Hepatitis B virus (HBV) replicates by the reverse transcription of the viral 3.5kb pregenomic RNA. Therefore the level of expression of this transcript in the liver is a primary determinant of HBV biosynthesis. In vivo neonatal transcription of the HBV 3.5kb pregenomic RNA is developmental regulated by hepatocyte nuclear factor 4α (HNF4α). In addition, viral biosynthesis in non-hepatoma cells can be supported directly by this nuclear receptor. However HBV transcription and replication can be supported by additional nuclear receptors including the retinoid X receptor α/peroxisome proliferator-activated receptor α (RXRα/PPARα), retinoid X receptor α/farnesoid X receptor α (RXRα/FXRα), liver receptor homolog 1 (LRH1) and estrogen-related receptors (ERR) in non-hepatoma cells. Therefore during neonatal liver development, HNF4α may progressively activate viral transcription and replication by binding directly to the proximal HNF4α recognition sequence within the nucleocapsid promoter. Alternatively, HNF4α may support viral biosynthesis in vivo indirectly by activating a network of liver-enriched nuclear receptors that, in combination, direct HBV 3.5kb pregenomic RNA transcription and replication.

In natural infection, hepatitis B virus (HBV) transcription and replication is essentially restricted to the hepatocytes in the livers of humans and a limited number of primates (15,19,33,36,41). HBV tropism is probably restricted at the level of entry by the viral receptor, which likely has a limited tissue distribution (10, 33). In addition, transcription of the viral genome limits HBV biosynthesis to cells expressing the nuclear receptors required for viral pregenomic RNA synthesis and replication (13,40). The nuclear receptors present in hepatocytes that regulate HBV transcription include both liganddependent and orphan nuclear receptors which lack known ligands (16,22,30,40). Long-chain fatty acids are ligands for peroxisome proliferator-activated receptor ␣ (PPAR␣), which links HBV biosynthesis to energy homeostasis (9). Bile acids are ligands for farnesoid X receptor ␣ (FXR␣), further linking HBV biosynthesis to lipid metabolism (29, 30). Hepatocyte nuclear factor 4␣ (HNF4␣) and estrogen-related receptor (ERR) are orphan nuclear receptors, which like PPAR␣ and FXR␣ can display alteration in transcriptional activities in response to the coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC1␣) and the corepressor small heterodimer partner (SHP) (1, 23). PGC1␣ is critical for the activation of liver gluconeogenesis and therefore couples HBV transcription and replication to liver carbohydrate metabolism and whole-body energy homeostasis (43). SHP expression is activated by bile acids via FXR␣ and tumor necrosis factor ␣ through AP1, leading to the inhibition of the activities of multiple nuclear receptors (6,11,14,24). Therefore, SHP may regulate HBV biosynthesis in response to changing lipid metabolism or inflammatory signals within the liver (28).Several nuclear receptors expressed in the liver have been shown to support HBV biosynthesis in nonhepatoma cell lines (see Fig. 1 to 6) (27a, 40). However, it is unclear which of these nuclear receptors are critical to supporting viral transcription and replication in hepatocytes in vivo. Conditional deletion of HNF4␣ in the liver of neonatal HBV transgenic mice demonstrated that this nuclear receptor was essential for viral biosynthesis (21). However, the early developmental loss of HNF4␣ is associated with decreased expression of a variety of additional nuclear receptors capable of supporting viral biosynthesis. Therefore, it is unclear if the loss in HBV transcription and replication observed in the liver-specific HNF4␣-null HBV transgenic mouse is due directly to the loss of HNF4␣ or to the indirect effects on other nuclear receptors (18). Similarly, it is apparent that hepatoma cells can support HBV biosynthesis, but it has not been established which transcription factors present in these cells, but not in nonhepatoma cells, are responsible for supporting viral pregenomic RNA synthesis (4,39,40).Given the importance of nuclear receptors and their associated coactivators and corepressors to liver energy homeostasis,

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (AT(1A)) receptor. The cytokines tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) were shown previously to upregulate AT(1A) receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-kappaB (NF-kappaB)-dependent transcription and also, to a lesser extent, altered mRNA splicing in the mechanism of receptor upregulation. Cytokine stimulation was found to increase AT(1A) heterogeneous nuclear RNA levels, which strongly suggests that mRNA upregulation occurs transcriptionally. The transcription factor NF-kappaB was previously deemed necessary for cytokine-induced AT(1A) receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-kappaB elements at -365 and -2540 bp. Both isolated elements were shown to bind NF-kappaB (using gel-shift assays) and to transactivate a minimal promoter (using reporter assays), although the element at -365 bp appeared stronger. Three splice variants of AT(1A) receptor mRNA that have different 5' untranslated regions were detected in rat tissues, namely, exons 1-2-3 (predominant), 1-2-3+6, and 1-3. Cytokine treatment of fibroblasts upregulated all splice variants, but exon 1-3 increased more than the others. This differential upregulation, albeit of modest magnitude, was statistically significant with IL-1beta treatment. Exon 2 contains an inhibitory minicistron and a predicted inhibitory hairpin structure. Luciferase reporter assays indicated that each splice variant translates at a different efficiency, with exon 1-2-3+6 (both minicistron and hairpin) < exon 1-2-3 (minicistron only) < exon 1-3 (neither minicistron or hairpin). These results provide evidence that cytokines increase AT(1) protein levels by altering both transcription and splicing.

Hepatitis B virus (HBV) replicates efficiently in hepatocytes in vivo and in hepatoma cells in culture because these cells have the appropriate composition of transcription factors to support the expression of the 3.5-kb viral pregenomic transcript, which is reverse transcribed to generate the relaxed circular genomic DNA present in infectious viral particles (4,11,26,27,29). In contrast, HBV transcription and replication does not occur in the majority of nonhepatic tissues in vivo or nonhepatoma cell lines in culture due to the absence of transcription of the 3.5-kb viral pregenomic RNA (11,27). However, complementation of nonhepatoma cells with several nuclear receptors but not other liver-enriched transcription factors leads to robust HBV transcription and replication (see Fig. 1 to 3) (18a, 27). These observations have led to the suggestion that transcriptional regulation of HBV biosynthesis is an important determinant of viral tropism, which may be just as significant as the presumptive cell type-specific expression of the viral receptor (22, 27).The molecular characterization of the various steps in the viral life cycle has been largely elucidated, utilizing human hepatoma cell lines (10, 24). In particular, the human hepatoma cell lines HepG2 and Huh7 have been utilized because they can support viral replication when transfected with genomic HBV DNA (4, 26). In addition, these two cell lines lack integrated HBV DNA which is commonly associated with other human hepatoma cell lines (4, 26, 28). As products from integrated HBV sequences might have unidentified effects on viral transcription and replication, the majority of studies aimed at understanding the various aspects of HBV transcription and replication have utilized HepG2 and Huh7 cells. The majority of observations using these two cell lines have been similar with regard to HBV biosynthesis, leading to the general assumption that they are essentially equivalent with respect to HBV biosynthesis (4,24,26). However, it is clear that these hepatoma cell lines are morphologically distinct and, consequently, probably display distinct patterns of gene regulation.Recent studies have indicated that 3.5-kb pregenomic HBV RNA expression and viral replication are regulated by a variety of nuclear receptors (see Fig. 1 to 3) (18a, 27). The observation that several nuclear receptors can potentially contribute to the level of viral biosynthesis raised the critical question of their relative importance both in vivo and in cell culture. In an attempt to address this issue, the effect of expressing the coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC1␣) and the corepressor small heterodimer partner (SHP) on HBV transcription and replication in both human hepatoma and nonhepatoma cells was quantitatively evaluated with a view to establishing the potential role of distinct nuclear receptors in regulating viral biosynthesis in the HepG2 and Huh7 cells. This analysis demonstrated that each nuclear receptor displayed a unique pattern of responsi...

In the human hepatoma cell line HepG2, retinoic acid, clofibric acid, and bile acid treatment can only modestly increase hepatitis B virus (HBV) biosynthesis. Utilizing the human embryonic kidney cell line 293T, it was possible to demonstrate that the retinoid X receptor ␣ (RXR␣) plus its ligand can support viral biosynthesis independently of additional nuclear receptors. In addition, RXR␣/peroxisome proliferator-activated receptor ␣ (PPAR␣) and RXR␣/farnesoid X receptor ␣ (FXR␣) heterodimeric nuclear receptors can also mediate ligand-dependent HBV transcription and replication when activated by clofibric acid and bile acid, respectively, independently of a requirement for the ligand-dependent activation of RXR␣. These observations indicate that there are at least three possible modes of ligand-mediated activation of HBV transcription and replication existing within hepatocytes, suggesting that multiple independent mechanisms control viral production in the livers of infected individuals. Hepatitis B virus (HBV) infection is primarily restricted to hepatocytes in the liver. This restriction is believed to occur at two distinct levels (1). The receptor(s) involved in viral entry is presumably present only on hepatocytes and governs species specificity (2). In addition, viral biosynthesis is restricted in a tissueand cell-type-specific manner because HBV transcription is dependent on liver-enriched transcription factors (3, 4). A variety of nuclear receptors have been shown to regulate HBV pregenomic 3.5-kb RNA synthesis and hence viral replication (5-7). Three of these nuclear receptors, retinoid X receptor (RXR), peroxisome proliferator-activated receptor (PPAR), and farnesoid X receptor (FXR), are ligand-dependent transcription factors that are activated by retinoids, peroxisome proliferators, and bile acids, respectively (8, 9). Therefore, it is apparent that the ligands for these nuclear receptors might be critical determinants of viral biosynthesis under both normal and pathophysiological conditions within the livers of infected individuals (10, 11).As the ligand-activated heterodimeric nuclear receptors RXR␣/PPAR␣ and RXR␣/FXR␣ regulate HBV pregenomic RNA synthesis by the recruitment of coactivators, it was of interest to evaluate the relative contributions of the individual heterodimeric partners to the overall level of viral transcription and replication (5, 6, 12). Characterization of the relative roles of individual polypeptides in the transcriptional activity of various heterodimeric nuclear receptors has been evaluated (13-15). This approach indicated that one partner might play a dominant role in controlling promoter activity depending on the nuclear receptors involved (13-15). Therefore, it was of interest to evaluate the effects of retinoids, peroxisome proliferators, and bile acids, alone or in combination, on HBV transcription and replication.In the current study, it is demonstrated that retinoids can activate HBV biosynthesis utilizing both RXR␣-containing homodimers and heterodimers. Alternativ...