Embryonic Liver Cells Research Articles

Pleiotrophin/Heparin-Binding Growth-Associated Molecule as a Mitogen of Rat Hepatocytes and Its Role in Regeneration and Development of Liver

Previously pleiotrophin (PTN) was identified among proteins secreted by Swiss 3T3 cells as a mitogen for cultured adult rat hepatocytes. The present study showed that the growth of rat hepatocytes was enhanced when cultured with rat hepatic stellate cells (HSCs). HSCs expressed PTN mRNA and secreted its protein in the co-cultures. Recombinant PTN enhanced the growth of hepatocytes in culture, suggesting that HSCs stimulate the growth of hepatocytes through the action of PTN. To know the biological role of PTN in the growth of hepatocytes in vivo, we examined the expression of PTN in four regeneration models of adult liver and embryonic liver of rat. The expression of PTN mRNA in the liver was markedly up-regulated by the treatment with D-galactosamine (GalN) or with acetylaminofluorene followed by partial hepatectomy. HSCs expressed PTN mRNA in response to GalN treatment and its protein was found on hepatocytes. The mRNA expression of N-syndecan, a PTN receptor, was up-regulated in GalN-treated hepatocytes. The mesenchymal cells in the septum transversum enclosing the embryonic liver, but not embryonic HSCs, expressed PTN mRNA. We suggest that PTN is secreted from activated adult HSCs and embryonic mesenchymal cells as a mitogen of parenchymal cells in adult and embryonic liver, respectively.

Expression of HNF4alpha isoforms in mouse liver development is regulated by sequential promoter usage and constitutive 3' end splicing.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is essential for the establishment and maintenance of liver-specific gene expression. The HNF4alpha gene codes for several isoforms whose developmental and physiological relevance has not yet been explored. HNF4alpha1 and HNF4alpha7 originate from different promoters, while alternative splicing in 3' leads to HNF4alpha2 and HNF4alpha8, respectively. HNF4alpha7/alpha8 were abundantly expressed in embryonic liver and fetal-like hepatoma cells. HNF4alpha1/alpha2 transcripts were up-regulated at birth and represented the only isoforms in adult-like hepatoma cells. In line with its expression profile, HNF4alpha7 activated more avidly than HNF4alpha1 reporter plasmids for genes that are expressed early. The expression patterns of both isoforms together with the differences observed in their transcriptional activities provide elements accounting for fine-tuning of the activity of HNF4alpha. The sequential expression of HNF4alpha7/alpha8 and HNF4alpha1/alpha2 during mouse liver development is the only modification in liver-enriched transcription factors thus far recorded, which parallels the transition from the fetal to the adult hepatic phenotype.

Serum after Partial Hepatectomy Stimulates iNOS Gene Transcription via Downstream NF-κB Site

It has been known that the expression of inducible nitric oxide synthase (iNOS) is up-regulated during hepatic regeneration. The present study characterized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) in vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-α (TNF-α) antibody in BNL CL.2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-κB-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of NF-κB complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcriptional activation of iNOS promoter by post-PH serum. Furthermore, site-directed mutational analysis of the two NF-κB sites individually or in combination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-κB site, but not by upstream NF-κB site. Taken together, these results suggest that the downstream NF-κB site acts as an essential component for the iNOS expression by post-PH serum during hepatic regeneration.

Does the developing liver inhibit early lung growth in congenital diaphragmatic hernia?

It has been hypothesised that the liver induces lung hypoplasia in congenital diaphragmatic hernia (CDH) by non-compressive intrathoracic growth rather than traditional mass herniation. Utilising a co-culture system, we tested the capacity of liver cells to inhibit lung growth by contact rather than compression. Heart, liver, and lungs were microdissected from normal rat embryos (n > 20 from at least three litters) on day 13.5 of gestation. Monolayer cultures of enzymatically dispersed livers and hearts were established at the same cell density. Lung primordia were cultured in direct contact with hepatic cells or partitioned from them by a permeable polytetrafluoroethylene membrane. This permits the contributions of diffusable factors and cell contact to be distinguished. Lungs were similarly cultured in direct contact with or partitioned from cardiac cells. Lungs cultured in isolation served as further controls. Daily inspection permitted assessment of in-vitro lung growth. Growth of lungs in direct contact with hepatic cells was equivalent to that of lungs partitioned from liver cells. Lungs in direct contact with cardiac cells and lungs partitioned from cardiac cells were also not inhibited compared to lungs cultured in isolation. Early lung development is thus not inhibited by humoral or contact-mediated interactions with embryonic liver cells. Lung hypoplasia in CDH is therefore unlikely to originate from contact inhibition with the developing liver. An intrinsic pulmonary defect may better explain hypoplastic lung development in CDH.

TRAIL and FasL induced apoptosis in hepatocytes, embryonal liver cells and hepatoma cell lines

TRAIL and FasL induced apoptosis in hepatocytes, embryonal liver cells and hepatoma cell lines

Trail and fasl induced apoptosis in hepatocytes, embryonal liver cells and hepatoma cell lines

Trail and fasl induced apoptosis in hepatocytes, embryonal liver cells and hepatoma cell lines

New retroviral vectors for tight regulated gene expression in hepatoma and embryonal liver cells

New retroviral vectors for tight regulated gene expression in hepatoma and embryonal liver cells

New retroviral vectors for tight regulated gene expression in hepatoma and embryonal liver cells

New retroviral vectors for tight regulated gene expression in hepatoma and embryonal liver cells

Enhancement of gene delivery by an analogue of α-MSH in a receptor-independent fashion

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)–DNA complexes with [Nle4,D-Phe7]-α-MSH(4–10), a pseudo-peptide analogue of α-melanocyte stimulating hormone (α-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an α-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine–DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an α-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle4,D-Phe7]-α-MSH(4–10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

Effect of Leptin and Insulin on Chick Embryonic Muscle Cells and Hepatocytes

In the present study we used the primary cultures of chick embryonic muscle and liver cells as a model for potential mutual combination effects of leptin and insulin, respectively. The influence of both hormones on the proliferation and protein synthesis was dose-dependent and related to the age of embryos from which the cells were isolated. Leptin (10 and 100 ng/well) increased the proliferation (estimated by DNA content and incorporation of labeled thymidine into DNA) and protein synthesis (determined by incorporation of labeled leucine into proteins) of muscle cells. The effect of leptin and insulin in muscle cells was similar. In younger embryo (11-day-old) the lower dose of leptin was more effective than the higher one compared to the insulin effect. Mutual effects of leptin and insulin were neither additive nor synergistic and were equivalent to the effects of individual hormones. In hepatocytes the influence of leptin was dependent on the age at which the cells were isolated (11- and 19-day-old embryos). The presence of insulin neither potentiated nor inhibited the effect of leptin.

Effect of gastrin and anti-gastrin antibodies on proliferation of hepatocyte cell lines.

Gastrin (G-17) and its precursor glycine-extended gastrin (G-17-gly) have been shown to be trophic to some gastrointestinal tumors. This in vitro study assessed the effect of G-17, G-17-gly, anti-gastrin antibodies (anti-G-17), and the CCK-B receptor antagonist PD135,158 on three hepatoma cell lines (PLC/PRF/5, HepG2 and MCA-RH7777) and an embryonic liver cell line (WRL68). The pancreatic adenocarcinoma cell line AR42J was used as a positive control. G-17 and G-17-gly caused significant proliferation of AR42J and WRL68 cell lines. G-17-gly but not G-17 induced significant proliferation of the PLC/PRF/5 cell line. Anti-G-17 and PD135,158 significantly inhibited unstimulated AR42J and WRL68 cell lines. Anti-G-17 also inhibited the proliferative effects of G-17 and G-17-gly on AR42J, WRL68, and PLC/PRF/5 cell lines, whereas PD135,158 inhibited the proliferative effect of G-17 only. G-17 and G-17-gly as well as anti-G-17 and PD135,158 had no effect on HepG2 and MCA-RH77777 cell lines. It is concluded that G-17-stimulated proliferation is mediated via the CCK-B receptor and G-17-gly via a separate, as yet uncharacterized, receptor. There may therefore be a role for gastrin in embryonic hepatocellular proliferation and perhaps also in the proliferation of some hepatocellular tumors.

Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells.

Nitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the murine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3 h of treatment with IFN-gamma and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-alpha gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-gamma and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP. Electrophoretic mobility shift assay showed that SNP inhibited IFN-gamma plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-gamma plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.

A combination of lactacystin and 12-O-tetradecanoylphorbol-13-acetate enhances the release of arachidonic acid from cells in cultures: release is reduced in confluent and serum deprived cells

Lactacystin, a selective inhibitor of some proteasome activities, in the presence of 12-tetradecanoyl phorbol-13-acetate stimulates the release of arachidonic acid from the phospholipids of bovine pulmonary artery endothelial, bovine embryonic thoracic aorta smooth muscle, rat liver and rat glial cells. The enhanced release from rat liver cells is inhibited by dexamethasone, aspirin, Na salicylate and the immunosuppressive fungal metabolite, gliotoxin. The effects of aspirin plus dexamethasone are not additive, but those of gliotoxin plus dexamethasone are additive. The stimulated and unstimulated release from all four cells are decreased when these cells are grown to confluence or when they are growth arrested by serum deprivation. The unstimulated as well as the stimulated release is partially inhibited by the cytosolic phospholipase A2inhibitor, methylarachidonyl fluorophosphonate. The enhanced release is reduced in the presence of the translation and transcription inhibitors, actinomycin and cycloheximide.

Nuclear activation and translocation of mitogen-activated protein kinases modulated by ethanol in embryonic liver cells

Activation and nuclear translocation of mitogen activated protein (MAP) kinases in ethanol-treated embryonic liver cells (BNLCL2) was investigated. The relative amount of MAPK proteins, MAP kinase activity and MAPK/LDH (lactate dehydrogenase) ratios were determined in nuclear and cytosolic fractions before and after serum stimulation. In ethanol-treated cells, serum-stimulated MAPK activation was potentiated in both cytosolic and nuclear fractions. Levels of both the p42 and p44 MAPK proteins increased in nuclear fractions from cells treated with ethanol alone for 24 h. Serum-stimulated nuclear translocation of both p42 and p44 MAPK was potentiated in ethanol-treated cells. Nuclear fractions from ethanol-treated cells had a modest increase in MAP kinase activity concurrent with the increased MAPK protein levels. The ratio of MAPK/LDH increased in nuclear fractions with increasing concentrations of ethanol and after serum stimulation. This further confirmed the nuclear translocation of MAPK and also demonstrated that it is not a non-specific effect of ethanol. These results demonstrate, for the first time, that in BNLCL2 liver cells ethanol treatment has dual effects. First, ethanol triggered nuclear translocation of MAPK without causing its activation. Second, it potentiated serum-stimulated activation and translocation of MAPK in the nucleus. These findings provide a novel mechanism through which ethanol may affect cellular and nuclear processes in liver cells.

Nitric Oxide Protects Murine Embryonic Liver Cells (BNL CL.2) from Cytotoxicity Induced by Glucose Deprivation

Abstract:We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon‐gamma and lipopolysaccharide. We used sodium nitroprusside and S‐nitroso‐L‐glutathione as exogenous nitric oxide‐generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose‐free media. Endogenous nitric oxide produced by treatment of the cells with interferon‐gamma and lipopolysaccharide protected the cells from glucose deprivation‐induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, NG‐monomethyl‐L‐arginine. Exogenous nitric oxide protected the cells from glucose deprivation‐induced cytotoxicity in a concentration‐dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scarvenger, 2‐phenyl‐4,4,5,5,‐tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3′,5′‐cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3′,5′‐cyclic monophosphate‐independent pathway.

Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation.

We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide. We used sodium nitroprusside and S-nitroso-L-glutathione as exogenous nitric oxide-generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media. Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L-arginine. Exogenous nitric oxide protected the cells from glucose deprivation-induced cytotoxicity in a concentration-dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway.

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-γ (Ifn-γ) or Ifn-γ and lipopolysaccharide in Bnl Cl.2 murine embryonic liver cells: role of hydrogen peroxide

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5–40 Gy) of radiation made BNL CL.2 cells responsive to interferon-γ alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) synergized with IFN-γ in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-α alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-γ or IFN-γ plus LPS was decreased by the addition of catalase, suggesting that H 2O 2 produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-γ or IFN-γ plus LPS. Furthermore, the treatment of nonγ-irradiated BNL CL.2 cells with H 2O 2 made the cells responsive to IFN-γ or IFN-γ plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-γ via the formation of H 2O 2 in BNL CL.2 murine embryonic liver cells.

Biallelic transcription of Igf2 and H19 in individual cells suggests a post-transcriptional contribution to genomic imprinting

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3,4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

Infectious Laryngotracheitis Virus, an Alpha Herpesvirus That Does Not Interact with Cell Surface Heparan Sulfate

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4°C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.

Interferon-Gamma Alone Triggers the Production of Nitric Oxide from Serum-Starved BNL CL.2, Murine Embryonic Liver Cells

A previous study has demonstrated that both interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-γ alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-γ. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-γ in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-γ- tumor necrosis factor-α (TNF-α), or LPS in combinations, only the combination of IFN-γ and LPS produced more NO than that produced by IFN-γ alone. The production of NO by the cells stimulated with IFN-γ or IFN-γ plus LPS was blocked by the addition of NG-monomethyl-L-arginine (NGMMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-γ alone or IFN-γ plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-γ and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.