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D Bernuau

Publications and source records attributed to D Bernuau.

At least 19 recordsLinked to original sources
PubMed2006

Physical and functional cooperation between AP-1 and beta-catenin for the regulation of TCF-dependent genes.

Stabilization of cytoplasmic beta-catenin is a hallmark of a variety of cancers. The stabilized beta-catenin is able to translocate to the nucleus, where it acts as a transcriptional activator of T-cell factor (TCF)-regulated genes. beta-Catenin may cross-talk with many signalling cascades to activate target genes. Whether beta-catenin cooperates with AP-1, another transcriptional complex activated during tumorigenesis is not fully clarified. We show that beta-catenin co-immunoprecipitates with c-Jun and c-Fos. GST pull-down experiments indicate a physical association of the armadillo repeat domain of beta-catenin with the DNA-binding domain of c-Jun and of the C-terminal domain of beta-catenin with the N-terminal domain of c-Fos. Promoter studies indicate that overexpression of AP-1 activates the transcription of two beta-catenin target genes, cyclin D1 and c-myc, by a mechanism independent of the AP-1 site, and fully dependent on the TCF-binding site. We further demonstrate that AP-1/beta-catenin synergism is involved during serum-induced cyclin D1 transcriptional activation. We identify a TCF-binding site on the cyclin D1 promoter which binds in vivo a complex induced by serum, containing beta-catenin, TCF4, c-Fos, c-Jun, JunB and JunD. This novel mechanism of interaction between two signalling cascades might contribute to the potentiation of malignancy.

Animals↗
PubMed2001

Functional cooperation between JunD and NF-kappaB in rat hepatocytes.

AP-1 and NF-kappaB are rapidly activated during liver regeneration. Whether these parallel inductions have potential functional implications is not known. Isolated rat hepatocytes were stimulated with two mitogens, epidermal growth factor or hepatocyte growth factor and with tumor necrosis factor alpha, a cytokine involved in the liver regenerative response in vivo and a strong inducer of NF-kappaB. All three cytokines increased AP-1 and NF-kappaB binding to their cognate cis-element and induced a 2.5-fold activation of NF-kappaB-dependent transcription. Inactivation of AP-1 by TAM67, a dominant negative mutant of AP-1 drastically inhibited basal and cytokine-induced NF-kappaB transactivation. Overexpression of Jun D, but not of the other Jun or Fos proteins increased by threefold NF-kappaB transactivation. Functional cooperation between JunD and p65 was demonstrated in a simple Gal-hybrid system. Finally, a twofold decrease in NF-kappaB transactivation was found in hepatocytes isolated from JunD(-/-) mice compared with hepatocytes from JunD(+/+) mice. Altogether these data demonstrate a functional cooperation of p65 with JunD, a major constituent of AP-1 in normal hepatocytes.

Animals↗
PubMed2000

Potentiation of Smad transactivation by Jun proteins during a combined treatment with epidermal growth factor and transforming growth factor-beta in rat hepatocytes. role of phosphatidylinositol 3-kinase-induced AP-1 activation.

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor beta (TGF-beta)-induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-beta treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-beta-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-beta potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-beta-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-beta treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-beta responses.

Animals↗
PubMed1999

Auxiliary liver transplantation: how to improve regeneration of the native liver by surgery.

The technical factors which could influence regeneration of the native liver (NL) in auxiliary liver transplantation (ALT) for fulminant hepatic failure (FHF) are not well known. We studied NL regeneration according to the location of graft anastomosis in the recipient's portal system (superior mesenteric vein versus portal vein), and graft weight (50% reduced-size versus full-size graft) in a rat model of ALT with 80% reduction of the NL, and graft arterialization. NL regeneration was significantly more obvious when the graft was anastomosed on the recipient's superior mesenteric vein, thus establishing venous flow to the NL from the pancreas, the spleen, and the stomach, and when a full-size graft was used. The influence of portal venous flow on NL regeneration, assessed by 3H[-thymidine incorporation, was measurable as early as day 2. Both technical variables in combination resulted in significantly greater regeneration (ratio weight of NL/body weight at day 30: 2.32 +/- 0.68% versus 1.21 +/- 0.63% respectively, P = 0.02). Early preservation of portal flow to the NL is advisable to maximize NL regeneration in ALT. In any case, this regeneration is not impeded by the use of large auxiliary grafts.

Animals↗
PubMed1999

Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes.

BACKGROUND/AIMS: Stimulation of activator protein-1 (AP-1), a Fos/Jun complex, is a key event in the cell response to growth factors. We have investigated whether hepatocyte growth factor (HGF) induces differential AP-1 responses in normal and transformed rat hepatocytes, the 7777 cells. METHODS: Primocultures of isolated hepatocytes or 7777 cells were stimulated with HGF. Gene expression was evaluated by ribonuclease protection assay and Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. Identification of the proteins bound to the probes was made by supershift assays with specific antibodies. Cells were electroporated with plasmids containing an AP-1-dependent chloramphenicol acetyl transferase (CAT) gene, and CAT activity was measured 24 h after treatment with medium alone or HGF. RESULTS: In both cell types, HGF triggered the same program of jun family mRNA activation, but distinct Fos/Jun proteins accumulated in the nucleus. HGF increased DNA-binding activity to the phorbol 12-O-tetradecanoate-13-acetate responsive element (TRE) in both cell types, but distinct TRE-binding proteins were recruited in the AP-1 dimers. HGF also increased consistently binding to a cAMP responsive element (CRE) in hepatocytes only. Finally, HGF triggered TRE- and CRE-dependent gene activations in hepatocytes but TRE-dependent gene activation alone in 7777 cells. CONCLUSIONS: HGF-induced AP-1 activation leads to the formation of distinct dimers with different functional capacities in normal and transformed hepatocytes. These data suggest the importance of qualitative abnormalities of the AP-1 complex for the establishment or maintainance of a transformed phenotype.

Animals↗
PubMed1998

Inducible expression of the alpha1-acid glycoprotein by rat and human type II alveolar epithelial cells.

Alpha1-acid glycoprotein (AGP) is a major acute phase protein in rat and human. AGP has important immunomodulatory functions that are potentially important for pulmonary inflammatory response. The liver is the main tissue for AGP synthesis in the organism, but the expression of AGP in the rat lung has not been investigated. We show that AGP mRNA was induced in the lung of dexamethasone-, turpentine-, or LPS-treated rats, whereas AGP mRNA was not detected in the lung of control rats. In the lung of animals treated intratracheally with LPS, in situ hybridization showed that AGP gene expression was restricted to cells located in the corners of the alveolus, consistent with an alveolar type II (ATII) cell localization. The inducible expression of the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII cells in primary culture: maximal expression required the presence of dexamethasone. IL-1 and the conditioned medium of alveolar macrophages acted synergistically with dexamethasone. Rat ATII cells secreted immunoreactive AGP in vitro when stimulated with dexamethasone or with a combination of dexamethasone and the conditioned medium of alveolar macrophages. In vivo, in the human lung, we detected immunoreactive AGP in hyperplastic ATII cells, whereas we did not detect AGP in the normal lung. We conclude that AGP is expressed in the lung in cases of inflammation and that ATII cells are the main source of AGP in the lung.

Acute-Phase Proteins↗
PubMed1997

Hepatocarcinogenesis in woodchuck hepatitis virus/c-myc mice: sustained cell proliferation and biphasic activation of insulin-like growth factor II.

Transgenic mice carrying the c-myc oncogene under control of woodchuck hepatitis virus (WHV) DNA sequences invariably develop hepatocellular carcinoma (HCC), despite a temporally limited expression of the transgene in the neonatal liver. To better characterize the different steps of the tumorigenic process, we analyzed the liver expression of the c-myc transgene and several growth-related genes by in situ hybridization and Northern blotting. In parallel studies, proliferated changes were investigated by detection of bromodeoxy-uridine-positive S-phase nuclei and apoptosis was evaluated by in situ nick end-labeling of DNA. During the neonatal period, high levels of c-myc messenger RNAs (mRNAs) were detected in all hepatocytes, and the expression of insulin-like growth factor II (IGF II) was frequently enhanced, correlating with increased cell proliferation. Despite elevated expression of the p53 gene, no change in liver cell apoptosis was observed. After weaning, c-myc transgene expression decreased to undetectable levels in all hepatocytes, whereas proliferation decreased but remained notably higher than in age-matched controls. The expression of c-fos, c-jun, and c-H-ras was highly variable during the preneoplastic period and in the tumors, with no consistent increase compared with controls. Resurgence of c-myc transgene expression was evidenced in all cells from hyperplastic lesions and carcinomas, accompanied with frequent focal reactivation of IGF II. Thus the strong proliferative stimulus induced by the combined effects of c-myc and IGF II in the neonatal liver might initiate a process characterized by persistent, dysregulated hepatocyte proliferation, in turn greatly increasing the risk of hepatocellular transformation.

Animals↗
PubMed1997

Presence of distinct AP-1 dimers in normal and transformed rat hepatocytes under basal conditions and after epidermal growth factor stimulation.

Activation of the transcriptional regulator AP-1, a dimeric complex formed of various combinations of Fos and Jun proteins, is a key step in the cellular response to mitogens. Because different dimers are believed to display different regulatory functions, we hypothesized that transformed cells that lack normal growth constraints might display AP-1 dimers that are different from those of normal cells. We therefore compared in primary and transformed rat hepatocytes (1) the composition of AP-1 dimers under basal conditions and (2) AP-1 induction by epidermal growth factor (EGF). Under basal conditions, AP-1 contained predominantly Jun homodimers in both cell types. However, whereas normal hepatocytes contained only JunD, both JunD and JunB were present in the AP-1 complex of 7777 cells. EGF treatment triggered almost identical programs of fos and jun gene activation at the messenger RNA (mRNA) level in both cell types, with an early accumulation of c-fos, c-jun, and junB mRNAs, but no change in junD mRNA levels. In both cell types, c-Fos and Fra-1 proteins increased after EGF treatment, but differences in the induction of Jun proteins were noted, with an increase of c-Jun in hepatocytes and an increase of JunB in 7777 cells. In both cell types, activation of AP-1 DNA binding activity by EGF was accompanied by the recruitment of Fra-1 into AP-1, detected earlier in 7777 cells than in hepatocytes, and with the transient appearance of c-Fos in 7777 cells only. Finally, EGF activated AP-1-dependent transcription in 7777 cells but not in hepatocytes. These data indicate important differences in the functional activity of AP-1 in transformed hepatocytes.

Animals↗
PubMed1996

Coexpression of periportal and perivenous enzymes in rat hepatocytes after experimental bile duct ligation: comparison with intrasplenically transplanted hepatocytes.

The coexpression of normally periportal and perivenous markers has been described in heterotopically transplanted hepatocytes. To determine whether such a coexpression might also occur in hepatocytes retaining their original intrahepatic location, we compared in bile-duct-ligated livers and intrasplenically transplanted hepatocytes, the expression and distribution of the predominantly periportal glucose-phosphatase, succinate dehydrogenase, and lactate dehydrogenase, the predominantly perivenous glutamate dehydrogenase, NADPH-dehydrogenase, and beta-hydroxybutyrate dehydrogenase, and the strictly perivenous glutamine synthetase. The coexpression of high levels of the two periportal markers glucose-6-phosphatase and lactate dehydrogenase and of the perivenous marker NADPH dehydrogenase was observed in two situations: in clusters of hepatocytes isolated within the ductular proliferation in bile-duct-ligated livers and the majority of intrasplenically transplanted hepatocytes. The expression of glutamine synthetase was different according to the site. The protein was observed in certain intrasplenically transplanted hepatocytes bordering the splenic vessels but was never detected in hepatocyte clusters found in bile-duct-ligated livers. Our study therefore suggests that the coexpression of periportal and perivenous markers in the same hepatocytes is likely to be a non-specific consequence of the loss of the normal connections of hepatocytes with the normal liver microcirculation.

Animals↗
PubMed1995

Direct solution hybridization of guanidine thiocyanate-solubilized cells for quantitation of mRNAs in hepatocytes.

The sensitivity of direct solution hybridization of hepatocytes solubilized in guanidium thiocyanate (GuSCN) for detecting alpha 1-acid glycoprotein and albumin mRNAs was studied. The sensitivity of detection was inversely correlated with the DNA concentration. Raising the hybridization temperature from 20 to 37 or 50 degrees C (with formamide) increased the hybridization efficiency three- to fourfold in cell lysates with a high DNA concentration (1 microgram/microliter), whereas the hybridization efficiency was already maximal at 20 degrees C in diluted samples. It was most important to normalize all hybridization reactions with an internal standard, such as sense mRNA, because of the great variation in hybridization efficiency from one cell preparation to another depending on the DNA concentration. Direct hybridization of GuSCN cell lysates labeled in vivo with [6-14C]orotic acid was more efficient than hybridizing equivalent amounts of purified [6-14C]-labeled RNA, perhaps because of greater mRNA integrity and/or better recoveries of mRNA in GuSCN cell lysates. Therefore, direct solution hybridization of GuSCN-solubilized hepatocytes, which avoids the problem of RNA purification, appears to be a rapid, sensitive, and reliable method for quantifying mRNA in hepatocytes.

Albumins↗
PubMed1995

The c-jun proto-oncogene down-regulates the rat alpha-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

Animals↗
PubMed1995

Distribution of albumin, alpha 1-inhibitor 3 and their respective mRNAs in periportal and perivenous rat hepatocytes isolated by the digitonin-collagenase technique.

The expression of albumin and alpha 1-inhibitor 3 genes was investigated in rat cell suspensions enriched in periportal (n = 10) and perivenous (n = 10) hepatocytes obtained by the digitonin-collagenase technique. The degree of enrichment of the cell suspensions was assessed: (1) by enzymic assays for the periportal marker alanine aminotransferase and for the perivenous marker glutamine synthetase; and (2) by their content of mRNAs for the periportal marker hepatic glutaminase and for glutamine synthetase. The existence of an antegrade intra-lobular gradient for albumin and alpha 1-inhibitor 3 mRNAs was demonstrated, with periportal:perivenous ratios of 2.33 and 3.80, respectively. However, no gradient was demonstrated for the respective protein contents with corresponding ratios of 0.98 and 1.21. A certain degree of overlap existed between periportal and perivenous suspensions for their content in albumin and alpha 1-inhibitor 3 mRNAs. A morphometrical analysis of the surface of digitonin-permeabilized hepatic tissue revealed that this overlap could be explained by a variable extent of permeabilization of the mediolobular zone from one rat to another and from one lobule to another in a given animal. These results suggest that while the digitonin-collagenase technique is well suited for studies in vitro of proteins expressed in sharp intra-lobular gradients or restricted to an intra-lobular compartment, it is not completely reliable for proteins distributed in continuous moderate intra-lobular gradients, such as albumin and alpha 1-inhibitor 3.

Acute-Phase Proteins↗
PubMed1994

Activation of ras oncogene in livers with cirrhosis.

Activation of cellular oncogenes and inactivation of anti-oncogenes have been postulated as important mechanisms during hepatocarcinogenesis. This study was conducted to detect abnormal levels of several proto-oncogenes (c-jun, c-fos, c-H-ras) and of the p53 and the alpha-fetoprotein gene in the liver during cirrhosis, a pathological process which predisposes to the development of hepatocarcinoma. Liver tissue from 11 patients with cirrhosis of different etiologies, and seven histologically normal liver fragments taken at the periphery of benign liver tumors of metastases were studied. Transcripts of the various oncogenes and of the alpha-fetoprotein gene were detected by in situ hybridization, and the p53 protein was revealed by immunocytochemistry. No overexpression of any of the mRNA tested or of the p53 protein was found in histologically normal liver in contact with benign or metastatic tumors. In contrast, 10 of the 11 specimens with cirrhosis (90.9%) displayed abnormally high levels of c-H-ras transcripts. Five samples with cirrhosis revealed a moderate increase in the level of c-fos mRNA. Only one case and two cases, respectively, exhibited increased levels of c-jun and alpha-fetoprotein mRNA. No cases were positive for the p53 antigen. Liver-cell proliferation, as assessed by immunocytochemistry with the Ki 67 monoclonal antibody, was low in both the group with cirrhosis and the control groups (0.49% and 0.55% positive cells, respectively). These data demonstrate that activation of c-H-ras mRNA is an almost constant finding in hepatocytes of livers with cirrhosis. This gene overexpression is not linked to hepatocellular proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1993

Activation of nuclear protooncogenes and alpha-fetoprotein gene in rat liver during the acute inflammatory reaction.

Nuclear protooncogene and alpha-fetoprotein gene expression is stimulated in hepatocytes during liver regeneration and by various growth factors in vitro. Metabolic adaptation of hepatocytes has been implicated in such gene reprogrammation. We examine here whether induction of an acute inflammation, a physiological situation of important metabolic adjustments, also triggers activation of nuclear oncogenes and of the AFP gene in rat liver. C-fos, c-jun and c-myc mRNA accumulated on Northern blots between 4-12 h of inflammation and the steady-state level of two small alpha-fetoprotein transcripts characteristic of the adult liver increased at 4 h and 24 h of inflammation. In situ hybridization showed accumulation of the mRNA of the four genes studied in all hepatocytes, without any zonal lobular heterogeneity. 3H-histoautoradiography and mitotic counts indicated an inhibition of DNA synthesis and mitosis, prolonged for at least 48 h after inflammation. Thus acute inflammation triggers the activation of nuclear protooncogenes and alpha-feto-protein gene in hepatocytes, but this activation is not followed by passage into the replicative cycle.

Acute-Phase Reaction↗
PubMed1993

Nuclear oncogenes and alpha-fetoprotein gene expression in hepatoma cell lines.

We have previously demonstrated a correlation between the kinetics of activation of the nuclear oncogenes c-fos, c-jun and c-myc and the alpha-fetoprotein (AFP) gene in adult rat hepatocytes proliferating in culture, which led us to raise the hypothesis of a possible regulation of the AFP gene by nuclear oncogenes. Whether the mechanisms of AFP gene expression in normal and transformed hepatocytes are similar is unknown. In this study we have searched for a possible correlation between the basal level of AFP gene expression, by several hepatoma cell lines that express the AFP gene differently, and the basal level of expression of the nuclear oncogenes c-fos, c-jun and c-myc. The analysis has been performed at the mRNA and protein levels. Our results demonstrate that cell lines that strongly express the AFP gene do not express higher levels of nuclear oncogenes than cell lines with weak expression of the AFP gene. These results, therefore, do not support a direct involvement of nuclear oncogenes on the basal level of AFP gene expression in hepatoma cell lines.

Animals↗
PubMed1993

Parenchymal innervation of normal and cirrhotic human liver: a light and electron microscopic study using monoclonal antibodies against the neural cell-adhesion molecule.

Hepatic innervation participates in the control of sinusoidal blood flow and in the regulation of certain metabolic functions of the liver. The study of the distribution of hepatic nerves has been hampered by the lack of adequate markers. We therefore tested the value of the neural cell-adhesion molecule (NCAM) as a probe for the study of parenchymal nerves in the normal and cirrhotic human liver. Four antibodies against various epitopes of NCAM were tested by light and electron microscopic immunohistochemistry: Leu19, ERIC-1, VC1.1, and HNK-1. Their reactivity was compared with that of antibodies against the following neural cell markers: S100 protein, neurofilaments, and neuron-specific enolase (NSE). The tissue reactivity of anti-NCAM antibodies was variable, suggesting a microheterogeneity of the NCAM molecule in the normal liver. Clones Leu19 and ERIC-1 proved to be the most sensitive of the anti-NCAM antibodies. Their sensitivity was superior to that of the antibodies directed against the other neural cell markers tested. In the normal liver, both Leu19 and ERIC-1 demonstrated a heterogeneous distribution of nerve fibers inside the hepatic lobule. Intralobular nerve fibers predominated in Zone 1. This might contribute to the constitution of distinct zonal microenvironments inside the hepatic lobule. In cirrhosis, no nerve fiber was detected inside parenchymal nodules; no nerve plexus was visible at the contact of proliferating neoductules. These alterations might contribute to the pathogenesis of the hemodynamic and metabolic disorders observed in cirrhosis.

Antibodies, Monoclonal↗
PubMed1993

Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.

Animals↗