transgenic mice expressing the rat HNF3␤ poly- peptide in their liver (8, 16).

Sodium-dependent uptake of bile acids across the hepatic basolateral membrane is rapidly and profoundly diminished during sepsis, thus contributing to the pathogenesis of sepsis- associated cholestasis. This effect is mediated by endotoxin or effector cytokines, which reduce expression of several hepatobiliary transporters, including the sodium-dependent bile acid transporter gene, ntcp. We test here the hypothesis that endotoxin treatment leads to impaired binding activity of ntcp promoter trans-acting factors, resulting in reduction of ntcp mRNA expression. After endotoxin administration, ntcp mRNA levels reached their nadir by 16 h, and nuclear run-on assays demonstrated a marked reduction in ntcp gene transcription. At 16 h after treatment, nuclear binding activities of two key factors that transactivate the ntcp promoter, hepatocyte nuclear factor (HNF) 1 and Footprint B binding protein (FpB BP), decreased to 44 and 47% of pretreatment levels, respectively, while levels of the other known ntcp promoter transactivator, signal transducer and activator of transcription 5, were unaffected. In contrast, the universal inflammatory response factors nuclear factor kappaB and activating protein 1 were both upregulated significantly. Examination of nuclear extracts obtained at sequential time points revealed that the

Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, […]

4 weeks of age, and then they were fed a high-fat diet for 4 weeks. The body weight and WAT mass of the epididymal fat pads of βHT- IRS2 mice were significantly higher than those of the control mice (Figure 8A). βHT-IRS2 mice developed type 2 diabetes, as fasting glucose levels were slightly but significantly higher than those of control mice (Figure 8B). In insulin tolerance tests, the glucose lowering effect of insulin was significantly lower in βHT-IRS2 mice than in control mice (Figure 8C). We performed histological and immunohistological analyses of pancreatic islets. The β cell mass of βHT-IRS2 mice was significantly reduced as compared with that of control mice (Figure 8D). We next investigated Pdx1 expression levels. Immunohistochemical studies revealed Pdx1 protein to be equally expressed in the nuclei of βHT-IRS2 mice and control mice (Figure 8E), despite the development of type 2 diabetes in the mutant mice. Taqman PCR revealed that the expression levels of Pdx1 mRNA in the βHT-IRS2 mice were slightly, but not signifi- cantly, lower than those in control mice (Figure 8F).

In this context, cell-based therapy has become the focus of intense investigation in recent years [3]. Therefore, various kinds of stem cells have been studied to examine their clinical applications in regenerative medicine [4]. To date, there are numerous studies describing the beneficial use of mesenchymal stem cells (MSCs) for liver cell therapy, mainly because of their multipotent potential, lack of ethical concerns, and risk of rejection [5, 6]. MSCs can be isolated from different kinds of tissues including bone marrow (the first known tissue as a source of MSCs) [7], umbilical cord blood, amniotic fluid as well as adipose tissue [8]. Of particular interest to researchers are the human adipose tissue- derived stem cells (hASCs) which for the first time were isolated and described by Zuk et al. In addition to their multilineage potential [9], hASCs have been reported to exhibit higher proliferation rate, more accessibility and

Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhib- ited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanisti- cally, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscrip- tionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mu- tation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Impor- tantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mecha- nisms.

tors have been shown to activate transcription in both ligand- dependent (1, 5, 32) and ligand-independent (31, 36) manners. Moreover, nuclear receptors exert diverse effects upon cell differentiation, morphogenesis, and carcinogenesis (2, 10, 12, 39). Known members of this superfamily include retinoic acid receptor a (RAR a ), RAR b , and RAR g ; RXR a , RXR b , and RXR g ; peroxisome proliferator-activated receptor (PPAR); hepatocyte nuclear factor-4 (HNF-4); chicken ovalbumin up- stream promoter transcription factor (COUP-TF); and recep- tors for thyroid hormone, estrogen, glucocorticoid, progester- one, vitamin D, androgen, and mineralocorticoid (19, 43). Recent studies have revealed that the liver-enriched nuclear receptors RXR a (17, 23) and HNF-4 (17) transactivate the HBV enhancer 1 by binding to the same site (Fig. 1B). COUP-TF appears to antagonize transcriptional activation by competing with RXR a and HNF-4 for binding to the same site on enhancer 1 (17). These findings suggest that RXRa is capable of regulating HBV gene expression by engaging in cooperative interactions with other nuclear receptors. While RXRa appears to be capable of functioning as a homodimer upon induction by 9-cis-retinoic acid (29, 46), it has been proposed that RXR a predominantly functions as a het- erodimeric partner, or auxiliary protein, for other nuclear receptors (8, 27, 45).

Hepatocellular carcinoma (HCC), which acc- ounts for about 95% of all primary liver can- cers, has become the third most frequent cause of cancer-related mortality worldwide [1, 2]. Each year, more than 700,000 patients are diagnosed with HCC and more than 600,000 cases dead due to this malignancy around the world [3]. Due to local invasion and intrahepatic metastasis, HCC patients have a high incidence of recurrence even after curative therapy and the 5-year survival rate of HCC patients is only approximately 5% [4]. Therefore, revealing the molecular mechanism for the tumorigenesis of HCC is indispensable for developing effective therapy.

occurrence of liver adenomatosis in six MODY3-affected patients from two unrelated large families, and a hot-spot germline mutation P291fs of HNF-1α was identified in the two pro-bands and 16 relatives from the two families. Con- sequently, MODY3-affected patients should be screened for liver adenomatosis as they carry significant risk as a result of HNF-1α mutations [15]. HNF-1α mutations are also linked with increased tissue glucose uptake, which can serve as a worsening factor in carcinomas. Ozaki et al. shed light on the potential mechanism of HNF-1α-inactivated HCAs (H-HCAs), and they found increased glucose uptake owing largely to GLUT2 and HK4 expression and G6PT1 inactivation [16]. Although the exact mechanism linking HNF-1α mutations with the development of diabetes is par- tially known, there are a number of studies that suspect structural mutations as a plausible explanation. The discus- sion in this section implicates HNF-1α mutations in accu- mulating lipids in the liver, promoting diabetes, and raising glucose uptake that can better help survive cancerous cells.

CNV: Copy number variants; COL3A1: Collagen type III, alpha 1; EDS: Ehlers- danlos-syndrome; FBN1: Fibrillin 1 gene; GBM: Glomerular basement membrane; HNF1 β : Hepatocyte nuclear factor-1 β gene; LHX1: Homeobox transcription factor 1 gene; MN: Membranous nephropathy; MRKH: Mayer- rokitansky-kuester-hauser-syndrome; NODAT: New onset diabetes mellitus after transplantation; NT-pro BNP: N-terminal pro brain natriuretic peptide; PLA2R: Phospholipase A2 receptor.

deposition, hepatocyte degeneration, nuclear pyknosis and mild necrosis.. Fat deposition, hepatocyte degeneration, focal necrosis, increase MMC.[r]

Klf4 was commonly expressed in nasopharyngeal carci- noma. Previously, KLF4 was found to be expressed in the cytoplasm of nasopharyngeal carcinoma, and it played a role as tumor suppressor gene [9]. Studies have shown that KLF4 was associated with H-Ras and PI3K, which are downstream to the EGFR pathway, mutations in the Ras pathway regulate the KLF4 expression [10], the PI3K mutation was accompanied by altered KLF4 expression [11]. H-Ras and PI3K were common mutations associated with cetuximab resistance, and contrary to our previ- ous research, KLF4 played the role of an tumor suppres- sor in these processes. The different functions of KLF4 were associated with its subcellular localization in cells and structure. The structural conformation of Klf4 had three typical C-terminus C2H2 zinc fingers (ZFs) as DNA binding regions and N-terminus activation regions [12], in the study of KLF4, nuclear localization sequence (NLS) has been shown to play a role in subcellular localization and is specifically expressed in the cytoplasm or nucleus

The aberrant expression of HNF1 β in tumors is associ- ated with epigenetic processes and epigenetic changes. In humans, one of the epigenetic mechanisms that regulate expression of genes is methylation of the clusters of CpG dinucleotides, called CpG islands. A probable mechanism of aberrant up-regulation of HNF1 β in ovarian clear cell carcinoma is hypomethylation of the HNF1 β CpG island [35]. Terasawa et  al. reported that methylation of the HNF1 β CpG island was rare in ovarian CCC, but com- mon in non-CCC ovarian cancers or various cancer cell lines [35]. Hypomethylation of the HNF1 β CpG island probably participates in the up-regulation of HNF1 β in ovarian CCC. Epigenetic inactivation of HNF1 β is also seen in colorectal, gastric, and pancreatic cancer cell lines, suggesting involvement of epigenetic inactivation of HNF1 β in tumorigenesis [41]. HNF1 β mutations are known to affect expression of downstream genes such as HNF4 α , PKHD1 and UMOD [20]. HNF4 α is upregu- lated by inducing HNF1 β expression, which suggests alterations in the hepatocyte nuclear factor network can be reversed by inducing HNF1 β through demethylation

Although the mode of action of oligonucleotides in humans compared to current therapies can be regarded as a great benefit, it can be a problem in situations when rapid reversal of the silenced protein is necessary. In some cases, singular treatment with recombinant pro- teins will be a solution, although correct dosing would be crucial. A more elegant solution would be to use an oligonucleotide antidote. Recently, it was shown in mice that upon prothrombin-specific ASO treatment, comple- mentary sense oligonucleotide antidote (SOAs) reversed the antithrombotic phenotype by specifically binding of ASOs intracellularly [75]. Even though these data are promising, therapeutic usefulness has to be proven for individual ASOs. Moreover, it has been shown that re- versal of siRNA therapy is feasible using complementary high affinity oligonucleotides as a “ synthetic target ” or decoy to abrogate silencing activity of antisense-loaded RISC (Reversir ™ , http://www.alnylam.com/web/assets/ Reversir_OTS_101315.pdf ). Currently, the strategy has been reported to be functional for reversal of the effect of an siRNA against coagulation factor IX. The major disadvantage of this oligonucleotide-specific approach is that it may take too long for the antidote to restore original levels of the initially silenced protein. For this reason, in the case of a sudden event such as a surgical intervention, an oligonucleotide-specific antidote may not be adequate and recombinant proteins or fresh frozen plasma treatment are still necessary.