c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1.

Aim: To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation.

Methods: IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses.

c-Fos accelerates hepatocyte conversion to a fibroblastoid phenotype through ERK-mediated upregulation of paxillin-Serine178 phosphorylation.

Transforming growth factor beta (TGF-beta) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-beta, EGF or a combination of both.

Jun D cooperates with p65 to activate the proximal kappaB site of the cyclin D1 promoter: role of PI3K/PDK-1.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the kappaB2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-kappaB proteins.

Beclin 1 mRNA strongly correlates with Bcl-XLmRNA expression in human hepatocellular carcinoma.

Beclin 1 physically associates with Bcl-x(L) and is considered as a haploinsufficient tumor suppressor. As the role of Beclin 1 in hepatocellular carcinoma (HCC) is unknown, we determined Beclin 1 mRNA expression in 27 pairs of tumoral/nontumoral (T/NT) liver samples. The Beclin 1 mRNA T/NT ratio was less than 0.

In vivo altered unfolded protein response and apoptosis in livers from lipopolysaccharide-challenged cirrhotic rats.

Background/aims: Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2alpha (eIF2alpha) to attenuate protein synthesis, including in NF-kappaB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFalpha-mediated apoptosis.

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.

Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells.

Aim: To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment.

Methods: Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry.

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta.

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation.

Disruption of MKK4 signaling reveals its tumor-suppressor role in embryonic stem cells.

The dual Ser/Thr kinase MKK4 and its downstream targets JNK and p38 regulate critical cellular functions during embryogenesis and development. MKK4 has been identified as a putative tumor-suppressor gene in human solid tumors of breast, prostate and pancreas. To clarify the mechanisms underlying the transforming potential of molecular defects targeting MKK4, we have generated totipotent embryonic stem (ES) cells expressing the dominant-negative mutant DN-MKK4(Ala), S257A/T261A.

Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis.

In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS.

Activation of NF-kappa B, AP-1 and STAT transcription factors is a frequent and early event in human hepatocellular carcinomas.

Background/aims: Hepatitis B and C viruses, two inducers of hepatocarcinomas, have been shown to activate AP-1, NF-kappa B and STAT in vitro, but no detailed information on the activity of these transcription factors in vivo have been provided.

Methods: We have measured the DNA binding activity of these transcription factors in the peri-tumoral and the tumoral parts of 15 primary liver cancers, of viral or non-viral etiologies, and in five hepatic metastases using electrophoretic mobility shift assays.

Results: AP-1, NF-kappa B and STAT binding activities were increased in the peritumoral tissue, compared with histologically normal livers in 73, 87 and 70%, respectively, of the cases.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.