Primary Cultures Of Fetal Hepatocytes Research Articles

227 DIFFERENTIATION OF HEPATOCYTE-LIKE CELLS FROM MOUSE EMBRYONIC STEM CELLS IN A MONOLAYER CULTURE SYSTEM

Embryonic stem (ES) cells can be differentiated in vitro into a variety of cell lineages. In vitro differentiation of ES cells, therefore, provides a model system for organogenesis as well as an innovative approach for regenerative medicine. However, current in vitro hepatic differentiation systems from ES cells require embryoid body formation, and such systems achieve quite low differentiation efficacy. In this study, in order to examine a system for preparation of significant numbers of hepatocytes from ES cells, mouse ES cells were directly differentiated into hepatocyte-like cells using monolayer culture conditions, and their hepatic characteristics were examined. ES cells were cultured on gelatin-coated plates for 3 days in ES cell medium containing leukemia inhibitory factor (100 units mL-1) and retinoic acid (10-8 M). After 3 days, the cells were harvested by trypsinization and cultured on gelatin-coated plates for 5 days in ES cell medium containing acidic fibroblast growth factor (aFGF; 100 ng mL-1), FGF4 (20 ng mL-1), and human growth factor (50 ng mL-1). After 5 days, the cells were passaged on type I collagen-coated plates for 2 days in ES cell medium containing Oncostatin M (10 ng mL-1), followed by additional culture in modified William's E medium containing 5% FBS and dexamethasone (10-7 M) for 7 days. For characterizing the differentiated ES cells, the cells were examined for albumin production by immunocytochemistry, and the expression profiles of hepatic genes by RT-PCR. For analyzing their hepatic function, the cells were examined for glycogen production by PAS staining, and their metabolic activities of ammonia elimination and urea synthesis were measured. After 10 days of culture, almost all cells showed albumin production by immunofluoresence. RT-PCR analysis showed expression of various genes specific for hepatocytes, such as ALB, AFP, TTR, and HNF3�, but not HNF4α, and genes specific for matured hepatocytes, such as PEPCK, TAT, G6P, and Cyp7a1. After 11 days of culture, glycogen production was partially detected in the hepatocyte-like cells by PAS staining. Although ammonia elimination activity of hepatocyte-like cells was shown to be significantly lower than that of the primary culture of fetal hepatocytes after 24 and 48 h of treatment (P < 0.05), its metabolic activity was acquired at the level of about 40% of that of fetal hepatocytes. Hepatocyte-like cells also showed lower activity of urea synthesis than fetal hepatocytes at 6, 12, and 24 h of treatment (P < 0.05), but this activity increased thereafter and reached about 70% of that of fetal hepatocytes after 48 h of treatment. These results demonstrated that monolayer culture conditions for ES cell differentiation could provide an in vitro system to produce significant numbers of hepatocytes. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

STAT3 Down-regulates the Expression of Cyclin D during Liver Development

As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential.

Hypoxia Regulates Insulin-Like Growth Factor-Binding Protein 1 in Human Fetal Hepatocytes in Primary Culture: Suggestive Molecular Mechanisms for in Utero Fetal Growth Restriction Caused by Uteroplacental Insufficiency

Hypoxia Regulates Insulin-Like Growth Factor-Binding Protein 1 in Human Fetal Hepatocytes in Primary Culture: Suggestive Molecular Mechanisms for in Utero Fetal Growth Restriction Caused by Uteroplacental Insufficiency

Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency.

Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

Expression of cyclooxygenase-2 promotes the release of matrix metalloproteinase-2 and -9 in fetal rat hepatocytes

Treatment of primary cultures of fetal hepatocytes with proinflammatory cytokines, lipopolysaccharide (LPS), and hepatocyte growth factor promoted the expression of cyclooxygenase-2 (COX-2) and the synthesis of high amounts of prostaglandins (PGs). Under these conditions, the active forms of the matrix metalloproteinases-2 and -9 (MMPs) were released to the extracellular medium. This process was inhibited when the synthesis of PGs was suppressed pharmacologically with COX-2 inhibitors. Addition to the cell cultures of PGE2 promoted the release of MMPs through a mechanism that involved the expression of COX-2 and the synthesis of additional PGs. Kinetic analysis of the secretion of MMPs in response to LPS and PGE2 showed a similar time course, with a lag period of 6 hours, which suggests that PGE2 does not act directly on the mechanism of MMP processing and release. Inhibitors of protein kinase A, p38 MAP kinase, phosphatidylinositol-3-kinase, and nuclear factor κB (NF-κB) activation impaired the release of MMPs in response to PGE2 challenge, indicating the involvement of multiple steps in the process. The ability of fetal hepatocytes to release MMPs in response to growth factors and inflammatory stimuli constitutes a model for the study of the extracellular matrix remodeling that accompanies most liver diseases. (HEPATOLOGY 2001;33:860-867.)

Fibronectin regulates morphology, cell organization and gene expression of rat fetal hepatocytes in primary culture

Background/Aims: The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to analyze how different extracellular matrix molecules modulate fetal hepatocyte morphology, growth and differentiation. Methods: We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-collagen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression. Results: Cell attachment was increased by all the extracellular matrix proteins to a similar extent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immunocytochemical analysis of the cells in these structures revealed high levels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures appeared either in the absence or in the presence of the mitogen. Cells cultured on fibronectin, regardless of whether epidermal growth factor was present or not, also presented the maximal levels of expression for liver specific genes, such as albumin or alpha-fetoprotein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-1α/HNF-1β ratio, when compared with those cells that were cultured on collagen or E-C-L extracellular matrix. Conclusions: These results suggest that fibronectin might play a differential role, as compared to other extracellular matrix proteins, in fetal hepatocyte organization and gene expression.

Glucocorticoid receptor down-regulates c-Jun amino terminal kinases induced by tumor necrosis factor alpha in fetal rat hepatocyte primary cultures.

The effect of dexamethasone on Jun N-terminal kinase (JNK) activity was assayed by using fetal hepatocytes in primary culture. The addition of tumor necrosis factor alpha (TNF-alpha) caused an increase in JNK in a dose- and time-dependent manner. We show that activation of JNK by this extracellular signal is inhibited by dexamethasone in a dose-dependent fashion. This inhibitory effect was observed in cells treated for 10 minutes with dexamethasone in the presence of protein phosphatase inhibitors such as orthovanadate or okadaic acid, or in cells previously treated with actinomycin D. Glucocorticoid receptor (GR) can be precipitated with the fusion protein, GST-c-Jun (1-79), bound to agarose beads. However, the inhibitory effect of glucocorticoids on JNK activity was also observed using ATF-2 as substrate. In addition, dexamethasone inhibits JNK phosphorylation induced by TNF-alpha. Finally, we show that GR can also be phosphorylated in tyrosine residues in response to TNF-alpha and epidermal growth factor (EGF) upon ligand-binding. Our results suggest that the anti-inflammatory effect of glucocorticoids on the inflammatory pathways induced by TNF-alpha can be explained, at least in part, by modulating JNK activity through a direct protein-protein interaction; the JNK phosphorylation and tyrosine-phosphorylation state of GR may be regulatory steps also involved in that effect.

Transforming Growth Factor-β (TGF-β) and EGF Promote Cord-like Structures That Indicate Terminal Differentiation of Fetal Hepatocytes in Primary Culture

When fetal hepatocytes were cultured in the presence of transforming growth factor-β (TGF-β1) and epidermal growth factor (EGF), some morphological changes were observed. Under these conditions, cells migrated, from typical clusters that hepatocytes adopt in culture, to form elongated, cord-like structures similar to the hepatic acinus organization. Immunocytochemical analysis of these cells revealed high levels of albumin and cytokeratin 18, phenotypic markers of parenchymal hepatocytes. Although some of the cells in the cord-like structures presented a cortical ring distribution of F-actin filaments, the cord also presented thick peripheral bundles and cells of the tips showed thin stress fibers oriented to the cell edges, typical of a migratory phenotype. In addition to these morphological effects, flow cytometric analysis of the cells revealed a larger size, granularity and intracellular lipid content (as a parameter related to liver metabolic function), in TGF-β + EGF-treated hepatocytes. Western blot analysis of the albumin levels revealed that both expression and secretion of albumin were increased in EGF + TGF-β-treated cells. Finally, all these changes were coincident with an enhancement in the DNA-binding activity for hepatocyte nuclear factors (HNF1, HNF3, and HNF4), as revealed in gel-shift experiments with nuclear extracts. We conclude that a cooperative action between TGF-β and EGF might modulate terminal maturation of fetal hepatocytes.

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca 2+-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c- jun and c- fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB. PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

CAMP and Ca2+Involvement in the Mitochondrial Response of Cultured Fetal Rat Hepatocytes to Adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both α- and β-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor beta in fetal rat hepatocytes in primary culture.

We have previously reported that transforming growth factor-beta (TGF-beta) induces apoptosis in fetal hepatocytes in primary culture. This effect was found to be associated with an increase in intracellular reactive oxygen species (ROS) and a lowering of total cellular reduced glutathione (GSH). In this study, we investigated whether protein synthesis plays a role in these TGF-beta-induced effects. When fetal hepatocytes were incubated in the presence of cycloheximide, a specific protein synthesis inhibitor, TGF-beta-induced apoptosis was completely blocked. The overall intracellular oxidized state of the cells, when measured using either 2',7'-dichlorofluorescein diacetate (DCFH) by laser-scanning confocal microscopy, or both DCFH and hydroethidine (DHE) by flow cytometric analysis, was increased transiently after the addition of TGF-beta. This increase was abolished by incubation of the cells in the presence of cycloheximide. Furthermore, the decrease in the total cellular GSH content induced by TGF-beta in these cells was not observed when cycloheximide was present. Cycloheximide effect was not associated with an enhancement of cysteine and restoration of cellular glutathione level, because inhibition of GSH synthesis with buthionine sulfoximine (BSO) did not prevent the cycloheximide protective effect. Experiments performed to check whether Fas (APO-1) ligand might be the protein that needs to be synthesized have indicated that this possibility can be excluded. All these findings suggest that apoptosis elicited by TGF-beta in fetal hepatocytes requires the synthesis of an unknown protein before ROS production, glutathione loss, and oxidative stress.

Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor beta in fetal rat hepatocytes in primary culture

We have previously reported that transforming growth factor-beta (TGF-beta) induces apoptosis in fetal hepatocytes in primary culture. This effect was found to be associated with an increase in intracellular reactive oxygen species (ROS) and a lowering of total cellular reduced glutathione (GSH). In this study, we investigated whether protein synthesis plays a role in these TGF-beta-induced effects. When fetal hepatocytes were incubated in the presence of cycloheximide, a specific protein synthesis inhibitor, TGF-beta-induced apoptosis was completely blocked. The overall intracellular oxidized state of the cells, when measured using either 2',7'-dichlorofluorescein diacetate (DCFH) by laser-scanning confocal microscopy, or both DCFH and hydroethidine (DHE) by flow cytometric analysis, was increased transiently after the addition of TGF-beta. This increase was abolished by incubation of the cells in the presence of cycloheximide. Furthermore, the decrease in the total cellular GSH content induced by TGF-beta in these cells was not observed when cycloheximide was present. Cycloheximide effect was not associated with an enhancement of cysteine and restoration of cellular glutathione level, because inhibition of GSH synthesis with buthionine sulfoximine (BSO) did not prevent the cycloheximide protective effect. Experiments performed to check whether Fas (APO-1) ligand might be the protein that needs to be synthesized have indicated that this possibility can be excluded. All these findings suggest that apoptosis elicited by TGF-beta in fetal hepatocytes requires the synthesis of an unknown protein before ROS production, glutathione loss, and oxidative stress. (Hepatology 1997 Oct;26(4):935-43)

Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5′ long terminal repeat controlling transcription of the β-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of β-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to β-galactosidase activity and cell survival. For maximum transfection of cells, 10 μg/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling β-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.

Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1-induced apoptosis in primary cultures of hepatocytes

Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death. (Hepatology 1996 May;23(5):1200-7)

Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1-induced apoptosis in primary cultures of hepatocytes.

Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death.

Epidermal growth factor, but not hepatocyte growth factor, suppresses the apoptosis induced by transforming growth factor-beta in fetal hepatocytes in primary culture

We studied whether the TGF-β-induced apoptosis in fetal hepatocyte primary cultures may be modulated by the presence of mitogenic stimuli, such as EGF or HGF. EGF prevented cell death, showing a dose dependence that was identical to that observed for its effect on DNA synthesis stimulation. HGF, in contrast, had no effect, even at high concentrations. EGF blocked apoptosis, since in the presence of this factor cells did not show DNA fragmentation. Moreover, EGF, but not HGF, blocked c- fos induction associated with the apoptotic process induced by TGF-β in these cells.

Apoptosis Induced by Transforming Growth Factor-β in Fetal Hepatocyte Primary Cultures: INVOLVEMENT OF REACTIVE OXYGEN INTERMEDIATES

Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes.

DNA adducts and P450 induction in human, rat and avian liver cells after exposure to polychlorobiphenyls

Polychlorinated biphenyls (PCBs) are industrial chemicals which have been detected in fish, birds and humans. They are known to exert marked effects on the liver. The induce hepatocellular carcinoma in rats and birds, and are suspected of being carcinogenic to humans. To better understand the genotoxic effects of PCBs, we used 32P-postlabelling to investigate DNA adduct formation, after exposure to PCBs (Aroclor 1254 and 3,′,4,4′-tetrachlorobiphenyl), in primary cultures of fetal hepatocytes from two animal species and in a human cell line (Hep G2). We also studied the induction of 7-ethoxyresorufin- O-deethylase (EROD) in these PCB-treated cells. The three cell types used are are known to express different cytochrome P450 families. The aim was to see whether a correlation could be established between EROD activity (a CYP1A1-related activity) and DNA adduct formation. DNA adducts were found in all three models after exposure to 50 μM 3,3′,4,4′-tetrachlorobiphenyl. The number of adducts was higher in quial hepatocytes (37 adducts per 10 9 nucleotides) than in rat hepatocytes or Hep G2 cells (20 adducts per 10 9 nucleotides in both cases). The major adduct was the same in all three cell types, but some adducts were found in only one or two species. These inter-species differences probably reflect metabolic differences leading to different ultimate carcinogens. Exposure to Aroclor 1254 failed to produce significant levels of DNA adduct, suggesting that pre-treated cells are required to magnify Aroclor 1254 metabolism. No correlation was found between adduct formation and the level of EROD induction.

Transforming growth factor β modulates growth and differentiation of fetal hepatocytes in primary culture

Fetal hepatocytes in primary culture are cells capable to carry out both proliferation and differentiation processes simultaneously. Previous studies have shown that these cells respond to mitogens, such as hepatocyte growth factor (HGF) or epidermal growth factor (EGF), inducing the expression of early genes, such as fos and myc. The transforming growth factor-beta (TGF-beta) family is one of the most influential groups of growth and differentiation factors. In this report, we show that TGF-beta 1 inhibits fetal hepatocyte proliferation, arresting these cells at G1 phase of the cell cycle. In addition, TGF-beta down-regulates the mitogen-induced myc early expression. However, TGF-beta has no effect on the expression of other protooncogenes, such as fos and H-ras. In addition to its inhibitory role on fetal hepatocyte growth, TGF-beta increases the mRNA levels of fibronectin, an extracellular matrix protein, and maintains the expression of some liver specific genes, such as albumin and alfafetoprotein, above control values. The analysis of the expression of some hepatocyte transcriptional factors has shown that TGF-beta increases HNF1 alpha and HNF1 beta mRNA levels. We conclude that TGF-beta may modulate liver growth and differentiation throughout fetal development.

Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes.

The biosynthesis of estrogens is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters through the use of alternative splicing mechanisms. Thus, transcripts containing various 5'-untranslated termini are present in human placenta and other fetal tissues, ovary, brain, and adipose stromal cells. Sequence corresponding to untranslated exon 1.4 is present in 5'-termini of transcripts expressed in adipose tissue and fetal liver, as well as adipose stromal cells in primary culture in the presence of dexamethasone and fetal calf serum (FCS). Identification of hormone-responsive, tissue-specific promoter regions, as well as growth factor-response elements upstream of exon 1.4, may provide insight into the regulation of estrogen biosynthesis in adipose tissue, which is implicated in the development of breast and endometrial cancer. The goals of the present study were to define the 1.4 promoter region with respect to the start of transcription and to characterize the region(s) responsible for conferring glucocorticoid responsiveness on aromatase expression. The transcription initiation site was identified by means of primer extension and S1 nuclease protection analyses. No TATA-like sequence was evident upstream of this site. Various deletion mutations of the upstream flanking region of exon 1.4 and including part of exon 1.4 were made using polymerase chain reaction or restriction enzyme digestion. The genomic fragments were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into adipose stromal cells and fetal hepatocytes in primary culture in medium containing FCS with or without dexamethasone. The -560/+10 base pair (bp) construct expressed CAT activity after a putative silencer element was deleted, and expression was induced by dexamethasone about 3-fold. Transfection of the -330/+170 bp construct, which contains an upstream glucocorticoid response element (GRE) as well as an Sp1-like sequence in untranslated exon 1.4, resulted in an 8-fold stimulation of expression of CAT activity by dexamethasone. The upstream GRE as well as the Sp1-like sequence in untranslated exon 1.4 were mutated separately, and together, to further confirm whether the GRE or Sp1 binding site play a role in the regulation of promoter 1.4-driven transcription. Mutation of either the GRE or Sp1 binding site, or both, in the -330/+170 bp construct, resulted in loss of dexamethasone-induced CAT reporter gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)