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1.
Redox Biol ; 40: 101841, 2021 04.
Article in English | MEDLINE | ID: mdl-33493901

ABSTRACT

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-ß) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-ß pathway. In fact, levels of active TGF-ß1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-ß1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.


Subject(s)
Liver Regeneration , Signal Transduction , Animals , Hepatocytes/metabolism , Liver/metabolism , Mice , NADPH Oxidase 4/genetics , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Transforming Growth Factor beta
3.
Oncogene ; 36(21): 3002-3014, 2017 05 25.
Article in English | MEDLINE | ID: mdl-27941881

ABSTRACT

Epithelial to mesenchymal transition is a common event during tumour dissemination. However, direct epithelial to amoeboid transition has not been characterized to date. Here we provide evidence that cells from hepatocellular carcinoma (HCC), a highly metastatic cancer, undergo epithelial to amoeboid transition in physiological environments, such as organoids or three-dimensional complex matrices. Furthermore, the NADPH oxidase NOX4 inhibits this transition and therefore suppresses efficient amoeboid bleb-based invasion. Moreover, NOX4 expression is associated with E-cadherin levels and inversely correlated with invasive features. NOX4 is necessary to maintain parenchymal structures, increase cell-cell and cell-to-matrix adhesion, and impair actomyosin contractility and amoeboid invasion. Importantly, NOX4 gene deletions are frequent in HCC patients, correlating with higher tumour grade. Contrary to that observed in mesenchymal cell types, here NOX4 suppresses Rho and Cdc42 GTPase expression and downstream actomyosin contractility. In HCC patients, NOX4 expression inversely correlates with RhoC and Cdc42 levels. Moreover, low expression of NOX4 combined with high expression of either RhoC or Cdc42 is associated with worse prognosis. Therefore, loss of NOX4 increases actomyosin levels and favours an epithelial to amoeboid transition contributing to tumour aggressiveness.


Subject(s)
Carcinoma, Hepatocellular/pathology , Epithelial-Mesenchymal Transition/genetics , Liver Neoplasms/pathology , NADPH Oxidases/physiology , Actomyosin/administration & dosage , Actomyosin/genetics , Actomyosin/metabolism , Carcinoma, Hepatocellular/genetics , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Down-Regulation/genetics , Gene Expression Profiling , Genes, Tumor Suppressor/physiology , Humans , Liver Neoplasms/genetics , NADPH Oxidase 4 , Neoplasm Invasiveness , Neoplasm Metastasis
4.
Biochim Biophys Acta ; 1853(10 Pt A): 2453-63, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26001768

ABSTRACT

Oval cells constitute an interesting hepatic cell population. They contribute to sustain liver regeneration during chronic liver damage, but in doing this they can be target of malignant conversion and become tumor-initiating cells and drive hepatocarcinogenesis. The molecular mechanisms beneath either their pro-regenerative or pro-tumorigenic potential are still poorly understood. In this study, we have investigated the role of the HGF/c-Met pathway in regulation of oval cell migratory and invasive properties. Our results show that HGF induces c-Met-dependent oval cell migration both in normal culture conditions and after in vitro wounding. HGF-triggered migration involves F-actin cytoskeleton reorganization, which is also evidenced by activation of Rac1. Furthermore, HGF causes ZO-1 translocation from cell-cell contact sites to cytoplasm and its concomitant activation by phosphorylation. However, no loss of expression of cell-cell adhesion proteins, including E-cadherin, ZO-1 and Occludin-1, is observed. Additionally, migration does not lead to cell dispersal but to a characteristic organized pattern in rows, in turn associated with Golgi compaction, providing strong evidence of a morphogenic collective migration. Besides migration, HGF increases oval cell invasion through extracellular matrix, a process that requires PI3K activation and is at least partly mediated by expression and activation of metalloproteases. Altogether, our findings provide novel insights into the cellular and molecular mechanisms mediating the essential role of HGF/c-Met signaling during oval cell-mediated mouse liver regeneration.


Subject(s)
Cell Movement/physiology , Epithelial-Mesenchymal Transition/physiology , Hepatocyte Growth Factor/metabolism , Liver/metabolism , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-met/metabolism , Stem Cells/metabolism , Actins/genetics , Actins/metabolism , Animals , Cadherins/genetics , Cadherins/metabolism , Hepatocyte Growth Factor/genetics , Liver/cytology , Mice , Mice, Knockout , Neuropeptides/genetics , Neuropeptides/metabolism , Occludin/genetics , Occludin/metabolism , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-met/genetics , Stem Cells/cytology , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism
5.
Cell Death Dis ; 5: e1326, 2014 Jul 17.
Article in English | MEDLINE | ID: mdl-25032849

ABSTRACT

Transforming growth factor-beta (TGF-ß) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-ß in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-ß receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-ß-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1(-/-) hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-ß, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-ß-induced survival signals in Cav1(-/-) hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-ß was impaired in Cav1(-/-) hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1(-/-) hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1(+/+) cells, which was not the case in Cav1(-/-) cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-ß treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-ß-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-ß pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-ß in liver cancer cells.


Subject(s)
ADAM Proteins/metabolism , Caveolin 1/metabolism , ErbB Receptors/genetics , Hepatocytes/metabolism , Transcriptional Activation , Transforming Growth Factor beta/metabolism , ADAM Proteins/genetics , ADAM17 Protein , Animals , Apoptosis , Caveolin 1/genetics , Cells, Cultured , Enzyme Activation , ErbB Receptors/metabolism , Female , Hepatocytes/enzymology , Male , Mice , Mice, Knockout , Phosphorylation , Reactive Oxygen Species/metabolism , Signal Transduction
6.
Hum Mol Genet ; 22(16): 3296-305, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23604518

ABSTRACT

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of the nervous system characterized by axonopathy in spinal cords and/or cerebral demyelination, adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFAs) in plasma and tissues. The disease is caused by malfunction of the ABCD1 gene, which encodes a peroxisomal transporter of VLCFAs or VLCFA-CoA. In the mouse, Abcd1 loss causes late onset axonal degeneration in the spinal cord, associated with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. We have formerly shown that an excess of the VLCFA C26:0 induces oxidative damage, which underlies the axonal degeneration exhibited by the Abcd1(-) mice. In the present study, we sought to investigate the noxious effects of C26:0 on mitochondria function. Our data indicate that in X-ALD patients' fibroblasts, excess of C26:0 generates mtDNA oxidation and specifically impairs oxidative phosphorylation (OXPHOS) triggering mitochondrial ROS production from electron transport chain complexes. This correlates with impaired complex V phosphorylative activity, as visualized by high-resolution respirometry on spinal cord slices of Abcd1(-) mice. Further, we identified a marked oxidation of key OXPHOS system subunits in Abcd1(-) mouse spinal cords at presymptomatic stages. Altogether, our results illustrate some of the mechanistic intricacies by which the excess of a fatty acid targeted to peroxisomes activates a deleterious process of oxidative damage to mitochondria, leading to a multifaceted dysfunction of this organelle. These findings may be of relevance for patient management while unveiling novel therapeutic targets for X-ALD.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adrenoleukodystrophy/metabolism , Fatty Acids/pharmacology , Mitochondria/metabolism , Oxidative Phosphorylation , Peroxisomes/metabolism , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/genetics , Animals , Brain/metabolism , Cells, Cultured , DNA, Mitochondrial , Fibroblasts/metabolism , Free Radicals/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/genetics , Oxidation-Reduction , Oxidative Stress , Spinal Cord/metabolism
7.
Cell Signal ; 24(2): 505-513, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22001397

ABSTRACT

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-ß)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.


Subject(s)
ErbB Receptors/metabolism , Hepatocytes/metabolism , Liver/metabolism , Proto-Oncogene Proteins c-met/genetics , Signal Transduction/genetics , Stem Cells/metabolism , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Deletion , Gene Expression , Heparin-binding EGF-like Growth Factor , Hepatocytes/cytology , Hepatocytes/drug effects , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Liver/cytology , Liver/drug effects , Mice , Mice, Knockout , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-met/deficiency , Quinazolines/pharmacology , Stem Cells/cytology , Stem Cells/drug effects , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolism , Tyrphostins/pharmacology
8.
Cell Death Differ ; 17(8): 1335-44, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20203689

ABSTRACT

Apoptosis induced by most stimuli proceeds through the mitochondrial pathway. One such stimulus is nutrient deprivation. In this study we studied death induced by glucose deprivation in cells deficient in Bax and Bak. These cells cannot undergo mitochondrial outer membrane permeabilization (MOMP) during apoptosis, but they undergo necrosis when treated with MOMP-dependent apoptotic stimuli. We find in these cells that glucose deprivation, rather than inducing necrosis, triggered apoptosis. Cell death required caspase activation as inhibition of caspases with peptidic inhibitors prevented death. Glucose deprivation-induced death displayed many hallmarks of apoptosis, such as caspase cleavage and activity, phosphatidyl-serine exposure and cleavage of caspase substrates. Neither overexpression of Bcl-xL nor knockdown of caspase-9 prevented death. However, transient or stable knockdown of caspase-8 or overexpression of CrmA inhibited apoptosis. Cell death was not inhibited by preventing death receptor-ligand interactions, by overexpression of c-FLIP or by knockdown of RIPK1. Glucose deprivation induced apoptosis in the human tumor cell line HeLa, which was prevented by knockdown of caspase-8. Thus, we have found that glucose deprivation can induce a death receptor-independent, caspase-8-driven apoptosis, which is engaged to kill cells that cannot undergo MOMP.


Subject(s)
Apoptosis , Caspase 8/metabolism , Glucose/physiology , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/genetics , Caspase 9/genetics , Caspase 9/metabolism , Cell Membrane Permeability/physiology , Gene Knockdown Techniques , HeLa Cells , Humans , RNA Interference , RNA, Small Interfering/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Serpins/metabolism , Viral Proteins/metabolism , bcl-2 Homologous Antagonist-Killer Protein/deficiency , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/deficiency , bcl-2-Associated X Protein/genetics
9.
Clin. transl. oncol. (Print) ; 11(4): 208-214, abr. 2009. tab, ilus
Article in English | IBECS | ID: ibc-123604

ABSTRACT

New advancements have been made in recent years in the understanding of the molecular mechanisms that govern human liver tumorigenesis. Experimental animal models have been widely used, especially mouse models. In this review we highlight some of the genetically engineered mouse models that have proved to be excellent tools to study the intracellular signalling pathways altered in hepatocarcinogenesis and establish potential correlations with data from humans, with special focus on hepatocellular carcinoma (HCC), the most common type of primary liver cancer. Information obtained from these animal models will help to design future therapeutic approaches to HCC, particularly those that explore drugs that specifically target the altered molecular pathways (AU)


Subject(s)
Animals , Male , Female , Animals, Genetically Modified , Carcinoma, Hepatocellular/genetics , Disease Models, Animal , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology
10.
Am J Clin Oncol ; 31(5): 481-7, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18838886

ABSTRACT

OBJECTIVES: Gemcitabine has well-recognized activity in the treatment of ovarian cancer. Fixed-dose rate (FDR) delivery has been proposed as a more rationale way to administer gemcitabine, to avoid saturation of the enzyme that catalyzes its intracellular transformation into the active metabolites, difluorodeoxycitidine biphosphate, and triphosphate. Our aim was to assess clinical activity of gemcitabine delivered by FDR infusion in patients with platinum resistant ovarian cancer. MATERIALS AND METHODS: Patients with platinum-resistant ovarian cancer received gemcitabine 1000 mg/m(2) over 120 minutes on days 1 and 8 of each cycle. Cycles were repeated every 3 weeks, and up to 6 cycles were delivered. RESULTS: Forty-eight patients were included in the study. Among 41 patients evaluable for response, 9 clinical responses (1 complete response and 8 partial responses) were observed, achieving a global response rate of 22%. Grade 3 to 4 hematological toxicity consisted of anemia (15% of patients), neutropenia (24%), and thrombopenia (10%). One patient died due to septic shock. The main grade 3 to 4 nonhematological toxicity was asthenia (7 patients, 17%). CONCLUSION: Activity of gemcitabine administered by FDR infusion in patients with platinum-resistant ovarian cancer seems similar to that achieved using 30-minute infusions, with higher toxicity.


Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm , Organoplatinum Compounds/adverse effects , Ovarian Neoplasms/drug therapy , Peritoneal Neoplasms/drug therapy , Adenocarcinoma, Clear Cell/drug therapy , Adenocarcinoma, Clear Cell/secondary , Adenocarcinoma, Mucinous/drug therapy , Adenocarcinoma, Mucinous/secondary , Adult , Aged , Aged, 80 and over , Carcinoma, Endometrioid/drug therapy , Carcinoma, Endometrioid/secondary , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/secondary , Deoxycytidine/administration & dosage , Female , Humans , Infusions, Intravenous , Middle Aged , Ovarian Neoplasms/pathology , Peritoneal Neoplasms/pathology , Prognosis , Ribonucleotide Reductases/antagonists & inhibitors , Survival Rate , Gemcitabine
11.
Acta pediatr. esp ; 61(3): 126-130, mar. 2003. tab
Article in Spanish | IBECS | ID: ibc-111034

ABSTRACT

Objetivo: Cuantificar sensibilidad (S), especificidad € y valores predictivos positivos (VPP) y negativos (VPN) para diversos puntos de corte de la eosinofilia en sangre y en moco nasal, de la IgE sérica total (en U/mL y en desviaciones estándar), como marcadores de sensibilización a alérgenos comunes en niños asmáticos. Métodos: Se midieron estos marcadores en 144 niños asmáticos, a quienes se realizó estudio alergológico individualizado. Como “patrón oro” de sensibilización se utilizó la positividad en pruebas cutáneas y/o RAST. Resultados: La eosinofilia absoluta en sangre presentaba un mejor punto de corte como sugestivo de alergia en ≥ 450 eosinófilos/mm3 , con S y E del 52 y el 71% y VPP del 61% y VPN del 63%. La eosinofilia porcentual en sangre, con el mejor punto de corte en ≥ 6%, tenía unos valores en el mismo orden del 56, 79, 70 y 67%. La eosinofilia en moco nasal, presentaba valores del 33, 89, 71 y 62% para un punto de corte de >25%. La IgE sérica total, con punto de corte en >100 U/mL, mostraba el 60, 92, 86 y 74%, y en desviaciones estándar, el mejor punto de corte era superior a +3 DE, con valores del 30, 89, 69 y 60%. Conclusiones: Aunque el valor diagnóstico de los tests es bajo, el conocimiento de sus valores predictivos para diversos puntos de corte permitirá una mejor evaluación de la actitud sobre indicación de estudio alergológico en el asma infantil (AU)


Aim: To quantify sensitivity, specifity and positive (PPV) and negative (NPV) predictive values, with several cut-off points, of eosinophlis in blood and nasal mucosa and of total serum IgE (in U/ml and standard deviations), as markers of sensitization to common allergens in asthmatic children. Methods: These markers were measured in 144 children with asthma, in whom and individualized allergologic testing was performed. Positive results in skin tests and/or RAST were used as the gold standard for sensitization. Results: A sensitivity and a specificity of 52% and 71%, respectively, were found for blood absolute eosinophilia, and the PPV blood absolute eosinophilia, and the PPV and NPV were 61% and 63%, using a cut-off point of ≥450 eosinophils/mm3. Using a cut-off point of ≥6% of blood eosinophils, these values were 56%, 79%, 70% and 67%, respectively. Nasal mucosa eosinophilia (cut-off point at 25%) had values of 33%, 89%, 71% and 62%, respectively. Total serum IgE showed values 60%, 92%, 86% and 74% respectively, for a cut –off point of >100 U/ml. In standard deviations, with a cut-off point of >+3 s.d., the sensitivity and specificity were 30% and 89%, and PPV and NPV were 69% and 60%. Conclusions: Although the diagnostic values of these tests are low, the knowledge of their predictive values for several cut-off points will allow a better evaluation for decision-making concerning when to perform allergologic tests in asthmatic children (AU)


Subject(s)
Child , Humans , Asthma/diagnosis , Asthma/prevention & control , Immunoglobulin E , Eosinophilia/complications , Eosinophilia/diagnosis , Eosinophilia/prevention & control , Allergens
12.
Hepatology ; 34(3): 548-56, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11526541

ABSTRACT

Most of the morphologic changes that are observed in apoptotic cells are caused by a set of cysteine proteases (caspases) that are activated during this process. In previous works from our group we found that treatment of rat fetal hepatocytes with transforming growth factor beta1 (TGF-beta1) is followed by apoptotic cell death. TGF-beta1 mediates radical oxygen species (ROS) production that precedes bcl-xL down-regulation, loss of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 (Herrera et al., FASEB J 2001;15:741-751). In this work, we have analyzed how TGF-beta1 activates the caspase cascade and whether or not caspase activation precedes the oxidative stress induced by this factor. Our results show that TGF-beta1 activates at least caspase-3, -8, and -9 in rat fetal hepatocytes, which are not required for ROS production, glutathione depletion, bcl-xL down-regulation, and initial cytochrome c release. However, caspase activation mediates cleavage of Bid and Bcl-xL that could originate an amplification loop on the mitochondrial events. An interesting result is that transmembrane potential disruption occurs later than the initial cytochrome c release and is mostly blocked by the pan-caspase inhibitor Z-VAD.fmk, indicating that the decrease in mitochondrial transmembrane potential (Delta(Psi)m) may be a consequence of caspase activity rather than the mechanism by which TGF-beta induces cytochrome c efflux. Finally, although Z-VAD.fmk completely blocks nucleosomal DNA fragmentation, it only delays cell death, which suggests that activation of the apoptotic program by TGF-beta in fetal hepatocytes inevitably leads to death, with or without caspases.


Subject(s)
Apoptosis/physiology , Caspases/physiology , Cytochrome c Group/metabolism , Hepatocytes/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Cells, Cultured , Down-Regulation/physiology , Enzyme Activation , Fetus , Hepatocytes/drug effects , Humans , Mitochondria, Liver/physiology , Rats , Rats, Wistar , Recombinant Proteins/pharmacology , Time Factors , bcl-X Protein
13.
FEBS Lett ; 499(3): 225-9, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11423121

ABSTRACT

We have previously found that transforming growth factor-beta (TGF-beta) induces an increase in radical oxygen species (ROS) production that mediates its apoptotic effects in fetal hepatocytes. In this paper we show that TGF-beta activates p38 mitogen-activated protein kinase (p38MAPK) and ROS may be responsible for this activation. Activation of p38MAPK occurs late, coincident with the maximal production of ROS, it is inhibited by radical scavengers and it is accentuated by the presence of glutathione synthesis inhibitors. However, p38MAPK does not appear to be involved in any of the apoptotic events: loss of Bcl-x(L) levels, cytochrome c release, cleavage of caspase substrates and loss of cell viability.


Subject(s)
Apoptosis , Hepatocytes/drug effects , Mitogen-Activated Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Cytochrome c Group/metabolism , Enzyme Activation/drug effects , Fetus/cytology , Hepatocytes/cytology , Hepatocytes/enzymology , Hepatocytes/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Wistar , bcl-X Protein , p38 Mitogen-Activated Protein Kinases
14.
J Hepatol ; 34(2): 192-201, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11281546

ABSTRACT

BACKGROUND: The metabolism of methionine plays an important role in regulating hepatic cellular function. Methionine adenosyltransferase (MAT) is the enzyme that catalyses the biosynthesis of S-adenosylmethionine (AdoMet) from ATP and methionine. Liver-specific MAT I/III levels are down-regulated in the regenerating rat liver after partial hepatectomy. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are two cytokines fundamental for liver regeneration. TNF-alpha stimulates sphingomyelin metabolism and ceramide generation in a variety of cell systems. AIMS: The role of exogenous cell-permeable ceramide in modifying MAT I/III mRNA levels and its association with TNF-alpha and IL-6 actions were investigated in rat hepatocytes and H35 hepatoma cells. RESULTS: C2-ceramide (N-acetylsphingosine) at 1-10 microM decreased MAT I/III expression. The effect was maximum after 2 h of treatment and it was maintained up to 24 h. MAT I/III protein levels also decreased. IL-6 (1-10 ng/ml) potentiated C2-ceramide effects in cultured hepatocytes while decreasing by itself MAT I/III levels with a similar time-response curve in both cell types. C2-ceramide actions were not associated with an increase in cell death. TNF-alpha was also a potent antagonist for MAT I/III expression, at 1-20 ng/ml decreased MAT I/III levels and induced endogenous ceramide generation. The decrease of MAT I/III mRNA levels (in all the cases) was not due to a decrease in mRNA half-life which suggests a regulation at the transcriptional level. Finally, the decrease in MAT I/III mRNA levels correlated to a decrease in MAT activity. CONCLUSION: This work demonstrates that short-chain ceramide can be used as a novel exogenous agonist that can modulate hepatic methionine metabolism in association with cytokines.


Subject(s)
Liver/drug effects , Liver/metabolism , Methionine Adenosyltransferase/genetics , Methionine Adenosyltransferase/metabolism , Sphingosine/pharmacology , Animals , Cells, Cultured , Gene Expression Regulation, Enzymologic/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Interleukin-6/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Liver Regeneration/drug effects , Liver Regeneration/physiology , Methionine/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sphingosine/analogs & derivatives , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology
15.
FASEB J ; 15(3): 741-51, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11259392

ABSTRACT

Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF-beta) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Deltapsim), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF-beta mediates ROS production that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3. TGF-beta induces a decrease in the protein and mRNA levels of bcl-xL, an antiapoptotic member of the Bcl-2 family. In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family. EGF maintains Bcl-xL, preventing Deltapsim collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl-xL levels, Deltapsim collapse, cytochrome c release, and activation of caspase 3; in contrast, the presence of glutathione synthesis inhibitors such as BSO accentuated the effect. The incubation of fetal hepatocytes in the presence of ter-butyl-hydroperoxide alone produces a decrease in bcl-xL. These results indicate that during the apoptosis mediated by TGF-beta in fetal hepatocytes, ROS may be responsible for the decrease in bcl-xL mRNA levels that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3, culminating in cell death.


Subject(s)
Apoptosis , Hepatocytes/cytology , Mitochondria/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Blotting, Western , Caspase 3 , Caspases/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Epidermal Growth Factor/metabolism , Hepatocytes/drug effects , Hepatocytes/physiology , Humans , Membrane Potentials/drug effects , Microscopy, Confocal , Mitochondria/drug effects , Models, Biological , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Pyrrolidines/pharmacology , RNA, Messenger/metabolism , Rats , Recombinant Proteins/metabolism , Thiocarbamates/pharmacology , bcl-2-Associated X Protein , bcl-X Protein
16.
Physiol Genomics ; 5(2): 53-65, 2001 Mar 08.
Article in English | MEDLINE | ID: mdl-11242589

ABSTRACT

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.


Subject(s)
3' Untranslated Regions/genetics , DNA/chemistry , DNA/genetics , Gene Expression Regulation , Microsatellite Repeats/genetics , Nucleic Acid Conformation , Receptors, Polymeric Immunoglobulin/genetics , 3' Untranslated Regions/metabolism , Animals , Base Sequence , Cell Line , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Enhancer Elements, Genetic/genetics , Genes, Reporter/genetics , Humans , Molecular Sequence Data , Nucleic Acid Denaturation , Organ Specificity , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Protein Biosynthesis , Rats , Reproducibility of Results , Species Specificity , Thermodynamics , Transcription, Genetic/genetics
17.
Hepatology ; 32(3): 528-35, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10960445

ABSTRACT

Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.


Subject(s)
Apoptosis/drug effects , Caspases/physiology , Cytochrome c Group/physiology , Epidermal Growth Factor/pharmacology , Liver/embryology , Phosphatidylinositol 3-Kinases/physiology , Transforming Growth Factor beta/pharmacology , Animals , Apoptosis/physiology , Caspase 3 , Cell Survival/drug effects , Cells, Cultured , Cytochrome c Group/antagonists & inhibitors , Down-Regulation , Embryo, Mammalian/physiology , Humans , Liver/cytology , Membrane Potentials/drug effects , Mitochondria/physiology , Mitogen-Activated Protein Kinases/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Wistar , bcl-X Protein
18.
J Hepatol ; 32(2): 242-50, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10707864

ABSTRACT

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


Subject(s)
Fibronectins/physiology , Gene Expression Regulation, Developmental/physiology , Liver/cytology , Liver/embryology , Animals , Cell Adhesion/physiology , Cell Division/physiology , Cells, Cultured , Extracellular Matrix Proteins/physiology , Fetus , Phenotype , Rats
19.
J Hepatol ; 31(5): 895-904, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10580588

ABSTRACT

BACKGROUND/AIMS: Loss of specific differentiation markers, adoption of a migrating morphology and progressive replacement of the cytokeratin network by vimentin intermediate filaments characterize the epithelial-mesenchymal transition of cultured neonatal rat hepatocytes. In a previous study (Hepatology 1997; 25: 598-606), we reported that this process can be differentially regulated by EGF and DMSO, two agents that affect hepatocyte growth and differentiation. The aim of the present study was to determine if growth activation or differential gene expression could explain the differences in EMT observed between these two factors. METHODS: We compared the effects of EGF, HGF, TGF-beta1 and DMSO on growth, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatocytes using thymidine incorporation, Northern blotting and Western blotting analysis. RESULTS: When TGF-beta1 or DMSO was added to the cultures supplemented with EGF and HGF, the mitogenic activity induced by these factors was inhibited. DMSO down-regulated c-myc and c-fos expression. mRNA levels of some liver-specific genes such as albumin, or liver-enriched transcription factors such as C/EBPdelta, HNF-4 and HNF-1beta were slightly different in cultures supplemented with DMSO or TGF-beta1. However, no differences were found when DMSO or TGF-beta1 was added to the cultures supplemented with EGF. Western blotting analysis showed that TGF-beta1 decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and vimentin. When DMSO or TGF-beta1 was added in combination with EGF or HGF, both factors maintained the increase in albumin and cytokeratin induced by the growth factors although DMSO, but not TGF-beta1, inhibited vimentin expression. CONCLUSIONS: Activation of vimentin expression produced in cultures supplemented with the mitogenic factors (EGF and HGF) is independent of the activation of cell growth, because DMSO but not TGF-beta1 can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expression in these cultures seems to be independent of the mRNA levels of transcription factors associated with the differentiated liver phenotype.


Subject(s)
Cell Differentiation/physiology , Epithelial Cells/cytology , Growth Substances/pharmacology , Liver/cytology , Mesoderm/cytology , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cells, Cultured , Dimethyl Sulfoxide/pharmacology , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/physiology , Gene Expression Regulation/drug effects , Genes, fos/drug effects , Genes, myc/drug effects , Hepatocyte Growth Factor/pharmacology , Keratins/genetics , Liver/drug effects , Liver/physiology , Mesoderm/drug effects , Mesoderm/physiology , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/genetics , Rats , Transcription Factors/genetics , Transcription, Genetic , Transforming Growth Factor beta/pharmacology , Vimentin/genetics
20.
Exp Cell Res ; 252(2): 281-91, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10527619

ABSTRACT

Transforming growth factor-beta (TGF-beta1) induces death of fetal hepatocytes by an apoptotic mechanism. However, even when very high concentrations and/or long-term exposure to the cytokine is used, 40-50% of cells always survive. The process of cell survival is coincident with changes in morphology and phenotype, with cells showing a fibroblastic appearance and eliciting an epithelial-fibroblastic transition. Surviving cells continue responding to TGF-beta in terms of growth control. Expression of liver-specific genes is very low in these cells; this effect is due to the decrease in their rate of transcription as soon as 2 h after the addition of the factor. Surviving cells present a decreased DNA binding activity for liver-enriched transcription factors, an increased DNA binding activity for AP-1, and a high expression of protooncogenes. These cells are immature hepatocytes since in the presence of the appropriate signal (i.e., epidermal growth factor), they can differentiate, organizing in cell clusters and increasing both liver-specific mRNA expression and liver-enriched transcription factor activity. In accord with these results, TGF-beta, secreted at high concentrations during liver carcinogenesis, might induce death of normal cells while providing a selective advantage for the survival of cells that are "partially transformed" or "less differentiated" and unresponsive to the factor.


Subject(s)
Apoptosis/drug effects , Liver/pathology , Transforming Growth Factor beta/pharmacology , Animals , Cell Differentiation , Cytoskeletal Proteins/metabolism , Humans , Liver/embryology , Liver/metabolism , Rats , Rats, Wistar , Recombinant Proteins/pharmacology
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