Hepatic Nuclear Factor 4α Research Articles

Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells.

Exo70, a member of the exocyst complex, is involved in cell exocytosis, migration, invasion and autophagy. However, the expression regulation and function of Exo70 in hepatocellular carcinoma are still poorly understood. In this study, we found Exo70 expression in human hepatoma cells was greatly reduced after knocking down hepatic nuclear factor 4α (HNF4α), the most important and abundant transcription factor in liver. This regulation occurred at the transcriptional level but not post-translational level. HNF4α transactivated Exo70 promoter through directly binding to the HNF4α-response element in this promoter. Cell cycle analysis further revealed that down-regulation of HNF4α and Exo70 was essential to berberine-stimulated G2/M cell cycle arrest in hepatoma cells. Moreover, knocking down either Exo70 or HNF4α induced G2/M phase arrest of hepatoma cells. Exo70 acted downstream of HNF4α to stimulate G2/M transition via increasing Cdc2 expression. Together, our results identify Exo70 as a novel transcriptional target of HNF4α to promote cell cycle progression in hepatoma, thus provide a basis for the development of therapeutic strategies for hepatocellular carcinoma.

Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells

Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14–deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14–deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14–deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14–deficient livers. We demonstrate that MMP-14–mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.

Ginsenoside Compound K suppresses the hepatic gluconeogenesis via activating adenosine-5'monophosphate kinase: A study in vitro and in vivo.

Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenoside. We have reported that CK presented anti-diabetic effect via diminishing the expressions of hepatic gluconeogenesis key enzyme. Here, we further explore the possible mechanism of CK on suppression hepatic gluconeogenesis via activation of adenosine-5'monophosphate kinase (AMPK) on type 2 diabetes mice in vivo and in HepG2 cells. Type 2 diabetes mice model was developed by high fat diet combined with STZ injection. 30mg/kg/d CK was orally administrated for 4weeks, the fasting blood glucose level and 2h OGTT were conducted, and the protein expression of AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) were examined. The mechanism of Compound K on hepatic gluconeogenesis was further explored in HepG2 hepatocytes. Glucose production, the protein expression of AMPK, PEPCK, G6pase and PGC-1α, hepatic nuclear factor 4α (HNF-4α) and forkhead transcription factor O1 (FOXO1) were determined after Compound K treatment at the presence of AMPK inhibitor Compound C. We observed that CK inhibited the expression of PEPCK and G6Pase in the liver and in HepG2 hepatocytes. Meanwhile, CK treatment remarkably increased the activation of AMPK, while decreasing the expressions of PGC-1α, HNF-4α and FOXO1. However, AMPK inhibitor Compound C could reverse these effects of CK on gluconeogenesis in part. The results indicated that the effect of CK on suppression hepatic gluconeogenesis might be via the activation the AMPK activity.

A novel nonsense mutation of the HNF1α in maturity-onset diabetes of the young type 3 in Asian population

We reported a novel nonsense mutation, R54X of the hepatic nuclear factor 1α (HNF1α) gene in a Chinese family. The mutation was identified in a 47 years old woman and her 19 years old daughter within a five-family members tested. Both the two patients were sensitive to insulin and glibenclamide treatments.

Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice

The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver.

Impairment of hepatic nuclear factor-4α binding to the Stim1 promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells.

The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca(2+) entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca(2+) entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.

Decreased nuclear receptor activity and epigenetic modulation associates with down-regulation of hepatic drug-metabolizing enzymes in chronic kidney disease.

Patients with chronic kidney disease (CKD) require many medications. CYP2C and CYP3A drug-metabolizing enzymes play a critical role in determining the pharmacokinetics of the majority of prescribed medications. These enzymes are transcriptionally regulated by the nuclear receptors pregnane X receptor (PXR) and hepatic nuclear factor 4α (HNF-4α). Expression of CYP2C and CYP3A is decreased in CKD; however, the mechanisms by which this occurs is unknown. We induced CKD in rats by 5/6 nephrectomy and used chromatin immunoprecipitation (ChIP) to determine nuclear receptor- and epigenetic alteration-mediated differences in the promoter region of the CYP2C and CYP3A genes. RNA polymerase II and HNF-4α binding was decreased 76 and 57% in the CYP2C11 promotor and 71 and 77% in the CYP3A2 promoter, respectively (P<0.05). ChIP also revealed a 57% decrease in PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). The decrease in PXR and HNF-4α binding was accompanied by diminished histone 4 acetylation in the CYP3A2 promoter (48%) and histone 3 acetylation in the CYP2C11 (77%) and CYP3A2 (77%) promoter loci for nuclear receptor activation (P<0.05). This study suggests that decreased nuclear receptor binding and histone acetylation may contribute to the mechanism of drug-metabolizing enzyme down-regulation and altered pharmacokinetics in CKD.

Med25 is required for estrogen receptor alpha (ERα)-mediated regulation of human CYP2C9 expression

The CYP2C subfamily of cytochrome P450 enzymes is an important class of drug metabolizing enzymes in human liver. CYP2C9 is the most abundant member of the human CYP2C subfamily in liver and metabolizes ∼15% of the therapeutic drugs as well as other xenobiotics and endogenous compounds. A number of nuclear receptors including xenobiotic-sensing receptors such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glucocorticoid receptor (GR) as well as liver enriched receptors hepatic nuclear factor 4α (HNF4α) and the estrogen receptor α (ERα) regulate CYP2C9 expression. Here, we show that Med25, a variable component of Mediator complex, enhanced ligand dependent ERα-mediated transcriptional activation of CYP2C9 promoter and interacts with activated ERα by 17β-estradiol through its C-terminal LXXLL motif. In conclusion, Med25 is identified as a new coactivator of ERα that is required for ERα-mediated regulation of CYP2C9 expression.

Exposure to omega-3 fatty acids at early age accelerate bone growth and improve bone quality

Omega-3 fatty acids (FAs) are essential nutritional components that must be obtained from foods. Increasing evidence validate that omega-3 FAs are beneficial for bone health, and several mechanisms have been suggested to mediate their effects on bone, including alterations in calcium absorption and urinary calcium loss, prostaglandin synthesis, lipid oxidation, osteoblast formation and inhibition of osteoclastogenesis. However, to date, there is scant information regarding the effect of omega-3 FAs on the developing skeleton during the rapid growth phase. In this study we aim to evaluate the effect of exposure to high levels of omega-3 FAs on bone development and quality during prenatal and early postnatal period. For this purpose, we used the fat-1 transgenic mice that have the ability to convert omega-6 to omega-3 fatty acids and the ATDC5 chondrogenic cell line as models. We show that exposure to high concentrations of omega-3 FAs at a young age accelerates bone growth through alterations of the growth plate, associated with increased chondrocyte proliferation and differentiation. We further propose that those effects are mediated by the receptors G-protein coupled receptor 120 (GPR120) and hepatic nuclear factor 4α, which are expressed by chondrocytes in culture. Additionally, using a combined study on the structural and mechanical bone parameters, we show that high omega-3 levels contribute to superior trabecular and cortical structure, as well as to stiffer bones and improved bone quality. Most interestingly, the fat-1 model allowed us to demonstrate the role of maternal high omega-3 concentration on bone growth during the gestation and postnatal period.

Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor

The clinical application of a bioartificial liver (BAL) depends on the availability of a human cell source with high hepatic functionality, such as the human hepatoma cell line HepaRG. This cell line has demonstrated high hepatic functionality, but the effect of BAL culture on its functionality in time is not known. Therefore, we studied the characteristics of the HepaRG-AMC-BAL over time, and compared the functionality of the HepaRG-AMC-BAL with monolayer cultures of HepaRG cells, normalized for protein (bioactive mass) and DNA (cell number).Histological analysis of 14-day-old BALs demonstrated functional heterogeneity similar to that of monolayer cultures. Hepatic functionality of the HepaRG-AMC-BALs increased during 2–3 weeks of culture. The majority of the measured protein-normalized hepatic functions were already higher in day 14 BAL cultures compared to monolayer cultures, including ammonia elimination (3.2-fold), urea production (1.5-fold), conversion of 15N-ammonia into 15N-urea (1.4-fold), and cytochrome P450 3A4 activity (7.9-fold). Lactate production in monolayer cultures switched into lactate consumption in the BAL cultures, a hallmark of primary hepatocytes. When normalized for DNA, only cytochrome P450 3A4 activity was 2.5-fold higher in the BAL cultures compared to monolayer cultures and lactate production switched to consumption, whereas urea production and 15N-urea production were 1.5- to 2-fold lower. The different outcomes for protein and DNA normalized functions probably relate to a smaller cell volume of HepaRG cells when cultured in the AMC-BAL. Cell damage was 4-fold lower in day 14 BAL cultures compared to monolayer cultures. Transcript levels of cytochrome P450 1A2, 2B6, 3A4 and 3A7 genes and of regulatory genes hepatic nuclear factor 4α and pregnane X receptor increased in time in BAL cultures and reached higher levels than in monolayer cultures. Lastly, metabolism of amino acids, particularly the alanine consumption and ornithine production of HepaRG-AMC-BALs more resembled that of primary hepatocytes than monolayer HepaRG cultures.We conclude therefore that BAL culture of HepaRG cells increases its hepatic functionality, particularly when normalized for biomass, both over time, and compared to monolayer, and this is associated with a reduction in cell damage, upregulation of both regulatory and structural hepatic genes, and changes in amino-acid metabolism. These results underline the potential of HepaRG cells for BAL application.

Hepatic nuclear factor 4α positively regulates complement C3 expression and does not interfere with TNFα-mediated stimulation of C3 expression in HepG2 cells

Complement C3 is involved in various protective and regulatory mechanisms of immune system. Recently it was established that C3 expression is regulated by nuclear receptors. Hepatic nuclear factor 4α (HNF4α) is a nuclear receptor critical for hepatic development and metabolism. We have shown that HNF4α is a positive regulator of C3 gene expression, realizing its effects through binding to two HNF4-response elements within the C3 promoter in HepG2 cells. TNFα is a well established positive regulator of C3 expression in hepatocytes during acute phase of inflammation. TNFα decreases the amount of HNF4α protein in HepG2 cells through NF-κB and MEK1/2 pathways thereby leading to a decrease in HNF4α bound to the C3 promoter. TNFα and HNF4α act in a synergetic way resulting in the potent activation of C3 transcription. These results suggest a novel mechanism of C3 regulation during acute phase response in HepG2 cells and display the mechanism of interaction of TNFα-induced pathways and HNF4α in transcriptional regulation of C3 gene.

Mechanical compaction directly modulates the dynamics of bile canaliculi formation

Homeostatic pressure-driven compaction is a ubiquitous mechanical force in multicellular organisms and is proposed to be important in the maintenance of multicellular tissue integrity and function. Previous cell-free biochemical models have demonstrated that there are cross-talks between compaction forces and tissue structural functions, such as cell-cell adhesion. However, its involvement in physiological tissue function has yet to be directly demonstrated. Here, we use the bile canaliculus (BC) as a physiological example of a multicellular functional structure in the liver, and employ a novel 3D microfluidic hepatocyte culture system to provide an unprecedented opportunity to experimentally modulate the compaction states of primary hepatocyte aggregates in a 3D physiological-mimicking environment. Mechanical compaction alters the physical attributes of the hepatocyte aggregates, including cell shape, cell packing density and cell-cell contact area, but does not impair the hepatocytes' remodeling and functional capabilities. Characterization of structural and functional polarity shows that BC formation in compact hepatocyte aggregates is accelerated to as early as 12 hours post-seeding; whereas non-compact control requires 48 hours for functional BC formation. Further dynamic immunofluorescence imaging and gene expression profiling reveal that compaction accelerated BC formation is accompanied by changes in actin cytoskeleton remodeling dynamics and transcriptional levels of hepatic nuclear factor 4α and Annexin A2. Our report not only provides a novel strategy of modeling BC formation for in vitro hepatology research, but also shows a first instance that homeostatic pressure-driven compaction force is directly coupled to the higher-order multicellular functions.

Metabolically Functional Hepatocyte-Like Cells from Human Umbilical Cord Lining Epithelial Cells

The primary hepatocyte is the best benchmark for drug biotransformation studies. However, due to the severe shortage of primary hepatocytes, there is a need for alternative reliable cell source. This study aims to isolate multipotent epithelial cells from the umbilical cord, differentiate these cells into hepatocyte-like cells (HLCs), and investigate the potential of using the differentiated cells for in vitro drug metabolism model. Human umbilical cord lining epithelial cells (UCLECs) were subjected to hepatic induction over a period of 28 days. HepG2 and cryopreserved human hepatocytes were used as control. Immunohistological staining was carried out for α-fetoprotein (AFP), albumin, cytokeratin 18 (CK18), and 19 (CK19). Glycogen storage ability was assessed through periodic acid-Schiff stain. Reverse transcription polymerase chain reaction was performed to examine gene expression of hepatic nuclear factor 4α (HNF4α) and cytochrome P450 isozymes 1A2, 2C9, 2D6, and 3A4. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) was utilized to analyze functional metabolic ability of the HLCs, where CYP3A4 was chosen as the study focus and testosterone as the drug substrate. After 28 days of induction, the fibroblastic phenotype of UCLECs changed to rotund polygonal shape resembling that of hepatocytes. Protein expression of AFP and CK19 was negative, while albumin and CK18 expression was upregulated. Gene expression of HNF4α, CYP1A2, CYP2D6, and CYP3A4 was observed but not for CYP2C9. After 4 h of incubation with testosterone, UPLC/MS/MS detected 2α-, 6β-, 15β-, and 16β-hydroxytestosterone. UCLECs are able to differentiate into HLCs that express liver-specific markers, and have functional metabolic capabilities.

The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells

PCSK9 (proprotein convertase subtilisin/kexin type9) plays an important role in control of plasma LDL (low-density lipoprotein) cholesterol metabolism by modulating the degradation of hepatic LDL receptor. Previous studies demonstrated that PCSK9 is a target gene of the SREBP2 [SRE (sterol-regulatory element)-binding protein 2] that activates PCSK9 gene transcription through an SRE motif of the promoter. In addition to SREBP2, HNF1α (hepatic nuclear factor 1α) positively regulates PCSK9 gene transcription in hepatic cells through a binding site located 28bp upstream from SRE. In the present study, we have identified a novel HINFP (histone nuclear factor P) recognition motif residing between the HNF1 motif and SRE that is essential for basal and sterol-regulated transcriptions of the PCSK9 promoter. Mutation of this motif lowers the basal promoter activity and abolishes the sterol-mediated repression as well as the SREBP2-induced activation of the PCSK9 promoter. We show further that the activity of SREBP2 in stimulating PCSK9 promoter activity is greatly enhanced by HINFP. Additional experiments suggest that HINFP and its cofactor NPAT (nuclear protein of the ataxia telangectasia mutated locus) form a functional complex, and NPAT may subsequently recruit the HAT (histone acetyltransferase) cofactor TRRAP (transformation/transactivation domain-associated protein) to facilitate the histone H4 acetylation of the PCSK9 promoter. Knockdown of HINFP, NPAT or TRRAP each markedly reduces the amount of acetylated histone H4 on the PCSK9 promoter region and lowers PCSK9 protein levels. Importantly, by utilizing co-immunoprecipitation assays, we have demonstrated a direct interaction between SREBP2 and HINFP and its cofactors NPAT/TRRAP. Taken together, these new findings identify HINFP as a co-activator in SREBP-mediated transactivation of PCSK9 gene expression.

Hepatocarcinogenesis in FXR−/− Mice Mimics Human HCC Progression That Operates through HNF1α Regulation of FXR Expression

Farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4) is a member of nuclear hormone receptor superfamily, which plays essential roles in metabolism of bile acids, lipid, and glucose. We previously showed spontaneously hepatocarcinogenesis in aged FXR(-/-) mice, but its relevance to human hepatocellular carcinoma (HCC) is unclear. Here, we report a systematical analysis of hepatocarcinogenesis in FXR(-/-) mice and FXR expression in human liver cancer. In this study, liver tissues obtained from FXR(-/-) and wild-type mice at different ages were compared by microarray gene profiling, histological staining, chemical analysis, and quantitative real-time PCR. Primary hepatic stellate cells and primary hepatocytes isolated from FXR(-/-) and wild-type mice were also analyzed and compared. The results showed that the altered genes in FXR(-/-) livers were mainly related to metabolism, inflammation, and fibrosis, which suggest that hepatocarcinogenesis in FXR(-/-) mice recapitulated the progression of human liver cancer. Indeed, FXR expression in human HCC was down-regulated compared with normal liver tissues. Furthermore, the proinflammatory cytokines, which were up-regulated in human HCC microenvironment, decreased FXR expression by inhibiting the transactivity of hepatic nuclear factor 1α on FXR gene promoter. Our study thereby demonstrates that the down-regulation of FXR has an important role in human hepatocarcinogenesis and FXR(-/-) mice provide a unique animal model for HCC study.

In Silico and In Vitro Identification of MicroRNAs That Regulate Hepatic Nuclear Factor 4α Expression

Hepatic nuclear factor 4α (HNF4A) is a nuclear transcription factor that regulates the expression of many genes involved in drug disposition. To identify additional molecular mechanisms that regulate HNF4A, we identified microRNAs (miRNAs) that target HNF4A expression. In silico analyses suggested that HNF4A is targeted by many miRNAs. We conducted in vitro studies to validate several of these predictions. With use of an HNF4A 3'-untranslated region (UTR) luciferase reporter assay, five of six miRNAs tested significantly down-regulated (∼20-40%) the luciferase activity. In HepG2 cells, miR-34a and miR-449a also down-regulated the expression of both the HNF4A protein and an HNF4A target gene, PXR (∼30-40%). This regulation appeared without reduction in HNF4A mRNA expression, suggesting that they must be blocking HNF4A translation. Using additional bioinformatic algorithms, we identified polymorphisms that are predicted to alter the miRNA targeting of HNF4A. Luciferase assays indicated that miR-34a and miR-449a were less effective in regulating a variant (rs11574744) than the wild-type HNF4A 3'-UTR. In vivo, subjects with the variant HNF4A had lower CYP2D6 enzyme activity, although this result was not statistically significant (p = 0.16). In conclusion, our findings demonstrate strong evidence for a role of miRNAs in the regulation of HNF4A.

Effect of simvastatin on the expression and regulation mechanism of apolipoprotein M

Apolipoprotein M (ApoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in the plasma. Statins have effects on many HDL-associated apolipoproteins. However, it is unknown whether statins have effects on ApoM. In the present study, we investigated the effects of simvastatin on ApoM expression and the underlying mechanism(s). Simvastatin up-regulated hepatic ApoM mRNA and protein expression in mice. In HepG2 cells, simvastatin significantly enhanced ApoM mRNA and protein expression in a dose-dependent manner. Simvastatin increased hepatic hepatocyte nuclear factor-1α (HNF-1α) mRNA and reduced liver X receptor-α (LXRα) mRNA expression in mice. The simvastatin-induced up-regulation of ApoM was blocked by an HNF-1α inhibitor (UCDA) or an LXRα agonist (TO901317) in HepG2 cells which indicates that this effect is mediated via the regulation of HNF-1α and LXRα. In conclusion, simvastatin significantly up-regulated ApoM expression in vivo and in vitro, which indicates that ApoM is another novel apolipoprotein regulated by simvastatin. The mechanism of this effect is related to the regulation of HNF-1α and LXRα.

Ontogenesis of phase I hepatic drug metabolic enzymes in sheep

Cytochrome P450 (CYP) enzymes are important for the metabolism of many drugs. While there is information on their identity and ontogeny in humans and rodents, similar data in sheep are lacking. In the present study, cDNA sequences of several CYP enzymes (CYP2A6, CYP2C19, CYP2D6) were cloned by rapid amplification of cDNA ends. In adult, newborn and fetal sheep the mRNA and protein levels of these CYPs and the regulatory factor, hepatic nuclear factor 4α (HNF4α) were determined in liver samples using real-time PCR and western blotting. The effect of antenatal glucocorticoid on these enzymes was also studied by i.v. infusion of cortisol (0.45 mgh(-1); 80 h) to another group of fetuses. The mRNA and protein levels of the CYPs and HNF4α were low or absent in the fetus, followed by increasing levels in the newborn and adult. Fetal cortisol administration significantly increased the mRNA and protein levels of CYP2D6. Moreover, the correlation observed between the CYP and HNF4α mRNA levels suggests a possible regulatory role for this transcription factor. The findings suggest that fetal and newborn lambs have a low ability to metabolise drugs that are substrates of these enzymes, and that this ability increases with advancing postnatal age, similar to the situation in humans.

Hepatocellular adenoma containing hepatocellular carcinoma in a male patient with familial adenomatous polyposis coli: Report of a case

Hepatocellular adenoma (HCA) is a benign condition occurring most frequently in young women using oral contraceptives. We herein report the first case of a 28-year-old man with familial adenomatous polyposis (FAP) who experienced hepatocellular carcinoma (HCC) within HCA. A preoperative computed tomography scan of the abdomen disclosed a tumor measuring 5.8 cm in diameter in the left lobe of the liver. A total proctocolectomy with construction of a linear stapled J-pouch, a hand-sewn ileoanal anastomosis and a liver tumor resection were performed. The pathology of the resected specimen confirmed the diagnosis of HCA containing HCC. Five cases of HCA have been reported that developed in patients with FAP, but this is the first case of HCA containing HCC. Hepatocellular adenomas occur due to the inactivation of hepatic nuclear factor 1α, but the mechanism underlying the malignant transformation from HCA to HCC could not be identified in this case.

Oxygen-regulated expression of the erythropoietin gene in the human renal cell line REPC

AbstractErythropoietin (EPO), the key hormone in red blood cell renewal, is mainly produced in the adult kidney. Anemia and hypoxia substantially enhance EPO expression to increase erythropoiesis. Investigations of the cellular physiology of renal EPO production have been hampered by the lack of an adequate human cell line. In the present study, we present the human kidney cell line REPC (for renal Epo–producing cells), established from an explanted human kidney exhibiting EPO gene expression and release of the EPO protein in an oxygen-dependent manner. Hypoxic induction of EPO mRNA showed the typical transient increase and peak in expression after 36 hours under continuous conditions of hypoxia. Bioactive EPO protein accumulated in the culture supernatant. The induction of EPO gene expression in REPCs critically depended on the activation of hypoxia-inducible transcription factors (HIFs). SiRNA treatment revealed that the expression of EPO was largely dependent on the activation of the transcription factor complex HIF-2. In addition, hepatic nuclear factor 4α was shown to be critically involved in hypoxia-induced renal EPO expression. Using the human kidney cell line REPC, we provide for the first time a powerful tool with which to study the cellular and molecular regulation of renal EPO production.