Non-liver Cell Lines Research Articles

AKR1D1 regulates glucocorticoid availability and glucocorticoid receptor activation in human hepatoma cells

Steroid hormones, including glucocorticoids and androgens, have potent actions to regulate many cellular processes within the liver. The steroid A-ring reductase, 5β-reductase (AKR1D1), is predominantly expressed in the liver, where it inactivates steroid hormones and, in addition, plays a crucial role in bile acid synthesis. However, the precise functional role of AKR1D1 to regulate steroid hormone action in vitro has not been demonstrated. We have therefore hypothesised that genetic manipulation of AKR1D1 has the potential to regulate glucocorticoid availability and action in human hepatocytes.In both liver (HepG2) and non-liver cell (HEK293) lines, AKR1D1 over-expression increased glucocorticoid clearance with a concomitant decrease in the activation of the glucocorticoid receptor and the down-stream expression of glucocorticoid target genes. Conversely, knockdown of AKR1D1 using siRNA decreased glucocorticoid clearance and reduced the generation of 5β-reduced metabolites. In addition, the two 5α-reductase inhibitors finasteride and dutasteride failed to effectively inhibit AKR1D1 activity in either cell-free or hepatocellular systems.Through manipulation of AKR1D1 expression and activity, we have demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells. These data suggest that AKR1D1 may have an important role in regulating endogenous (and potentially exogenous) glucocorticoid action that may be of particular relevance to physiological and pathophysiological processes affecting the liver.

The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers

Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancer. In liver cancer, PHB1 acts as a tumor suppressor, but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-Seq analysis, we found interleukin-8 (IL-8) to be one of the most highly up-regulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and nonliver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that IL-8 mRNA levels were increased, whereas PHB1 mRNA levels were decreased, in the tumors compared with adjacent nontumorous tissues. Notably, HCC patients with high IL-8 expression have significantly reduced survival. An inverse correlation between PHB1 and IL-8 mRNA levels is found in HCCs with reduced PHB1 expression. To understand the molecular basis for these observations, we altered PHB1 levels in liver cancer cells. Overexpression of PHB1 resulted in lowered IL-8 expression and secretion. Silencing PHB1 increased c-Jun N-terminal kinase (JNK) and NF-κB activity, induced nuclear accumulation of c-JUN and p65, and enhanced their binding to the IL-8 promoter containing AP-1 and NF-κB elements. Conditioned medium from PHB1-silenced HepG2 cells increased migration and invasion of parental HepG2 and SK-hep-1 cells, and this was blocked by co-treatment with neutralizing IL-8 antibody. In summary, our findings show that reduced PHB1 expression induces IL-8 transcription by activating NF-κB and AP-1, resulting in enhanced IL-8 expression and release to promote tumorigenesis.

Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

Identification of Slug and SOX7 as transcriptional repressors binding to the hepatitis B virus core promoter

The Hepatitis B Virus (HBV) may gain entry into non-liver cells but does not actively replicate in them. We investigated the possibility that these cells possess mechanisms that block HBV core promoter (HBVCP) transcription, specifically absent in liver cells, which together with other liver-specific mechanisms, such as sodium-taurocholate cotransporting polypeptide-mediated entry, enable liver cells to effectively produce HBV. Liver and non-liver cell lines were screened for their capacity to activate the HBVCP and synthesize pre-genomic RNA (pgRNA). Transcription regulators differentially expressed between cells with active or inactive HBVCP were determined by human transcriptome array. Slug (SNAI2) and SRY-related HMG box 7 (SOX7) transcriptional repressors were identified and shown to bind specifically to the HBVCP by electrophoretic mobility shift assay. The resultant inhibitory effect on HBVCP transcription was validated using luciferase reporter and assays for pgRNA, HBcAg and cccDNA accumulation in cells with HBV replicon and HBV infection models. To further confirm their specific activity, short peptide mimetics generated from Slug zinc-finger domains and SOX7 HMG-box were generated. The HBVCP was found to be active in liver and selected non-liver cells. These cells have low/negligible expression of Slug and SOX7, which inhibit HBVCP transcription specifically by binding at the pgRNA initiator site and competitively displacing hepatocyte nuclear factor 4α, respectively. Overexpression of Slug and/or SOX7 specifically reduced HBVCP transcription, significantly diminishing pgRNA synthesis, HBcAg and cccDNA accumulation in HBV-infected primary human hepatocytes. Similar results were obtained with Slug and SOX7 stapled peptides individually, which were even more potent in combination. Slug and SOX7 are transcriptional repressors that bind specifically to the HBVCP. Their absence or weak expression in liver cells contribute to the favorable host environment for the active and efficient production of HBV. Hepatitis B virus (HBV) replication occurs efficiently in human liver because of the specificity of viral uptake receptors and presence of numerous liver-enriched transcription activators. Herein, we show that the specific lack of transcriptional inhibitory mechanisms in liver cells also contribute to effective HBV production. HBV replication is kept low in non-liver cells as transcriptional repressors Slug and SRY-related HMG box 7 (SOX7) actively bind to the transcriptional initiator and displace transcription activators, respectively, within the HBV core promoter.

Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [14C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [14C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

Human Apolipoprotein E is necessary and sufficient for production of infectious HCV particles in human non-liver cell lines

HCV has a narrow tissue tropism. Accordingly, efficient replication is only observed in the liver and in cultured human liver cell lines. The molecular determinants that are responsible for the restricted tropism of HCV are incompletely understood. Recent evidence indicates that HCV assembly is tightly coupled to the synthesis of human lipoproteins that occurs in liver cells. Specifically, Apolipoprotein B (ApoB), ApoE, and microsomal triglyceride transfer protein (MTP), which are all essential for production of very low density lipoproteins (VLDL) have been implicated in HCV assembly. To define the minimal liver cell-specific requirements for production of infectious HCV progeny, we attempted to reconstitute assembly of HCV particles in human non-liver cell lines. To this end, we created stable HeLa (human cervical cancer) and Huh-7.5 (human hepatocarcinoma) cells replicating selectable subgenomic HCV RNA replicons. Co-expression of viral structural proteins reconstituted particle production only in Huh-7.5 replicon cells, but not in HeLa replicon cells. Importantly, ectopic expression of ApoE was necessary and sufficient to allow production of infectious HCV trans-complemented particles (HCVtcp) in HeLa cells. Comparison between Huh-7.5 and HeLa derived particles revealed no gros differences between the density distribution of intra- and extracellular particle. Using a similar approach, we could also observe ApoE-dependent production of infectious HCVtcp in several other non-liver cell lines. Enhancement of HCV replication by introduction of miR-122 into ApoE-expressing 293T and HeLa cells was sufficient for production of full-length HCV particles. More detailed analyses of particle composition and receptor usage of these particle types are currently ongoing. Collectively, these data indicate that ApoE may be the sole liver cell-specific cofactor for assembly of infectious HCV particles. In turn, ApoE may be major determinant limiting HCV tissue tropism.

Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

Abstract 2039: Applying genomic approaches to identify novel targets for human hepatocellular carcinoma

Abstract Hepatocellular Carcinoma (HCC), a primary malignant cancer of the liver, is the 5th most common cancer worldwide and the 3rd most common cause of cancer mortality. This aggressive cancer has a poor prognosis with a median survival of 6 months, and overall 5 year survival rate of about 14%, which falls to 2% for metastatic disease. Currently only one drug, Sorafenib, is FDA approved for the treatment of HCC, and has been shown to prolong survival approximately 3 months. However, the adverse side effects of the drug significantly diminish patient quality of life. Consequently, there is an urgent need for the discovery of novel gene targets selective for HCC, which can guide the development of new therapies in HCC. High-throughput RNAi technology was applied in order to identify novel gene targets. Small interfering RNA (siRNA) inhibits gene expression at a post transcriptional level by preventing mRNA translation of protein products. Through systematic knockdown of individual genes, potential drug targets that inhibit cellular proliferation can be identified. This approach was applied to a panel of 8 HCC cell lines (SNU449, SNU182, SNU387, SNU475, SNU423, Hep3B, HepG2, and PLC/PRF/5). Initially, assay development was extensively conducted in order to optimize conditions specific for each cell line including doubling time, seeding density, FBS concentration, DMSO toxicity, and transfection reagent concentration (≥95% transfection efficiency and ≤20% toxicity). Following assay optimization, each cell line was screened against 953 genes (711 validated kinase, 36 MAPK, and 206 Phosphatase) containing 4 unique siRNA sequences per gene. Cell lines were run in duplicate plates containing siRNA, negative controls (Buffer, Non-Silencing, All Star Non-Silencing, and GFP), and positive controls (UBB and ACDC) generating over 70,000 data points. Quality control was conducted following data collection to evaluate cell viability, toxicity, transfection efficiency, coefficient of variation (CV) of controls, Z’ factor, and screen reproducibility prior to moving onto hit selection. “Hits” will then be selected as siRNA that induce over 60% cell death/inhibition of growth relative to negative controls. In order to reduce false positive rates from siRNA “off-target” effects, targets will then be prioritized as genes with 2 or more siRNA sequences showing phenotypic inhibition of cell proliferation. Eight HCC cell lines have been screened against this custom siRNA set thus far with hits currently being identified. Ultimately, targets will be evaluated in follow up studies, assigning priority to hits where lethality is observed across multiple HCC cell lines while not affecting non-liver and normal liver cell lines, thereby validating liver-specific targeting. Therefore, the discovery of potential HCC targets for novel therapies would potentially impact a broader population of patients with HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2039. doi:10.1158/1538-7445.AM2011-2039

Novel tumour‐specific promoters for transcriptional targeting of hepatocellular carcinoma by herpes simplex virus vectors

Hepatocellular carcinoma (HCC) is a cancer of poor prognosis, with limited success in patient treatment, which it makes an excellent target for gene therapy and viral oncolysis. Accordingly, herpes virus simplex type-1 (HSV-1) is one of the most promising viral platforms for transferring therapeutic genes and the development of oncolytic vectors that can target, multiply in, and eradicate hepatoma cells via their lytic cycle. Enhanced efficacy and specificity of HSV-1-based vectors towards HCC may be achieved by using HCC-specific gene promoters to drive selective viral gene expression and accomplish conditional replication and/or to control the expression of therapeutic genes. However, careful verification of promoter function in the context of the replication-competent HSV-1 vectors is required. The present study aimed to identify novel HCC-specific promoters that could efficiently direct transgene expression to HCC cells and maintain their activity during active viral replication. Publicly available microarray data from human HCC biopsies were analysed in order to detect novel candidate genes induced primarily in HCC compared to normal liver. HCC specificity and promoter activity were evaluated by RT-PCR and chromatin immunoprecipitation. Additionally, transcriptional activity of promoters was further evaluated in the context of HSV-1 genome, using luciferase assays in cultured cells and animal models. Eight HCC-specific genes were characterised in this study: Angiopoietin-like-3, Cytochrome P450, family 2, subfamily C, polypeptide 8, Vitronectin, Alcohol dehydrogenase 6-class V, Apolipoprotein B, Fibrinogen beta chain, Inter-alpha-globulin-inhibitor H3 and Inter-alpha-globulin-inhibitor H1. Specific HCC expression and active gene transcription were confirmed in human liver and non-liver cell lines and further evaluated in primary neoplastic cells from hepatitis C and B virus (HCV- and HBV)-associated HCC patients. High promoter activity and specificity in the presence of HSV-1 infection and from within the viral genome, was validated, both in vitro and in vivo. We identified and experimentally characterized novel hepatoma-specific promoters, which were valuable for cancer-specific gene therapy, using HSV-1 vectors.

Study on the characteristics and hepatotropism of the negative regulatory element of the HBVnt453-250

Objective To analyze the characteristics and hepatotropism of this negative element HBVnt453-250 sequence.Methods pHBv453-250,pHBV250-453.plucHBV453-250 and plucHBV250-453 were constructed,with luciferase and enhanced green fluorecence protein(EGFP)gene as the reporter gene,respectively.After transfection of HepG2 cells with these plasmids,luciferase assays,real-time PCR and western blot assays were used to detect the gene transcription and expression level.The SV40 promoter of pGL3 control and pHBV453-250 were replaced by the cytomegalovirus early promoter,resulting in plasmids pCMVcontrol(luc)and pCMV453-250(luc).Results Compared with pHBV453-250,the mutant plasmids.with the inhibitory element inserted in difierent site or inverted orientation.exerted similar downregulation of Juciferase gene transcription and expression.Western blot analysis demonstrated the similar repression when EGFP was used as the reporter gene.By transfeeted to HepG2 cell line,the plasmid pCMV453-250(1UC)could reduce lneiferase activity(36.56%)compared with pCMLcontrol(luc).When the plasmids plueHBV453-250 and plucHBV250-453 were transfected to non-liver cell lines(A549,HeLa),luciferase gene was expressed weakly,compared with that of pGL3control(P＜0.05).Conclusion The inhibitory effect of HBVnt453-250 sequence acted in both orientation-and position-independent manners,and had no promoter selectivity and funotioned in hepatocyte-independent manner. Key words: Hepatitis B virus; Plus strand; Negative regulatory element; Hepatotropism

The Hepatitis C Virus RNA 3′-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.

Interference of hepatitis B virus gene expression and replication by siRNA

Introduction: Hepatitis B virus (HBV) is a public health problem in the world. HBV-infected carriers, over 300 million people worldwide, are at high risk of developing end-stage liver diseases such as cirrhosis and have also dramatically increased the risk of liver cancer. Current treatments for HBV infections are not efficient. The enormous disease burden caused by the HBV infections makes the development of novel therapies critical. Molecular therapies for HBV can be directed at reducing viral load by interfering with the life cycle of the viruses. Objective: To explore the potential use of RNA interference (siRNA) as a therapeutic approach to inhibit HBV viral gene expression and replication. Methods: We first chose the target sequences for siRNA along the coding region of S antigen of HBV adw strain (HBsAg). A selected target sequence contained AA leader followed 19-nt. Double-stranded RNA oligos containing the 19-nt with added TT at 3′ ends were synthesized. DNA constructs containing the siRNA were also made in a vector called pSUPERretro. Effects of these siRNA or DNA construct on the expression of HBsAg were examined by anti-HBsAg Western blot on cell lysates of a variety of cell lines. Results: We have designed a series of small interfering RNA (siRNA) targeted to various regions of HBV genome and first tested the efficacy of these siRNAs in suppression of HBV gene expression. Three of four siRNAs directed to the coding region of HBsAg (siHBsAg) were very effective in knocking down the expression of co-transfected HBsAg in a variety of cell lines, including hepatocellular carcinoma HepG2 and Hep3B cells as well as non-liver cell lines such as HeLa and 293T cells. siHBsAg-mediated gene suppression is in a dose-dependent manner with 1 nM of siHBsAg1 being sufficient to inhibit HBsAg accumulation in cells. We are currently testing the ability of these siRNAs in inhibiting HBV viral replication in cell lines carrying whole HBV genome and animal models. Conclusion: siRNA-directed gene silencing is effective in inhibiting HBV gene expression, suggesting that it can be potentially used as a successful therapeutic approach to the prevention and treatment of HBV in the foreseeable future.

Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver.

Protein S is a vitamin-K-dependent plasma glycoprotein that serves as a cofactor for activated protein C in the protein C anticoagulation pathway, which regulates blood coagulation by inactivating factors Va and VIIIa. Mechanisms regulating the expression of the protein S gene remain unknown to date. The aim of this study was to characterize the cis-acting DNA elements of the human protein S gene in liver. We found that liver cell lines (HepG2 and PLC) transcribed the human protein S gene to mRNA, whereas non-liver cell lines (HEK293 and HeLa cells) either transcribed the gene weakly or not at all. Isolation and analysis of tissue-specific gene expression in HepG2 and HeLa cells of the 5'-flanking region from -6183 to +294 of the protein S gene indicated that the consensus binding motifs to HNF3 and Sp1 or MAZ transcription factors in the flanking region are essential for protein S gene expression. Exogenous expression of the Sp1 gene augmented the protein S-reporter gene expression in HepG2 or PLC cells but had no effect in HeLa cells. Taken together, we would conclude that transcription factors of HNF3, MAZ, and Sp1 are required for high-level expression of the protein S gene in hepatic cells, but in non-hepatic cells such as HeLa cells, an unknown factor(s) binds to the Sp1 region and disturbs the action of Sp1 and MAZ.

Involvement of hepatocyte nuclear factor-4 in the expression of the growth hormone receptor 1A messenger ribonucleic acid in bovine liver.

The GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5'-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5'-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5'-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5'-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5'-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5'-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.

An Upstream Repressor Element That Contributes to Hepatocyte-Specific Expression of the Rat Serum Amyloid A1 Gene

Serum amyloid A (SAA) is a major acute-phase protein whose expression can be dramatically induced in response to tissue damage, infection, and inflammation. Its expression is highly tissue-specific, restricted almost exclusively to liver hepatocytes. We have shown that a 320-bp fragment of the rat SAA1 promoter could confer liver-cell-specific expression on a reporter gene when transfected into cultured cells. Here we report the identification of a 29-bp regulatory element that possesses inhibitory activities on SAA1 promoter in HeLa cells but has no such effects in liver cells. Moreover, this regulatory element has properties of a transcriptional repressor; in that, it can function with a heterologous promoter and is independent of orientation and distance from the transcription initiation site. Protein binding studies showed that this regulatory element can form specific protein–DNA complexes with nuclear proteins from several nonliver cell lines (HeLa, 10T1/2, and C2) and placenta. However, the same DNA–protein complex was not detected in extracts from liver or liver-derived cell lines (HepG2 and Hep3B). Taken together, our results demonstrate the presence of a DNA-binding protein, termed tissue-specific repressor, found only in nonhepatocytes which may function to repress SAA1 gene expression by interacting with a repressor element. Thus, liver-specific expression of the SAA1 gene is apparently regulated by both positive and negative regulatory elements and their interacting transcription factors to ensure that it is expressed only in suitable cell types.

Promoters for synthesis of the pre-C and pregenomic mRNAs of human hepatitis B virus are genetically distinct and differentially regulated.

Two similar, yet functionally distinct genomic RNAs are transcribed from the DNA genome of the human hepatitis B virus. The pre-C RNAs encode the precore protein which is proteolytically processed to yield e antigen. The pregenomic RNAs encode both the nucleocapsid protein and reverse transcriptase and serve as the templates for viral DNA replication. To determine whether synthesis of these two RNAs is directed from a single or a closely spaced pair of promoters, we introduced point and insertion mutations into the basal elements of the promoter that directs their synthesis. Transcription from these mutants was examined both in cell-free transcription systems derived from hepatoma (HepG2) and nonliver (HeLa) cell lines and by transient transfection of hepatoma cell lines (Huh7 and HepG2). The data from these experiments indicated that synthesis of the pre-C and pregenomic RNAs is directed by two distinct promoters and that the basal elements of these two promoters partially overlap, yet are genetically separable, with each consisting of its own transcriptional initiator and a TATA box-like sequence situated approximately 25 to 30 bp upstream of its sites of initiation. A 15-bp insertion was found to be sufficient to physically separate these two promoters. Furthermore, these two promoters can be differentially regulated, with the transcriptional activator Sp1 specifically activating transcription from the pregenomic promoter and the hepatocyte nuclear factor 4 specifically repressing transcription from the pre-C promoter. Thus, we conclude that the promoters used in synthesis of the pre-C and pregenomic mRNAs are genetically distinct and separately regulated.

Orphan Nuclear Receptor HNF-4 Binds to the Human Coagulation Factor VII Promoter

The human coagulation protease factor VII plays a pivotal role in the initiation of the coagulation cascade by both the extrinsic and the intrinsic pathway. Although the gene, encoding factor VII, is expressed predominantly in the liver, the mechanisms underlying this tissue-specific expression have not been elucidated. In this study, we have analyzed the contribution of 5 kilobases upstream of the ATG translational initiation codon upon hepatic factor VII gene transcription. Transient transfection assays of a set of nested deletions in both liver and non-liver cell lines, HepG2 and HeLa respectively, indicate that several regions are involved in liver-specific expression. A slight negative effect on factor VII promoter activity in HepG2 cells is mediated by sequences upstream of position -1212. DNase I protection experiments reveal six footprints, FPVII1 through FPVII6, within the proximal 714 base pairs but a minimal promoter of 165 base pairs containing only FPVII3-6 is sufficient to confer liver-specific expression in HepG2 cells. Interestingly, FPVII6, at position -14 to +10 on the sense strand, would indicate that an as yet unknown transcription factor covers the ATG translational initiation codon. Gel retardation experiments show that the liver-enriched transcription factor HNF-4 binds specifically to footprint FPVII4 at position -71 to -49. Furthermore, a T --> A transversion, that in the HNF-4 binding site of factor IX causes a severe bleeding disorder, was introduced into the HNF-4-binding site of factor VII and reduced promoter activity by 20-50%. Coordinate HNF-4-mediated regulation of several blood protease genes as well as genes involved in lipid metabolism might account for the positive correlation of these factors with increased risk of occlusive heart diseases.

Orphan receptor HNF-4 and bZip protein C/EBP alpha bind to overlapping regions of the apolipoprotein B gene promoter and synergistically activate transcription

As the sole protein component of low density lipoproteins, apolipoprotein B (apoB) plays an important role in cholesterol metabolism. Previously, we found that the proximal promoter region of apoB (-81 to -52 relative to the start site) played a critical role in hepatocyte-specific gene expression and that that region contained overlapping binding sites for nuclear factors AF-1 (-81 to -62) and C/EBP (-69 to -52) (Metzger, S., Leff, T., and Breslow, J. L. (1990) J. Biol. Chem. 265, 9978-9983). In this study, we show that HNF-4, a member of the steroid hormone receptor superfamily, binds the AF-1 site on the apoB promoter and through it activates transcription in transient transfection assays in both liver and non-liver cell lines, HepG2 and HeLa, respectively. Mutational analysis of the AF-1/HNF-4 binding site indicated a correlation of HNF-4 binding and transcriptional activity. In addition, transient co-transfection experiments with HNF-4 and C/EBP alpha expression vectors showed that the two factors can synergistically activate transcription to levels more than 3-fold above the sum of either factor alone. Finally, using gel retardation analysis we show that purified HNF-4 and C/EBP proteins can concurrently occupy their overlapping binding sites on the apoB promoter in vitro. However, since the same system showed a lack of cooperative binding, we argue that an alternative mechanism is responsible for the synergistic effect of HNF-4 and C/EBP alpha on apoB gene transcription.

Regulation of hepatitis B virus gene expression

Human hepatitis B virus (HBV) mainly infects hepatocytes. HB viral gene expression has been demonstrated to be liver-specific using DNA transfection methods. This liver-specific gene expression is regulated by promoter/enhancer elements. HBV contains two enhancer elements. Enhancer element I has been studied in detail at the DNA-protein level. This is further substantiated by DNA transfections of liver and non-liver cell lines with expression plasmids containing enhancer elements controlling the transcription of reporter genes. Genetic analysis of the enhancer elements defined the minimal sequences which play a key role in the regulation of enhancer function. One of the factors binding in this region is RXR alpha. Using only the DNA binding domain of the liver-specific RXR alpha expressed in E. coli, we demonstrated binding of RXR alpha to the putative retinoic acid receptor response element (RARE) in the HBV enhancer. Our studies implicate a potentially important role of retinoic acid and its receptor in the liver-specific regulation of HBV gene expression and the disease pathogenesis associated with infection.

Regulatory sequences of duck hepatitis B virus C gene transcription

The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements.