Liver Transcription Research Articles

Characterization of three growth hormone-responsive transcription factors preferentially expressed in adult female liver.

Plasma GH profiles regulate the sexually dimorphic expression of cytochromes P450 and many other genes in rat and mouse liver; however, the proximal transcriptional regulators of these genes are unknown. Presently, we characterize three liver transcription factors that are expressed in adult female rat and mouse liver at levels up to 16-fold [thymus high-mobility group box protein (Tox)], 73-fold [tripartite motif-containing 24 (Trim24)/transcription initiation factor-1alpha (TIF1alpha)], and 125-fold [cut-like 2 (Cutl2)/cut homeobox 2 (Cux2)] higher than in adult males, depending on the strain and species, with Tox expression only detected in mice. In rats, these sex differences first emerged at puberty, when the high prepubertal expression of Cutl2 and Trim24 was extinguished in males but was further increased in females. Rat hepatic expression of Cutl2 and Trim24 was abolished by hypophysectomy and, in the case of Cutl2, was restored to near-female levels by continuous GH replacement. Cutl2 and Trim24 were increased to female-like levels in livers of intact male rats and mice treated with GH continuously (female GH pattern), whereas Tox expression reached only about 40% of adult female levels. Expression of all three genes was also elevated to normal female levels or higher in male mice whose plasma GH profile was feminized secondary to somatostatin gene disruption. Cutl2 and Trim24 both responded to GH infusion in mice within 10-24 h and Tox within 4 d, as compared with at least 4-7 d required for the induced expression of several continuous GH-regulated cytochromes P450 and other female-specific hepatic genes. Cutl2, Trim24, and Tox were substantially up-regulated in livers of male mice deficient in either of two transcription factors implicated in GH regulation of liver sex specificity, namely, signal transducer and activator of transcription 5b (STAT5b) and hepatocyte nuclear factor 4alpha (HNF4alpha), with sex-specific expression being substantially reduced or lost in mice deficient in either nuclear factor. Cutl2 and Trim24 both display transcriptional repressor activity and could thus contribute to the loss of GH-regulated, male-specific liver gene expression seen in male mice deficient in STAT5b or HNF4alpha. Binding sites for Cutl1, whose DNA-binding specificity is close to that of Cutl2, were statistically overrepresented in STAT5b-dependent male-specific mouse genes, lending support to this hypothesis.

Circadian Rhythms: Per2bations in the Liver Clock

A master circadian clock resides in the brain and is required to synchronize the clocks in peripheral tissues such as the liver. Until now, it has been unclear how the central clock synchronizes the peripheral ones. New work points to one of the core clock genes, mPer2, as an essential link in this chain.

Hepatic ENPP1 expression is induced in diabetic rabbits

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an inhibitor of the insulin receptor. Variants of ENPP1 are associated with infantile arterial calcification, obesity, and insulin resistance. To study the functional relevance of this protein in vivo, we cloned rabbit ENPP1 and studied its regulation in experimentally induced diabetes mellitus. We amplified and sequenced the complete coding sequence of rabbit ENPP1 gene out of a liver cDNA library using redundant primers deduced from other species. Next, we performed quantitative PCR of ENPP1 to study the tissue distribution of ENPP1 expression and its regulation in an alloxan-dependent diabetes model. The putative rabbit ENPP1 protein contains 873 amino acids and is highly conserved when compared with human ENPP1 (90% amino acid identity). Particularly high levels of ENPP1 mRNA expression were found in adipose tissue. Quantitative PCR analysis revealed a significant upregulation of ENPP1 transcription in liver (p = 0.025) and brain (p = 0.034) of diabetic rabbits compared with controls. Hepatic ENPP1 expression is induced in diabetic rabbits when compared with controls. This approximately twofold upregulation of ENPP1 mRNA in rabbit liver parallels previous findings in patients with type 2 diabetes mellitus. We provide further molecular information about ENPP1 as a potential pharmacologic target and characterize its regulation in an insulin-dependent diabetes mellitus animal model.

Hepatocyte nuclear factor-4α plays pivotal roles in the regulation of mouse carboxylesterase 2 gene transcription in mouse liver

The mouse carboxylesterase 2 isozyme, mCES2, is thought to play important roles in lipid metabolism and is expressed in the liver, kidney, and small intestine at high levels. In this study, we examined the molecular mechanisms controlling this tissue-specific expression of mCES2, and demonstrated that hepatocyte nuclear factor-4α (HNF-4α) could enhance transcription of the mCES2 gene in vitro and in vivo. It was found that effects of HNF-4α on the level of mCES2 promoter activity were repressed by small heterodimer partner (SHP) and chenodeoxycholic acid (CDCA) in luciferase assays. Accordingly, mCES2 gene transcription was repressed by CDCA treatment in mouse immortalized hepatocytes. Our results suggested that this repression resulted from the combined effects of both inhibition of HNF-4α transactivation ability by SHP and reduction of HNF-4α expression level. These findings show that HNF-4α plays an important role in the regulation of mCES2 gene transcription.

Core transcriptional regulatory circuitry in human hepatocytes

We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator–gene interactions. Autoregulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.

Pooling samples within microarray studies: a comparative analysis of rat liver transcription response to prototypical toxicants

Combining or pooling individual samples when carrying out transcript profiling using microarrays is a fairly common means to reduce both the cost and complexity of data analysis. However, pooling does not allow for statistical comparison of changes between samples and can result in a loss of information. Because a rigorous comparison of the identified expression changes from the two approaches has not been reported, we compared the results for hepatic transcript profiles from pooled vs. individual samples. Hepatic transcript profiles from a single-dose time-course rat study in response to the prototypical toxicants clofibrate, diethylhexylphthalate, and valproic acid were evaluated. Approximately 50% more transcript expression changes were observed in the individual (statistical) analysis compared with the pooled analysis. While the majority of these changes were less than twofold in magnitude ( approximately 80%), a substantial number were greater than twofold (approximately 20%). Transcript changes unique to the individual analysis were confirmed by quantitative RT-PCR, while all the changes unique to the pooled analysis did not confirm. The individual analysis identified more hits per biological pathway than the pooled approach. Many of the transcripts identified by the individual analysis were novel findings and may contribute to a better understanding of molecular mechanisms of these compounds. Furthermore, having individual animal data provided the opportunity to correlate changes in transcript expression to phenotypes (i.e., histology) observed in toxicology studies. The two approaches were similar when clustering methods were used despite the large difference in the absolute number of transcripts changed. In summary, pooling reduced resource requirements substantially, but the individual approach enabled statistical analysis that identified more gene expression changes to evaluate mechanisms of toxicity. An individual animal approach becomes more valuable when the overall expression response is subtle and/or when associating expression data to variable phenotypic responses.

Starvation and Feeding a High-Carbohydrate, Low-Fat Diet Regulate the Expression Sterol Regulatory Element-Binding Protein-1 in Chickens

In mammalian liver, the mature form of sterol regulatory element-binding protein-1c (SREBP-1c) is an important activator of a wide array of genes involved in triacylglycerol biosynthesis. Starvation and feeding a high-carbohydrate, low-fat diet modulate the concentration of mature SREBP-1c primarily by a pretranslational mechanism. It is not known whether alterations in nutritional status regulate the concentration of SREBPs in nonmammalian species. In this study, we found that in previously starved chicks, feeding a high-carbohydrate, low-fat diet stimulated a robust increase (14-fold at 5 h of feeding) in the concentration of mature SREBP-1 in liver. Feeding a high-carbohydrate, low-fat diet also increased the concentration of precursor SREBP-1 and SREBP-1 messenger RNA in chick liver; however, the magnitude of this effect was substantially lower than that observed for mature SREBP-1. DNA binding experiments demonstrated that 3 protein complexes containing SREBP bound the acetyl-CoA carboxylase-alpha (ACCalpha) sterol regulatory element (SRE) in chick liver and that the binding activity of 2 of these complexes was increased by consumption of a high-carbohydrate, low-fat diet. Additional analyses showed that feeding a high-carbohydrate, low-fat diet had no effect on the concentration of mature SREBP-2 and the binding of SREBP-2 to the ACCalpha SRE in chick liver. These results indicate that alterations in the concentration of mature SREBP-1 play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACCalpha transcription in chick liver and that diet-induced changes in mature SREBP-1 concentration in chick liver are mediated primarily by a posttranslational mechanism.

Sexually dimorphic P450 gene expression in liver-specific hepatocyte nuclear factor 4alpha-deficient mice.

Hepatocyte nuclear factor (HNF) 4alpha is a liver-enriched nuclear receptor that plays a critical role in regulating the expression of numerous hepatic genes, including members of the cytochrome P450 (CYP) superfamily, several of which are expressed in a sex-dependent manner. Presently, we use a liver-specific Hnf4alpha-deficient mouse model to investigate the role of HNF4alpha in regulating liver-enriched transcription factors and sexually dimorphic Cyps in liver in vivo. Real-time PCR analysis of RNA isolated from livers of wild-type and Hnf4alpha-deficient mice revealed the following: 1) HNF4alpha exerts both positive regulation (Hnfalpha, C/ebpalpha, and C/ebpbeta) and negative regulation (Hnf3alpha and the HNF4alpha coactivator Pgc-1alpha) on liver transcription factor expression; 2) a strong dependence on HNF4alpha characterizes several male-predominant Cyps (2d9 and 8b1), female-predominant Cyps (2b10, 2b13, 3a41, and 3a44) and Cyps, whose expression is sex independent (3a11, 3a25); 3) HNF4alpha confers a unique, positive regulation of two male-expressed genes (Cyp4a12 and GSTpi) and a negative regulation of several female-predominant genes (Cyp2a4, Cyp2b9, Hnf3beta, and Hnf6), both of which are manifest in male but not female mouse liver. These trends were confirmed at the protein level by Western blot analysis using antibodies raised to Cyp2a, Cyp2b, and Cyp3a family members. Thus, HNF4alpha is an essential player in the complex regulatory network of liver-enriched transcription factors and the sexually dimorphic mouse Cyp genes that they regulate. HNF4alpha is proposed to contribute to the sex specificity of liver gene expression by positively regulating a subset of male-specific Cyp genes while concomitantly inhibiting the expression of certain female-specific Cyps and liver transcription factors, by mechanisms that are operative in male, but not female, mouse liver.

Progression of HCC in mice is associated with a downregulation in the expression of hepatocyte nuclear factors.

Hepatocyte nuclear factors (HNF) play a critical role in development of the liver. Their roles during liver tumorigenesis and progression of hepatocellular carcinomas (HCC) are, however, poorly understood. To address the role of HNFs in tumor progression, we generated a new experimental model in which a highly differentiated slow-growing transplantable mouse HCC (sgHCC) rapidly gives rise in vivo to a highly invasive fast-growing dedifferentiated variant (fgHCC). This in vivo model has allowed us to investigate the fundamental mechanisms underlying HCC progression. A complete loss of cell polarity, a decrease in cell-cell and cell-extracellular matrix (ECM) adhesion, elevation of telomerase activity, and extinction of liver-specific gene expression accompanies tumor progression. Moreover, cells isolated from fgHCCs acquired the ability to proliferate rapidly in culture. These alterations were coupled with a reduced expression of several liver transcription factors including HNF4, a factor essential for hepatocyte differentiation. Forced re-expression of HNF4alpha1 in cultured fgHCC cells reversed the progressive phenotype and induced fgHCC cells to re-establish an epithelium and reform cell-ECM contacts. Moreover, fgHCC cells that expressed HNF4alpha1 also re-established expression of the profile of liver transcription factors and hepatic genes that are associated with a differentiated hepatocyte phenotype. Importantly, re-expression of HNF4alpha1 in fgHCC reduced the proliferation rate in vitro and diminished tumor formation in congenic recipient mice. In conclusion, loss of HNF4 expression is an important determinant of HCC progression. Forced expression of this factor can promote reversion of tumors toward a less invasive highly differentiated slow-growing phenotype.

Impact of dimethyl sulfoxide on expression of nuclear receptors and drug-inducible cytochromes P450 in primary rat hepatocytes

Dimethyl sulfoxide (DMSO) is reported to induce hepatocyte redifferentiation. The impact of DMSO on liver transcription factors, cytochromes P450 (CYPs), and nuclear receptors regulating CYP expression was assayed in primary rat hepatocytes by QPCR. CYP 2B1, 3A1, and 4A1 mRNAs were reduced to 10–30% of initial liver levels without DMSO and restored at or above liver levels by DMSO treatment. In contrast, CYP1A1 mRNA increased ∼5-fold during the course of culture, independent of DMSO. DMSO enhanced expression of the nuclear receptors CAR, PXR, and PPARα 2- to 5-fold, which may contribute to the increase in basal CYP expression. Without DMSO, liver transcription factors were decreased (HNF4, C/EBPα), largely unchanged (HNF1α, HNF3α, and C/EBPβ) or elevated (HNF3β, HNF6) compared to intact liver. DMSO largely restored hepatic levels of HNF4 and C/EBPα, partially suppressed the elevated levels of HNF6, increased HNF1α ∼2-fold, and had little effect on HNF3α, HNF3β, and C/EBPβ. Overall, DMSO helped maintain normal hepatic transcription factor patterns and basal CYP and nuclear receptor profiles, suggesting that hepatocytes cultured with DMSO may be useful for CYP metabolic studies under conditions where the endogenous liver phenotype is preserved.

Rapid transcriptional suppression of rat cytochrome P450 genes by endotoxin treatment and its inhibition by curcumin.

Down-regulation of constitutive hepatic cytochrome P450 (P450) mRNAs by bacterial endotoxin (lipopolysaccharide, LPS) or other inflammatory stimuli has been documented extensively, but the contribution of transcriptional suppression to this effect is poorly understood. Here, we demonstrate that the rates of transcription of the CYP2C11, CYP3A2, and CYP2E1 genes are reduced to 20, 30, and 10% of control levels, respectively, in rat liver within 1 to 2 h of injection of LPS (1 mg/kg). The magnitude and rapidity of these effects indicate that transcriptional suppression is a primary reason for the decline in P450 mRNAs. Injection of curcumin significantly inhibited the rapid transcriptional suppression of CYP2E1, and blocked that of CYP3A2. These effects seemed to be independent of inhibition of nuclear factor-kappaB (NF-kappaB) activation by curcumin, because induction of known NF-kappaB-regulated genes was not attenuated. One hour after LPS injection, the DNA-binding activities of hepatocyte nuclear factor (HNF)1alpha, HNF3beta, and HNF4alpha were reduced to 73, 72, and 53%, respectively, of control values. The nuclear abundances of Sp1, liver-enriched transcriptional inhibitory protein (LIP), HNF1alpha, and HNF3beta were unchanged, whereas the abundance of HNF4alpha was reduced to 87% of control levels. We conclude that changes in Sp1 or LIP do not contribute significantly to the early suppression of P450 transcription in the acute phase rat liver. Although changes in DNA-binding activities of HNF1alpha, HNF3beta, and HNF4alpha are too small individually to explain the observed changes in P450 transcription, the role of each factor in concert with other factors remains to be determined.

Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter.

Rat aflatoxin B(1) aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B(1). The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is approximately 8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to approximately 2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of approximately 900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.

Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus. Potential role in the glucocorticoid response.

Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.

Transcriptional regulation of the apolipoprotein AI gene.

Since the original description of the gene encoding apolipoprotein AI (apoAI) considerable progress has been made in identifying the regulatory regions and corresponding transcription factors that regulate its transcription in liver and intestinal cells. Nuclear receptors (in particular HNF-4) have emerged as the dominant (although by no means the exclusive) transcriptional activators that drive high levels of apoAI gene expression. In this review, some of the mechanisms (including interactions with recently described coactivators, as well as synergism with additional transcription factors bound to proximal and distal enhancers) that may underlie this process are discussed. Furthermore, apoAI gene expression has long been known to be subject to a variety of developmental, hormonal, dietary and pharmacological stimuli. The review thus also describes selected examples of these phenomena with the aim of highlighting how the operative transcriptional mechanisms are likely to be continually modified while maintaining the appropriate output.

A Free Radical Scavenger, Edaravone, Attenuates Steatosis and Cell Death via Reducing Inflammatory Cytokine Production in Rat Acute Liver Injury

Background/Aims: Reactive oxygen radicals play an important role in various forms of liver injury. In this study, we evaluated the efficacy of edaravone, a newly synthesized free radical scavenger, in its clinical dosage on an experimental model of acute liver injury in rats.Methods: The clinical dose of edaravone (3 mg/kg) was intravenously administered immediately and 3 h after intraperitoneal administration of carbon tetrachloride (CCl4) in rats. Histological evaluation including apoptosis and cytokine profiles were examined.Results: Fatty degeneration and necrosis with marked elevation of serum alanine aminotransferase and lactate dehydrogenase levels developed after CCl4 administration were significantly reduced by edaravone. In addition, the apoptotic index assessed by TUNEL method was significantly lowered in the edaravone treated group. Serum and liver transcription levels of interleukin-6, tumor necrosis factor-α, interleukin-4, and interleukin-10 were increased following CCl4 administration, and they were attenuated by edaravone treatment. The formation of malondialdehyde, 4-hydroxynonenal adduct and one of the markers for oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, was also inhibited by edaravone treatment.Conclusion: Edaravone has a remarkable protective effect on acute liver injury caused by oxygen radicals through not only attenuating the membrane lipid peroxidation, but also inhibiting the production of inflammatory cytokines. We theorize that edaravone may have a clinical benefit in the treatment of various liver injuries.

Phosphoenolpyruvate Carboxykinase Overexpression Selectively Attenuates Insulin Signaling and Hepatic Insulin Sensitivity in Transgenic Mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.

Diminished Hepatic Response to Fasting/Refeeding and Liver X Receptor Agonists in Mice with Selective Deficiency of Sterol Regulatory Element-binding Protein-1c

Two treatments, fasting/refeeding and administration of liver X receptor (LXR) agonists, elevate the mRNA for sterol regulatory element-binding protein-1c (SREBP-1c) and enhance lipid synthesis in liver. These treatments do not affect the mRNA for SREBP-1a, an alternative transcript from the same gene. Through homologous recombination, we eliminated the exon encoding SREBP-1c from the mouse genome, leaving the SREBP-1a transcript intact. On a normal diet, livers of SREBP-1c(-/-) mice manifested reductions in multiple mRNAs encoding enzymes of fatty acid and triglyceride synthesis, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). In contrast, SREBP-1c(-/-) livers showed a compensatory increase in hepatic SREBP-2 mRNA, accompanied by increased mRNA levels for cholesterol biosynthetic enzymes. In fasted/refed animals, ACC and FAS mRNAs rose, but not to the same extent as in wild-type livers. The refeeding-induced increase in SREBP-1c(-/-) mice was greater than in mice lacking SREBP cleavage-activating protein (SCAP), in which all nuclear SREBPs are absent. Thus, SREBP-2 and/or SREBP-1a can substitute partially for SREBP-1c in permitting an insulin-mediated increase in ACC and FAS mRNAs. In contrast, mRNAs for several other lipogenic enzymes (glucose-6-phosphate dehydrogenase, malic enzyme, glycerol-3-phosphate acyltransferase, and stearoyl-CoA desaturase-1) showed a complete failure of the normal inductive response to refeeding, indicating specific reliance on SREBP-1c. Moreover, these mRNAs, as well as multiple other lipogenic mRNAs, showed a markedly blunted response to the LXR agonist T090137, indicating an essential role of SREBP-1c in the LXR response.

GH is a regulator of IGF2 promoter-specific transcription in human liver.

The regulation of the insulin-like growth factor-II gene (IGF2) is complex and involves the usage of four promoters resulting in different 5' untranslated regions, but with a common translated product. The IGF2 gene product is a mitogenic and survival factor that has been suggested to be important for a normal fetal development and cancer. In this paper we present evidence suggesting that the human IGF2 gene is regulated by GH, and that this regulation occurs in a promoter-specific way. Three lines of evidence support this finding. First, in vivo data from patients treated with GH (one injection or daily injections for 5 consecutive days) showed an increase in the IGF2 P2 promoter derived transcript after acute treatment, and of the P4 promoter transcript after short-term treatment while the P1 promoter derived transcript did not show any significant change. Secondly, isolated human liver cells treated with GH for 2 h displayed an upregulation of the P2 promoter derived transcript. Thirdly, employing transfection experiments in GH-receptor positive CHO cells with P2 and P4 promoter-luciferase constructs, an upregulation by GH was evident, while a P1 promoter construct was unresponsive. We suggest that GH may be a physiological regulator of IGF2 in humans.

Augmented hepatic interferon gamma expression and T-cell influx characterize acute hepatitis progressing to recovery and residual lifelong virus persistence in experimental adult woodchuck hepatitis virus infection

Woodchucks infected with woodchuck hepatitis virus (WHV) have profiles of liver disease and age-dependent rates of progression to chronic hepatitis (CH) comparable with those seen in human hepatitis B. The mechanism of recovery from acute hepadnaviral infection or its evolution to chronicity remains unknown, although the liver immune responses are expected to play an important role. To determine the dynamics of intrahepatic cytokine expression and T-cell involvement, and to assess their value in predicting the outcome of acute hepatitis (AH) in the adult onset of WHV infection, we evaluated liver transcription of interferon gamma (IFN-γ); tumor necrosis factor α (TNF-α); interleukins (IL)-2, -4, and -6; and the T-cell influx in relation to disease histologic severity and virus load in serial liver biopsies collected during the life span of experimentally infected woodchucks. Our results show that recovery from viral AH in adulthood is preceded by a significantly greater hepatic expression of IFN-γ and CD3, an increased TNF-α transcription, lower hepatic WHV load, and a greater degree of liver inflammation than those in acute infection with CH outcome. Furthermore, we have learned that the elevated IFN-γ, TNF-α, and CD3 expression in the liver endures for years not only in CH, but also, although to a lesser extent, in apparently completely resolved infection. This is consistent with our previous findings that residual WHV replication and remnant liver inflammation continue for life after recovery from AH. This study indicates that antiviral cytokines, in particular IFN-γ, may play a central role in the long-term control of occult hepadnavirus persistence in the liver. (HEPATOLOGY 2001;34:1049-1059.)

Chromosomal assignments of mammalian genes with an acute inflammation-regulated expression in liver.

A set of acute inflammation-regulated genes expressed in liver has been assigned to rat, mouse, and human chromosomes by detecting species-specific PCR amplicons in rat(x)mouse or mouse(x)hamster somatic cell hybrids or radiation hybrids or by in silico matches of corresponding rat cDNAs to various libraries of previously assigned rat, mouse, or human genes or expressed-sequence tags. This allowed us to assign 24, 22, and 21 inflammation-regulated genes to rat, mouse, and human chromosomes, respectively. From these assignments as well as those previously determined for a larger set of genes with an acute inflammation-regulated transcription in liver, we further investigated whether such genes are clustered onto given chromosomes. A cluster was found on rat Chromosome (Chr) 6q with a conserved synteny on mouse Chr 12 and human Chr 14q13-q32, and another cluster previously reported on human Chr 1q has been extended with five further genes. Our data suggest that during an acute inflammation, a higher-order regulation may control some liver-expressed genes that share a given chromosome area.