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Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na + -taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology.

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background & Aims: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro., Methods: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation., Results: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo., Conclusions: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The development of human cell models stably expressing functional properties of the in vivo cells they are derived from for predicting toxicity of chemicals is a major challenge. For mimicking the liver, a major target of toxic chemicals, primary hepatocytes represent the most pertinent model. Their use is limited by interdonor functional variability and early phenotypic changes although their lifespan can be extended not only by culturing in a 2D dimension under sophisticated conditions but also by the use of synthetic and natural scaffolds as 3D supporting templates that allow cells to have a more stable microenvironment. Hepatocytes derived from stem cells could be the most appropriate alternative but up to now only liver progenitors/hepatoblasts are obtained in vitro. A few hepatocyte cell lines have retained a variable set of liver-specific functions. Among them are the human hepatoma HepaRG cells that express drug metabolism capacity at levels close to those found in primary hepatocytes making them a suitable model for both acute and chronic toxicity studies. New screening strategies are now proposed based on miniaturized and automated systems; they include the use of microfluidic chips and cell chips coupled with high content imaging analysis. Toxicogenomics technologies (particularly toxicotranscriptomics) have emerged as promising in vitro approaches for better identification and discrimination of cellular responses to chemicals. They should allow to discriminate compounds on the basis of the identification of a set of markers and/specific signaling pathways.

Functional expression of both sinusoidal and canalicular hepatic drug transporters was investigated in the highly differentiated human hepatoma HepaRG cell line and also, for comparison, in primary human hepatocytes and in the hepatoma HepG2 cell line. Using RT-qPCR assays, differentiated HepaRG cells were found to display a pattern of transporter expression close to that found in primary human hepatocytes, i.e. they exhibit substantial mRNA levels of the influx transporters OCT1, OATP-B, OATP-C and NTCP, and of the secretion transporters MRP2, MRP3, BSEP and P-glycoprotein. By contrast, expression of influx transporters was not present or very weak in HepG2 cells. Drug transport assays allowed to detect functional activities of OCT1, OATPs/OAT2, NTCP, MRPs and P-glycoprotein in differentiated HepaRG cells as in primary human hepatocytes whereas HepG2 cells only showed notable MRP and P-glycoprotein activities. In addition, expression of canalicular transporters in HepaRG cells was found to be up-regulated by known inducers of transporters such as rifampicin, phenobarbital and chenodeoxycholate acting on P-glycoprotein, MRP2 and BSEP, respectively. HepaRG cells thus exhibit functional expression of both sinusoidal and canalicular drug transporters and have retained regulatory pathways controlling transporter levels. These data, associated with the known high expression of drug metabolizing enzymes in HepaRG cells, highlight the interest of such hepatoma cells for analysing hepatic drug detoxification pathways.

Background/aims: Dendritic cells (DCs), which play a critical role during immune response, could present alternative differentiation patterns depending on tissue microenvironment. Our aim was to examine the influence of hepatic microenvironment on human monocyte differentiation into DCs., Methods: Cytology, immunophenotyping, cytokine production and T-cell activation were analyzed in DCs differentiated from human monocytes co-cultured with rat liver epithelial cells (RLEC) or human cells from various tissue origins and compared to control DCs obtained on plastic with GM-CSF/IL-4., Results: RLEC environment promotes DC differentiation in the presence of IL-4 without GM-CSF. These DCs evidence similar expression of MHC-II, co-stimulatory and adhesion molecules than control DCs, but distinct lineage markers defining a CD11c+/CD14+/CD123+ DC subset. This phenotype is common to DCs from RLEC and human liver environment and differs from that obtained with skin or intestine environments. Functionally, they produce IL-10 but not IL-12p70 and favor IL-4/IL-10 secretion by T-cells rather than IFN-gamma., Conclusions: Our results confirm that tissue niches modulate DC differentiation and demonstrate that hepatic environment influences monocyte differentiation into a DC subset directing Th2 response, a key data for understanding the specialized immune response in liver. They also make RLEC co-culture system useful for studying liver DC functions.

Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me(2)SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes and in vivo thioacetamide-induced liver failure, we showed that Me(2)SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me(2)SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST M1/2 and GST Alpha1/2 dropped under apoptotic conditions; furthermore transfection of GST M1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me(2)SO-protected hepatocytes. Together, these results demonstrate that Me(2)SO-mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection.

Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.

We hypothesized that glutathione transferases could be induced and may participate to cellular defenses against the oxidative stress occurring during liver regeneration. Here, we evidenced that murine GSTA1 (mGSTA1), A4, Pi, and Mu are up-regulated during mouse liver regeneration, exhibiting a biphasic pattern of induction correlating early G(1) phase and G(1)/S transition of the cell cycle. Using confocal microscopy immunolocalization and subcellular fractionation, mGSTA4 was demonstrated in both mitochondria and cytosol and found preferentially increased in cytosol during liver regeneration. In addition, mGSTA4 was induced in vivo and in cultured hepatocytes by tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and epidermal growth factor (EGF), factors that play crucial roles in hepatocyte survival and proliferation during liver regeneration. However, the mitogenic effect of EGF was not responsible for the induction of mGSTA4. In transient transfections, IL-6 and EGF, but not TNFalpha, transactivated the human GSTA4 (hGSTA4) promoter cloned upstream of the luciferase reporter gene suggesting that IL-6 and EGF up-regulated hGSTA4 at a transcriptional level, whereas TNFalpha could rather act at a post-transcriptional level. The inhibition of phosphoinositide 3-kinase, p38 MAPK, and MEK/ERK signaling pathways, using specific inhibitors, prevented EGF-dependent induction of mGSTA4 and transactivation of hGSTA4 promoter. Altogether, these data favor the conclusion that, in regenerating hepatocytes, several GST isoforms are induced and that cytokines TNFalpha and IL-6 and survival factor EGF positively regulate mGSTA4 via survival signaling pathways.

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1- to 3-day-old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny., (Copyright 2001 Wiley-Liss, Inc.)

gamma-Glutamyl transpeptidase (GGT), a major enzyme of glutathione (GSH) homeostasis, is often used as a biliary marker to follow the differentiation of hepatic precursor cells. The expression of the GGT gene is driven by different promoters and yields multiple mRNAs, depending on the cell type or the stage of differentiation. In the present study, we analyzed the GGT mRNA expression pattern by quantitative reverse transcriptase-polymerase chain reaction or by in situ hybridization i) in the liver, in vivo, at early stages of development; ii) in oval cells, which proliferate and differentiate into hepatocytes in response to galactosamine injury in vivo; and finally, iii) during hepatoblast differentiation, in vitro. We show that GGT gene transcription originates from promoters P3, P4, and P5 in rat hepatic precursor cells. Differentiation of these cells induces profound alterations in GGT gene expression, leading to extinction of promoters P4 and P5, when they differentiate into the hepatocytic pathway, and to extinction of promoters P3 and P5 when they differentiate into the biliary pathway. This diversity in GGT mRNA expression provides unique molecular probes to follow hepatic precursor cell differentiation. Furthermore, the identification of factors governing GGT P5 and P4 promoter expression should provide further insight into the molecular events that occur as the liver precursor cell differentiates into the hepatic lineages.

In the adult liver, the plasma membrane Liver Regulating Protein (LRP) has been found to participate in the recognition signals between hepatocytes and rat liver epithelial cells (RLECs) and to play a critical role in the functional stability of hepatocytes. Here, we report the involvement of LRP in hematopoiesis. First, LRP was evidenced in the hematopoietic fetal rat liver and in adult rat bone marrow. Then, the involvement of LRP in adult rat and human hematopoiesis was investigated in coculture of hematopoietic cells with RLECs. In the rat, RLECs sustain long-term production of hematopoietic cells and committed progenitors as well as bone marrow stroma cells. These effects can be specifically blocked by addition of anti-LRP antibodies to both culture systems. The supportive activity of RLECs on human hematopoiesis was assessed by comparison to the reference MS-5 cell line from both unifractioned and purified CD34(+)CD38(-) hematopoietic cells. Taken together, our results argue for the involvement of LRP in heterotypic interaction signals mediated by RLEC and bone marrow stromal cells which lead to hematopoietic primitive progenitors development. They might lead to interesting applications in fundamental research, pharmacology and therapeutics.

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis., (Copyright 2000 Academic Press.)

Primary human hepatocytes were used to develop a culture model for in vitro propagation of hepatitis C virus (HCV). Production of positive- strand full-length viral RNA in cells and culture supernatants was monitored by PCR methods targeting three regions of the viral genome: the 5' NCR, the 3' X-tail and the envelope glycoprotein E2. De novo synthesis of negative-strand RNA was also demonstrated. Evidence for a gradual increase in viral components over a 3 month period was obtained by two quantitative assays: one for evaluation of genomic titre (quantitative PCR) and one for detection of the core antigen. Production of infectious viral particles was indicated by passage of infection to naive hepatocyte cultures. Reproducibility of the experiments was assessed using cultures from three liver donors and eleven sera. Neither the genotype, nor the genomic titre, nor the anti-HCV antibody content, were reliable predictive factors of serum infectivity, while the liver donor appeared to play a role in the establishment of HCV replication. Quasispecies present in hepatocyte cultures established from three different liver donors were analysed by sequencing hypervariable region 1 of the E2 protein. In all three cases, the complexity of viral quasispecies decreased after in vitro infection, but the major sequences recovered were different. These data strongly suggest that human primary hepatocytes are a valuable model for study of persistent and complete HCV replication in vitro and for identification of the factors (viral and/or cellular) associated with successful infection.

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.

Ribavirin, a guanosine analog, used in combination with interferon alpha (IFN-alpha) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 micromol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 micromol/L ribavirin, was completely restored by the addition of 80 micromol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.

Stromal cell lines from bone marrow and other blood-forming organs including fetal liver have been found to support hematopoiesis. In this paper, we demonstrate that rat liver biliary epithelial cells (RLEC), most likely originating from primitive bile ductules, are able to support long-term hematopoietic cell growth as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) production. RLEC have previously been shown to express a cell surface molecule named liver-regulating protein (LRP), which is involved in the long-term maintenance of hepatocyte functions in a coculture system. In addition, LRP-like molecules have been found in spleen, thymus, lymph nodes, and peripheral blood cells. In the present study, we found that hematopoietic cells and several stromal cell types from bone marrow were LRP-positive, and immunoprecipitation revealed polypeptides similar to those found in RLEC. We then investigated the biological role of LRP on hematopoiesis using short-term RLEC and bone marrow stromal cell culture systems. Addition of specific anti-LRP antibody to both systems reduced hematopoietic cell proliferation and committed progenitor production, whereas it did not directly affect the clonal proliferation and maturation of these progenitors in methylcellulose assays. Moreover, using diffusible chamber cultures that suppress direct contacts with hematopoietic cells, we observed low cell growth and no effect of monoclonal antibody (mAb) L8 treatment. All these results strongly argue for a cell proximity signal mediated by RLEC and bone marrow stromal cells and for the involvement of LRP-like molecules in this signal in liver and bone marrow hematopoietic function.

Cell-based therapy could represent an alternative treatment to orthotopic liver transplantation in acute liver failures and for the correction of genetic defects of various enzymatic functions. Several recent studies indicate that hepatocytes injected either in the spleen or in portal vein can restore liver-specific function(s) in animal model systems. Alternatively, an extracorporal hybrid bioartificial liver might provide liver-specific functions, maintain the patient alive and allow spontaneous recovery of the patient's own liver, or act as a bridge toward liver transplantation in acute liver failures. Various drawbacks of devices such as flat culture substrates, hollow-fiber bioreactors or microcarriers led us to develop a reliable extracorporeal bioartificial liver based on alginate-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect which results in the lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We isolated porcine hepatocytes by collagenase perfusion of the liver and cells were immobilized within alginate beads which were subsequently inoculated in a bioreactor. Porcine hepatocytes expressed liver-functions at high levels, particularly those involved in detoxification and biotransformation processes; they were immunoisolated from immunoglobins and could be cryopreserved. This system represents a promising tool for the design of an extracorporeal bioartificial liver in human beings.

Several novel differentiated cell lines have been derived from a human hepatocarcinoma named HBG. Analysis of their functional properties evidenced a gradual differentiation process as they became confluent and a remarkable stability of the whole quiescent population for at least 6 weeks. However, when replated at low density after several weeks of quiescence, the differentiated cells were able to rapidly reverse to active proliferation, accompanied by transient dedifferentiation. Demonstration that the differentiated hepatic cells were growth-arrested in G1 phase was provided by the increased number of cells with 2C DNA content and decreased expression of S-phase markers. Characteristic features of oncogenes and cell cycle genes were defined during the differentiation process: (a) a biphasic expression of c-myc, with the latter wave covering the quiescence period; (b) opposite kinetics of c-Ki-ras and of N-ras expression with a pattern of changes paralleling that of c-myc; and (c) a decrease of cyclin D1 protein expression and of the cyclin D1-associated kinase activity. The mechanisms by which quiescent differentiated cells might reinitiate active proliferation were analyzed by studying several genes involved in cell growth and death regulation. We found: (a) a point mutation and loss of the specific activity of the tumor suppressor gene p53 without alteration of the apoptotic response to transforming growth factor beta1; (b) a gradual decrease of retinoblastoma protein, which was constantly present, mainly in a hyperphosphorylated form; and (c) an increase of cyclin-dependent kinase inhibitor p27 expression in confluent differentiating cells, as expected, whereas, surprisingly, a disappearance of the p21 protein was observed in parallel. These data may reflect specific mechanisms of cell cycle regulation in liver parenchymal cells through which these cells can proceed to control their reversible differentiation program.

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver-specific mRNAs and DNA-binding activity of ubiquitous and liver-enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3-methylcholanthrene-inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA-binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF-1 and HNF-4-like DNA-binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1-like DNA-binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer-binding protein and HNF-3-like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis-B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis-B virus replication and virion production, in agreement with the notion that HNF-1 activity is necessary for viral replication. We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis-B virus replication.

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Regenerating liver, hepatocyte primary cultures and differentiated hepatoma cell lines are widely used to study the proliferation/differentiation/apoptosis equilibrium in liver. In hepatocytes, priming factors (TNF alpha, IL6) target G0/G1 transition while growth factors (HGF, EGF, TGF alpha) control a mid-late G1 restriction point. A characteristic pattern of cdk/cyclin expression is observed in hepatocytes, presumably related to their ability to proliferate a limited number of times and to undergo a reversible differentiation. Interestingly, cell-cell interactions between hepatocytes and liver biliary cells in co-cultures, result in a cell cycle arrest in mid G1 of hepatocytes which are insensitive to mitogens. Apoptosis exists in hepatocytes but is still poorly documented. However, hepatoma cell lines stimulated by TGF beta undergo cell death in a p53-independent pathway. In conclusion, the interplay of growth and apoptosis regulators and cell-cell interactions control the proliferation/differentiation/apoptosis balance which is a specific feature of hepatocytes.

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene spermatocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and spermatids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interaction-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocultures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte- and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.

Embryonic (E) 12 rat liver epithelial cells constitute a population of bipotential progenitor cells which can differentiate along the hepatocyte (Hep) or biliary epithelial cell (BEC) lineage in primary culture. In the present study, E12 cells were seeded on fibronectin-coated substratum and exposed to sodium butyrate (SB) for various exposure times, and the emergence of the Hep or BEC phenotype was monitored by following the variations in albumin production and assessing the appearance of the two surface-exposed markers HES6 and BDS7. Continuous exposure to SB resulted into a major reduction in albumin production and, at Day 9 postseeding, few cells coexpressed BDS7 and albumin. When cells were exposed to SB for 5 days and then cultured for an additional 5 days without SB, they massively express BDS7, but very little HES6. Moreover, the reverse sequence, i.e., 5 days without SB followed by 5 days with it, led to the appearance of many cells expressing both HES6 and BDS7. These results indicate that progenitors committed preferentially along the Hep lineage still have the option to switch to BECs, at a transitional stage that we refer to as a "differentiation window."

Id proteins are known as negative regulators of differentiation in various cell types. In this report, we show that the Id-1 gene was down-regulated during the development of rat liver. No Id-1 transcripts were detected in terminal differentiated hepatocytes. We have studied Id-1 expression in proliferating hepatocytes using an in vivo model of liver regeneration after partial hepatectomy and an in vitro growth factor-stimulated hepatocyte culture system. Strong activation of Id-1 was observed in mid-late G1 of the hepatocyte cell cycle at a time corresponding to a mitogen restriction point. These observations suggest that Id-1 is involved in the control of proliferation and differentiation in liver cells.

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.

The liver epoch like other tissue epochs occurs after that different events have induced heterogeneity in embryonic cells which results in distinct evolutionary processes. These events and those of organogenesis like "induction" are deeply dependent on cell-cell communications. Cell-cell interactions involve either soluble factors (hormones, growth factors), extracellular matrix or plasma membrane proteins responsible for cell-cell recognition and/or adhesion. All these plasma membrane signals are transduced to the nucleus and modulate the expression of groups of genes. To be functionally stable along the adult stage the liver has to maintain an ordered activity of cell renewal. This balance between proliferation and differentiation is, at least in part, controlled by cell-cell communications. Therefore, it is not surprising that intercellular communications are altered during hepatocarcinogenesis. They involve changes in the distribution of junctions, in the amounts of extracellular matrix components and/or growth factors which all result in modifying the differentiation/proliferation balance. Cell culture models have been used for these different studies; new in vitro systems should be set up in the near future by taking advantage of the targeted hepatocarcinogenesis in transgenic mouse.

In normal adult liver, hepatocytes are arrested in G0, and they rapidly respond to a mass loss by a definite number of divisions. Thus, taking advantage of the in vivo regenerative capacity of the liver following partial hepatectomy, we have analyzed both expression and activation of p34cdc2 (= cdk1) and p33cdk2 through the cell cycle, particularly during the long lasting G1 phase and in the G1/S transition. While p33cdk2 is constantly expressed during the cell cycle, p34cdc2 is completely absent in resting hepatocytes and remains unexpressed for up to 20 h after partial hepatectomy, a time period corresponding to the G1 phase and G1/S transition, and then accumulates in the S, G2, and M phases. No histone H1 kinase activity is detected during the G1 phase, while two peaks of p34cdc2 kinase activity are observed during the S and M phases and only one peak of p33cdk2 kinase activity in the S phase. p34cdc2 forms complexes with both cyclins A and B while p33cdk2 is associated with cyclin A only. Surprisingly, cyclins E and D1 are present in resting liver and with modest variation throughout the cell cycle. Taken together, our data provide evidence that the pattern of G1-associated proteins in hepatocytes during liver regeneration is distinct from that described in other cell types.

Interleukin 4 (IL4) has been shown to exhibit anti-inflammatory effects by inhibiting the secretion by monocytes of proinflammatory cytokines such as interleukin 1 (IL1), interleukin 6 (IL6), and tumor necrosis factor (TNF) and by inducing the secretion of the IL1 receptor antagonist. We investigated the role of this cytokine on the production of acute-phase proteins in primary human hepatocyte cultures. Cells were exposed to either IL4 and/or IL6, the most potent mediator of hepatic acute phase proteins. IL4 led to decreased production of haptoglobin, C-reactive protein and albumin while alpha 1-antitrypsin and fibrinogen remained unaffected. These inhibitory effects of IL4 were also observed at the mRNA level. In addition, IL4 inhibited the IL6-induced production of haptoglobin although it had no effect on the induced C-reactive protein and fibrinogen. Our results demonstrate that IL4 can affect the production of a subset of acute-phase proteins by human hepatocytes and can antagonize some of the effects of IL6. These observations reinforce the notion that IL4 can be considered as an anti-inflammatory cytokine.

Stathmin is a 19 kDa cytoplasmic phosphoprotein proposed to act as a relay for signals activating diverse intracellular regulatory pathways. After two-thirds partial hepatectomy, the concentration of stathmin reached a peak between 48 and 72 hours, comparable to the levels observed in neonatal liver, at about 10 times the basal adult level. Stathmin then decreased to basal levels within 7 days, more rapidly than during postnatal tissue development (7 weeks), with no detectable change in its phosphorylation state. Interestingly, the mRNA for stathmin reached a peak much earlier than the protein, at 24 hours posthepatectomy, and decreased to a still detectable level until 96 hours after hepatectomy. Altogether, the present results further support the generatility of the implication of stathmin in regulatory pathways of cell proliferation and differentation during normal tissue development and posttraumatic regeneration.

The liver epoch like other tissue epochs, occurs after that different events have induced heterogeneity in embryonic cells which result in distinct evolutionary processes. These events and those of organogenesis like "induction", are deeply dependent on cell-cell communications. Cell-cell interactions involve either soluble factors (hormones, growth factors), extracellular matrix or plasma membrane proteins responsible for cell-cell recognition and/or adhesion. All these plasma membrane signals are transduced to the nucleus and modulate the expression of groups of genes. To be functionally stable along the adult stage the liver has to maintain an ordered activity of cell renewal. This balance between proliferation and differentiation is at least in part, controlled by cell-cell communications. Therefore, it is not surprising that intercellular communications are altered during hepatocarcinogenesis. They involve changes in the distribution of junctions, in the amounts of extracellular matrix components and/or growth factors which all result in modifying the differentiation/proliferation balance. Cell culture models have been used for these different studies; new in vitro systems should be set up in the near future by taking advantage of the targeted hepatocarcinogenesis in transgenic mouse.

We have previously succeeded in infecting normal human hepatocyte primary cultures with hepatitis B virus (HBV). However, infection was subject to individual variations even in the presence of dimethyl sulfoxide (DMSO), which appeared to increase the amounts of viral DNA associated with the cells. In this study, we have defined conditions which enhance hepatitis B virus penetration into the cells, and we show that, under these conditions, infection of hepatocytes is always possible, regardless of their individual origin. We have found that addition of polyethylene glycol (PEG) to the cultures maintained in the presence of 2% DMSO at the time of infection markedly increased the infection process and made it highly reproducible. Moreover, both the tissue and species specificity were preserved. This increased HBV infection was correlated to increased amount of internalized HBV DNA and to enhanced attachment of the virions. From these results it may be assumed that PEG could favor a better interaction between virions and cells, resulting in an activated internalization of bound viral particles. Data also show that adult human hepatocyte primary cultures, which are not equally susceptible to HBV infection, are consistently capable of viral replication when the viral genome has entered the cells. This suggests that the main limitation of the in vitro HBV infection lies in the ability of human hepatocytes to specifically bind the viral particles.

The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.

Cytokeratins CK 8 and CK 18 are the two keratins expressed in the liver. They are known to undergo extensive changes in expression with alteration of the hepatocyte phenotype in vitro. In this study, we have investigated the variation in levels of these two cytokeratins in hepatocytes selected from different situations in vivo. The amounts of corresponding transcripts were compared; cytokeratin 8 and 18 mRNAs were present at similar levels in hepatocytes freshly isolated from adult liver and, unexpectedly, from 17-day-old foetuses and newborn rats, whereas they were markedly higher in regenerating hepatocytes isolated early after partial hepatectomy. In order to investigate whether the different factors that can promote hepatocyte differentiation also produce a similar set of cytoskeletal changes, we have analysed both the expression and the distribution of cytokeratins in hepatocytes under different culture conditions allowing modulation of differentiation. Establishment of cell-cell contacts and addition of glucocorticoids were used as two modulating factors. Coculturing hepatocytes with rat liver epithelial cells (RLEC), which favours active expression of liver-specific genes, resulted in a gradual decline of cytokeratin mRNAs, whereas pure hepatocyte cultures, which exhibit rapid phenotypic changes, expressed increasing levels of CK 8 and CK 18 transcripts. Furthermore, intracellular CK distribution was dramatically modified in parallel: the CK-positive material formed a fine network of fibrils uniformly distributed in the cytoplasm of hepatocytes in pure culture, whereas in cocultured cells CK immunofluorescence appeared principally located at the cellular periphery and it was regularly arranged in long fibrils just beneath the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal human adult hepatocytes were examined for their ability to synthesize and secrete factor V using primary culture. The culture medium contained both factor V and factor Va as determined by bioassay and activation experiments. Immunoprecipitation of newly synthesized labelled factor V showed the presence of both native factor V (m.w. 330,000) and two fragments of respective molecular weight 300,000 and 265,000. Northern blot analysis revealed the presence of a single 7 kb factor V mRNA in cultured human hepatocytes as in liver biopsies, together with fibrinogen beta and albumin transcripts. Relative levels of factor V, fibrinogen beta and albumin mRNAs differed when the cells cultured, suggesting that expression of the three corresponding genes might in part be independently regulated. Furthermore, addition of glucocorticoids enhanced factor V and fibrinogen beta mRNA levels 1.6- and 5-fold respectively, but did not significantly increase that of albumin. These results provide evidence that human hepatocytes actively participate in the synthesis of plasma factor V and constitute a valuable model to study the common and specific regulations involved in the control of the expression of this gene in human liver.

Insulin-like growth factor II is believed to play an important role in fetal growth and development. The insulin-like growth factor II gene expression is tissue specific and developmentally regulated. We have previously shown an enhanced level of insulin-like growth factor II messenger RNA and protein in human hepatocellular carcinomas. This led to the suggestion that hepatocytes might be involved in insulin-like growth factor II expression. However, previous studies based on in situ hybridization only showed insulin-like growth factor II messenger RNA in liver sinusoidal cells. This paper reports on the analysis of the expression of insulin-like growth factor II and insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs in vivo in isolated rat hepatocytes at various stages of development and in vitro in adult rat hepatocytes primary culture. Our study indicates that isolated rat hepatocytes synthesize insulin-like growth factor II messenger RNA with a switch between fetal and adult messenger RNA profiles occurring 21 days after birth. In addition, adult rat hepatocytes in culture expressed insulin-like growth factor II messenger RNA and protein. Insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs were also detected. Therefore these results are consistent with the hypothesis that insulin-like growth factor II acts as an autocrine growth factor for hepatocytes in addition to having a paracrine effect. They also indicate that primary culture of hepatocytes is a good model for further studies on insulin-like growth factor II gene regulation.

Laminin deposition is increased in fetal liver and in a variety of liver diseases, including the development of carcinoma. We investigated the role of the hepatocyte in the synthesis of both laminin and its 32- to 67-kd receptor in normal adult, fetal, and diethylnitrosamine-treated rat livers, and in adult rat hepatocyte primary cultures. Laminin was localized by immunoelectron microscopy in the endoplasmic reticulum of hepatocytes in fetal-derived and in 18-month-old diethylnitrosamine-treated rat livers. Steady-state mRNA levels for the three chains of laminin (A, B1, and B2) and the laminin receptor (LBP-32) were examined. Northern-blot analyses showed that hepatocytes at all stages lacked the A-chain mRNA. B1-chain mRNA was undetectable in normal adult hepatocytes, while significant levels of B1-chain mRNA were found in fetal hepatocytes and in adult hepatocyte primary culture. In hepatocytes from diethylnitrosamine-treated rats, B1-chain mRNAs were abundant and were present mainly in nodular formations rather than in the nontumorous areas. B2-chain mRNAs were barely detectable in either normal adult or fetal hepatocytes. In diethylnitrosamine-treated rats, the steady-state B2-chain mRNA level was higher in nodules than in nontumorous areas. In primary culture, B2-chain mRNAs were present as early as 4 hours after adult hepatocyte seeding, and dramatically increased during the following 2 days. Only low levels of laminin-receptor (LBP-32) mRNAs were present in normal adult hepatocytes, whereas the levels were high in the fetal and in the tumor-containing livers. In diethylnitrosamine-treated rats, LBP-32 mRNAs were more abundant in nodular formations rather than in nontumorous areas. In hepatocyte primary culture, the expression of the LBP-32 mRNA dramatically increased during the first 24 hours. These results show that in hepatocytes, expression of laminin chains and its receptor LBP-32 are not coordinated and depends on the maturation of the cells. In addition, they suggest that the expression of B1 and B2 chains in adult hepatocytes is related to changes of the normal phenotype and/or the pericellular environment.

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.

We have studied tyrosine aminotransferase in the liver of adult and old rats. Thermostability and trypsin action were not modified, and no charge differences have been found by isoelectric focusing between 'adult' and 'old' enzymes. Inducibility by glucocorticoids was increased in vivo in these 27- to 31-month-old rats, but not in vitro, in cultured hepatocytes. Moreover, we have shown the absence of 'cross-reacting material' for tyrosine aminotransferase in senescent rat livers. The rapid turnover of this enzyme may explain the apparent absence of alterations during aging.