The numerous functions of the liver are controlled primarily at the transcriptional level by the concerted actions of a limited number of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 1␣ [HNF1␣], -1␤, -3␣, -3␤, -3␥, -4␣, and -6 and members of the c/ebp family). Of these, only HNF4␣ (nuclear receptor 2A1) and HNF1␣ appear to be correlated with the differentiated phenotype of cultured hepatoma cells. HNF1␣-null mice are viable, indicating that this factor is not an absolute requirement for the formation of an active hepatic parenchyma. In contrast, HNF4␣-null mice die during embryogenesis. Moreover, recent in vitro experiments using tetraploid aggregation suggest that HNF4␣ is indispensable for hepatocyte differentiation. However, the function of HNF4␣ in the maintenance of hepatocyte differentiation and function is less well understood. To address the function of HNF4␣ in the mature hepatocyte, a conditional gene knockout was produced using the Cre-loxP system. Mice lacking hepatic HNF4␣ expression accumulated lipid in the liver and exhibited greatly reduced serum cholesterol and triglyceride levels and increased serum bile acid concentrations. The observed phenotypes may be explained by (i) a selective disruption of very-low-density lipoprotein secretion due to decreased expression of genes encoding apolipoprotein B and microsomal triglyceride transfer protein, (ii) an increase in hepatic cholesterol uptake due to increased expression of the major high-density lipoprotein receptor, scavenger receptor BI, and (iii) a decrease in bile acid uptake to the liver due to down-regulation of the major basolateral bile acid transporters sodium taurocholate cotransporter protein and organic anion transporter protein 1. These data indicate that HNF4␣ is central to the maintenance of hepatocyte differentiation and is a major in vivo regulator of genes involved in the control of lipid homeostasis.The adult liver executes numerous functions that are essential for metabolic homeostasis including plasma protein synthesis; carbohydrate, lipid, and amino acid metabolism; and xenobiotic metabolism. The majority of these functions are performed by hepatocytes. Hepatocyte-enriched transcription factors control transcription of genes that are preferentially expressed in liver (5). Our understanding of the role of liverenriched transcription factors in gene expression has largely been developed using transfection assays in cultured cells. Cisacting elements for transcription factor binding have been characterized using this approach in conjunction with in vitro DNA binding assays. However, many genes have regulatory elements for several transcription factors, and it becomes difficult to assess which factor predominates in vivo. Moreover, it is becoming increasingly clear that the in vitro regulation of a gene is not always reflected by the in vivo situation. For example, numerous hepatic genes are transactivated by hepatocyte nuclear factor 3␤ (HNF3␤) in vitro but deletion of HNF3␤ in the mature hepatocyte using ...

The 86 kDa immediate early IE2 protein of human cytomegalovirus (HCMV) can activate transcription of both viral and cellular genes and can repress transcription from its own promoter. Using two in vivo assays, we provide evidence of a functional interaction between IE2 and the retinoblastoma (RB) protein: IE2 alleviates RB‐induced repression of a promoter bearing E2F binding sites and RB alleviates IE2‐mediated repression of its own promoter. These functional effects are likely to be a result of a direct contact between IE2 and RB, which we can demonstrate both in vitro and in HCMV‐infected cells. The interaction between IE2 and RB shows similar characteristics to the interaction between RB and E1A. First, binding to IE2 requires an intact RB pocket domain. Secondly, the binding is sensitive to the phosphorylation state of RB, because cyclin A‐CDK‐induced phosphorylation of RB diminishes IE2 binding. Thirdly, the IE2 domain required for RB binding is separate to the domains necessary for TBP and TFIIB binding. Our results demonstrate that large and small DNA viruses have a common interface with the host cell, namely the association with the RB tumour suppressor protein.

CYP2D6 is a highly polymorphic human gene responsible for a large variability in the disposition of more than 100 drugs to which humans may be exposed. Animal models are inadequate for preclinical pharmacological evaluation of CYP2D6 substrates because of marked species differences in CYP2D isoforms. To overcome this issue, a transgenic mouse line expressing the human CYP2D6 gene was generated. The complete wild-type CYP2D6 gene, including its regulatory sequence, was microinjected into a fertilized FVB/N mouse egg, and the resultant offspring were genotyped by both polymerase chain reaction and Southern blotting. CYP2D6-specific protein expression was detected in the liver, intestine, and kidney from only the CYP2D6 humanized mice. Pharmacokinetic analysis revealed that debrisoquine (DEB) clearance was markedly higher (94.1 +/- 22.3 l/h/kg), and its half-life significantly reduced (6.9 +/- 1.6 h), in CYP2D6 humanized mice compared with wild-type animals (15.2 +/- 0.9 l/h/kg and 16.5 +/- 4.5 h, respectively). Mutations in hepatic nuclear factor 4alpha (HNF4alpha), a hepatic transcription factor known to regulate in vitro expression of the CYP2D6 gene, could affect the disposition of CYP2D6 drug substrates. To determine whether the HNF4alpha gene modulates in vivo pharmacokinetics of CYP2D6 substrates, a mouse line carrying both the CYP2D6 gene and the HNF4alpha conditional mutation was generated and phenotyped using DEB. After deletion of HNF4alpha, DEB 4-hydroxylase activity in CYP2D6 humanized mice decreased more than 50%. The data presented in this study show that only CYP2D6 humanized mice but not wild-type mice display significant DEB 4-hydroxylase activity and that HNF4alpha regulates CYP2D6 activity in vivo. The CYP2D6 humanized mice represent an attractive model for future preclinical studies on the pharmacology, toxicology, and physiology of CYP2D6-mediated metabolism.

Liver carnitine palmitoyltransferase I catalyzes the transfer of long-chain fatty acids into mitochondria. L-CPT I is considered the rate-controlling enzyme in fatty acid oxidation. Expression of the L-CPT I gene is induced by starvation in response to glucagon secretion from the pancreas, an effect mediated by cAMP. Here, the molecular mechanisms underlying the induction of L-CPT I gene expression by cAMP were characterized. We demonstrate that the cAMP response unit of the L-CPT I gene is composed of a cAMP-response element motif and a DR1 sequence located 3 kb upstream of the transcription start site. Our data strongly suggest that the coactivator PGC-1 is involved in the regulation of this gene expression by cAMP in combination with HNF4␣ and cAMP-response element-binding protein (CREB). Indeed, (i) cotransfection of CREB or HNF4␣ dominant negative mutants completely abolishes the effect of cAMP on the L-CPT I promoter, and (ii) the cAMPresponsive unit binds HNF4␣ and CREB through the DR1 and the cAMP-response element sequences, respectively. Moreover, cotransfection of PGC-1 strongly activates the L-CPT I promoter through HNF4␣ bound at the DR1 element. Finally, we show that the transcriptional induction of the PGC-1 gene by glucagon through cAMP in hepatocytes precedes that of L-CPT-1. In addition to the key role that PGC-1 plays in glucose homeostasis, it may also be critical for lipid homeostasis. Taken together these observations suggest that PGC-1 acts to coordinate the process of metabolic adaptation in the liver.

Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.

Hepatocyte nuclear factor 4␣ (HNF4␣) regulates the expression of many genes preferentially expressed in liver. HNF4␣-null mice die during embryogenesis precluding the analysis of its function in the adult. To circumvent this problem, liver-specific HNF4␣-null mice were produced. Mice lacking hepatic HNF4␣ expression exhibited increased serum ammonia and reduced serum urea. This disruption in ureagenesis may be explained by a marked decrease in expression and activity of hepatic ornithine transcarbamylase (OTC). To determine the molecular mechanisms involved in transcriptional regulation of the mouse OTC gene, the OTC promoter region was analyzed. Sequence analysis revealed the presence of two putative HNF4␣-binding sites in the mouse OTC promoter region. By using transient transfection analysis, it was established that high levels of promoter activity were dependent on both HNF4␣-binding sites and the expression of HNF4␣. Furthermore, the proximal HNF4␣-binding site was found to be more important than the distal one for transactivating OTC promoter. These data demonstrate that HNF4␣ is critical for urea homeostasis by direct regulation of the OTC gene in vivo.The liver plays an important role in the metabolism of carbohydrates, proteins, lipids, vitamins, and hormones, the synthesis and excretion of bile acids, the production of blood coagulation factors and plasma proteins, and the detoxification of various xenobiotics. Most of these functions are achieved by hepatocytes that account for over 60% of the liver mass. The expression of genes preferentially expressed in liver is controlled by liver-enriched transcription factors (LETF)

Polymorphonuclear leukocytes (PMNL) have been shown to harbour human cytomegalovirus (HCMV) in viraemic patients, but to date PMNL of asymptomatic healthy subjects have not been examined directly to determine whether this is a normal site of HCMV persistence. Using the polymerase chain reaction (PCR), paired DNA samples prepared from adherent peripheral blood mononuclear cells (PBMC), which are known to be a site of persistence of HCMV, and PMNL of 10 healthy adults were analysed. All of seven individuals who were HCMV seropositive, and one of three who were seronegative gave a reproducible signal for HCMV DNA in their adherent PBMC, whereas none of the paired PMNL DNA samples gave a positive result. The remaining two seronegative subjects showed no HCMV DNA in either the PBMC or PMNL samples. In every case where PCR for HCMV was negative, PCR amplification of a control human gene was used to show there was no inability to amplify the DNA. We conclude that within the leukocyte population of normal asymptomatic HCMV carders, PMNL do not appear to harbour persistent HCMV whereas adherent PBMC in the same subjects are a site of persistence.We and others have previously used the polymerase chain reaction (PCR) to demonstrate the presence of human cytomegalovirus (HCMV) in leukocytes of seropositive and some seronegative healthy carriers (Taylor-Wiedeman et al., 1991; Stainer et al., 1989). In these normal subjects, peripheral blood monocytes (PBMC) were the principal site of persistence (TaylorWiedeman et al., 1991). However, only PBMC were studied and analysis of polymorphonuclear leukocytes (PMNL) from healthy carriers was not addressed.Detection of HCMV in the PMNL fraction of peripheral blood has been reported in patients infected with human immunodeficiency virus (HIV), other immunocompromised patients and immunocompetent patients acutely ill with HCMV (Saltzman et al., 1988;Dankner et al., 1990;Fiala et al., 1975;Rinaldo et al., 1977; Revello et al., 1992;Gerna et al., 1991). Consequently, we were interested in determining whether the granulocyte might also represent a normal site of persistence which would be consistent with the clinical observations that HCMV is present in PMNL of viraemic patients. To determine this, DNA from PMNL cells and adherent PBMC from the same blood sample for each of 10 healthy subjects was prepared and examined by PCR. In this report we show that PMNL are not a source of persistent virus in the leukocyte population of normal asymptomatic HCMV carriers.Subjects were all healthy adult volunteers whose serological status was determined using a competitive ELISA (CompEnz-CMV, Northumbria Biologicals). One subject's serum gave a negative result in ELISA but DNA from adherent PBMC was PCR-positive. A second serum sample, examined 2 months later using an HCMV latex agglutination assay (Becton Dickinson), showed the individual to have remained seronegative. Lymphoprep (Nycomed) gradient separations yielded PBMC, banding at the plasma interface, and PMNLenriched fractions lying at the top ...

During liver development, hepatocytes undergo a maturation process that leads to the fully differentiated state. This relies at least in part on the coordinated action of liver-enriched transcription factors (LETFs), but little is known about the dynamics of this coordination. In this context we investigate here the role of the LETF hepatocyte nuclear factor 6 (HNF-6; also called Onecut-1) during hepatocyte differentiation. We show that HNF-6 knockout mouse fetuses have delayed expression of glucose-6-phosphatase (g6pc), which catalyzes the final step of gluconeogenesis and is a late marker of hepatocyte maturation. Using a combination of in vivo and in vitro gain-and loss-of-function approaches, we demonstrate that HNF-6 stimulates endogenous g6pc gene expression directly via a synergistic and interdependent action with HNF-4 and that it involves coordinate recruitment of the coactivator PGC-1␣. The expression of HNF-6, HNF-4, and PGC-1␣ rises steadily during liver development and precedes that of g6pc. We provide evidence that threshold levels of HNF-6 are required to allow synergism between HNF-6, HNF-4, and PGC-1␣ to induce time-specific expression of g6pc. Our observations on the regulation of g6pc by HNF-6 provide a model whereby synergism, interdependency, and threshold concentrations of LETFs and coactivators determine time-specific expression of genes during liver development.