Isabel Fabregat

Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor β in fetal hepatocytes

Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF‐ß) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Δψm), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF‐ß mediates ROS production that precedes the loss of Δψm, the release of cytochrome c, and the activation of caspase 3. TGF‐ß 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 Δψm collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl‐xL levels, Δψm 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‐ß in fetal hepatocytes, ROS may be responsible for the decrease in bcl‐xL mRNA levels that precedes the loss of Δψm, the release of cytochrome c, and the activation of caspase 3, culminating in cell death.—Herrera, B., Alvarez, A. M., Sanchez, A., Fernandez, M., Roncero, C., Benito, M., Fabregat, I. Reactive oxygen species (ROS) mediates the mitochondrial‐dependent apoptosis induced by transforming growth factor ß in fetal hepatocytes. FASEB J. 15, 741‐751 (2001)

Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive oxygen intermediates.

Transforming growth factor-β (TGF-β), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-β concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-β, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-β-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-β (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-β in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-β in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-β in fetal hepatocytes.

The Epithelial Mesenchymal Transition Confers Resistance to the Apoptotic Effects of Transforming Growth Factor β in Fetal Rat Hepatocytes

Resumen del poster presentado al 50th Inner Ear Biology Workshop, celebrado en Alcala de Henares-Madrid (Espana) del 10 al 13 de septiembre de 2013.Resumen del trabajo presentado al 15o Congreso Nacional de la Sociedad Espanola de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013.Resumen del poster presentado al CIBERDEM Annual Meeting, celebrado en Cerdanyola del Valles, Barcelona (Espana) del 11 al 13 de mayo de 2016.-- et al.Resumen del trabajo presentado al XXXVIII Congreso de la Sociedad Espanola de Ciencias Fisiologicas (SECF), celebrado en Zaragoza del 13 al 16 de septiembre de 2016.Poster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los dias 3 al 6 de julio de 2013 en Berlin (Alemania)Memoria presentada para optar al grado de Doctor por la Licenciada en Biologia Angela Prieto Folgado y realizada en el Instituto de Investigaciones Biomedicas Alberto Sols.La realizacion de este trabajo ha sido posible gracias a la financiacion otorgada por el FIS al proyecto de investigacion 96/1803.Grant Funding Source: Supported by the Fondo de Investigaciones Sanitarias (PI0011406) to MF.The chemotherapeutic study of a limited series of steroidal sapogenins from several endemic species of the flora of the Canary Islands is presented here. On the whole, they possess a very weak antibacterial activity, a slight antifungal effect and one of them, vespertilin, displays interesting cytostatic activity (ID50 = 5 micrograms/ml). A pharmacodynamic screening carried out on this product mainly revealed very slight toxicity, antihistaminic activity and a light tranquilizing effect. The data obtained justify further research.The purpose of this study was to characterize the role of ions other than Ca2+ in hepatic responses to alpha 1-adrenergic stimulation. We report that the alpha 1-adrenoreceptor activation of hepatic functions is accompanied by extracellular acidification and an increase in intracellular pH. These effects are dependent on extracellular Na+ concentration and are inhibited by the Na+/H+ antiporter blocker 5-(N-ethyl-N-isopropyl) amiloride under conditions that preclude antagonistic effects on agonist binding. Thus, the activation of plasma membrane Na+/H+ exchange is an essential feature of the hepatic alpha-adrenoreceptor-coupled signaling pathway. The following observations indicate that the sustained hepatic alpha 1-adrenergic actions rely on a functional coupling between the plasma membrane Na+/H+ and Na+/Ca2+ exchangers, resulting in the stimulation of Ca2+ influx. 1) Inhibition of the Na+/K(+)-ATPase does not prevent the alpha 1-adrenergic effects. However, alpha 1-adrenoreceptor stimulation fails to induce intracellular alkalinization and to acidify the extracellular medium in the absence of extracellular Ca2+. 2) A non-receptor-induced increase in intracellular Na+ concentration, caused by the ionophore monensin, stimulates Ca2+ influx and increases vascular resistance. 3) Inhibition of Na+/Ca2+ exchange prevents, in a concentration-dependent manner, most of the alpha 1-agonist-induced responses. 4) The actions of Ca(2+)-mobilizing vasoactive peptide receptors or alpha 2-adrenoreceptors, which produce neither sustained extracellular acidification nor release of Ca2+, are insensitive to Na+/H+ exchange blockers.Poster presentado en la VII Reunion Anual de la Red Tematica de Investigacion Cooperativa en Cancer (RTICC), celebrada en Salamanca el 24 de septiembre de 2014Resumen del trabajo presentado al VI Meeting de la Red Espanola de Canales Ioniocs (RECI), celebrado en Santiago de Compostela del 6 al 8 de septiembre de 2017.Tesis Doctoral presentada por Laura Jimenez Perez para optar al grado de doctor por la Universidad de Valladolid, Departamento de Bioquimica y Biologia Molecular y FisiologiaPoster presentado en la VII Reunion Anual de la Red Tematica de Investigacion Cooperativa en Cancer (RTICC), celebrada en Salamanca el 24 de septiembre de 2014Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.Esta Tesis Doctoral fue realizada en el Centro Andaluz de Biologia del Desarrollo por la licenciada Briseida Beli Cacho Valadez para optar al grado de Doctor por la Universidad Pablo de Olavide.Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.Trabajo presentado en el XI Simposi de Neurobiologia: Future technical advances, organizado por la Socitat Catalana de Biologia, en Barcelona, los dias 12 y 13 de noviembre de 2018El estudio de la relacion entre componentes de la dieta y la salud/enfermedad utiliza metodos de valoracion de la ingesta dietetica, del estatus nutricional y de marcadores de funcion o de efecto. En concreto, en el estudio de los carotenoides y la salud ocular, interesa el estudio de dos carotenoides sin actividad provitamina A, la luteina y la zeaxantina, por su posible papel en la optimizacion de la funcion visual y en la prevencion de enfermedades cronicas asociadas a la edad, y de tres carotenoides con actividad provitamina A: -caroteno, -caroteno y -criptoxantina, por ser precursores de retinol, nutriente del que depende el ciclo visual para una vision normal. En el presente trabajo se ha llevado a cabo el estudio de los carotenoides de la dieta mas relevantes para la salud ocular humana considerando de forma simultanea parametros relacionados con la ingesta, el estatus y la funcion visual, asi como diversas variables que pueden modificar el estatus nutricional, como son la concentracion de lipidos en sangre, y la bioaccesibilidad de los carotenoides a partir de alimentos de amplio consumo...Fetal rat hepatocytes treated with transforming growth factor beta (TGF-beta) die by apoptosis. However, a subpopulation of them survives and undergoes an epithelial mesenchymal transition (EMT). This transition also occurs upon incubation with fetal bovine serum. We have isolated the subpopulations that undergo EMT (TGF-beta-treated-fetal hepatocytes: TbetaT-FH; serum-treated-fetal hepatocytes: ST-FH) and show that they present high levels of vimentin and Snail expression and lack cytokeratin 18 and E-cadherin. Both TbetaT-FH and ST-FH cells require mitogens to grow and maintain the response to TGF-beta in terms of growth inhibition. However, they lack differentiation markers such as the liver-enriched transcription factors hepatocyte nuclear factor 4 (HNF-4) or HNF-1alpha and express the progenitor marker OV-6. Interestingly, the EMT process confers them resistance to the apoptotic effect of TGF-beta, with cells showing higher levels of active AKT and Bcl-x(L) than fetal hepatocytes. In summary, these cells are refractory to the apoptotic effects of TGF-beta, showing characteristics of liver progenitors and of some hepatocellular carcinoma cells.Memoria de tesis presentada por Luis Vazquez Fonseca, Licenciado en Bioquimica para optar al grado de Doctor. Esta Tesis Doctoral ha sido realizada bajo el programa de doctorado de Biotecnologia y Tecnologia Quimica en el grupo de investigacion del CIBERER U729 en el Centro Andaluz de Biologia del Desarrollo, Area de Biologia Celular del Departamento de Fisiologia, Anatomia y Biologia Celular de la Universidad Pablo de Olavide y bajo la direccion del Dr. Carlos Santos Ocana y el Dr. Placido NavasResumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Poster presentado al 17o Congreso Nacional de la Sociedad Espanola de Neurociencia, celebrado en Alicante del 27 al 30 de septiembre de 2017.The mutations at the bithorax locus produce a transformation of anterior haltere into anterior wing. The bx1 allele presents unusual features when compared with other bx alleles. The phenotype of bx1 homozygotes is temperature sensitive but only with regard to the distal and not to the proximal transformation, thus suggesting two different components in the bithorax transformation. The phenotype of bx1 homozygotes is stronger than that of bx1 over the deletion of the gene, suggesting a trans interaction of the bx1 chromosomes which results in mutual partial inactivation. We show by temperature shift and clonal analysis experiments that the decision on whether to differentiate haltere or wing structures is taken at the end of the proliferation period of the mutant disc.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Poster presentado al XXVII Congreso Nacional de la Asociacion Espanola de Genetica Humana celebrado en Madrid del 10 al 12 de abril de 2013.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Poster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los dias 3 al 6 de julio de 2013 en Berlin (Alemania)Resumen del trabajo presentado al Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio de 2019.Poster presentado en el XII European Meeting on Glial Cells in Health and Disease, celebrado los dias 15 a 18 de julio de 2015 en Bilbao (Espana)Trabajo presentado en el XL Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular. FEBS3+1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular, celebrado en Barcelona (Espana), del 23 al 26 de octubre de 2017Resumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Trabajo presentado en el XII GEIRLI Meeting: New trends in redox biology: a multidisciplinary approach, celebrado en Barcelona (Espana), los dias 4 y 5 de julio de 2019Treatment of nucleosomal particles with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by a biphasic release of histones H2A plus H2B; one H2A.H2B dimer is more easily released than the other. This behavior allows the preparation of nucleosomal particles containing only one H2A.H2B dimer, which were complemented with 125I-labeled H2A.H2B. These reconstituted particles, which contain one labeled and one unlabeled H2A.H2B dimer, were treated with the amount of reagent needed to release one of the two H2A.H2B dimers. Radioactivity was equally distributed between residual particles and released proteins, which is consistent with equivalent binding sites in the nucleosomal particle for H2A.H2B dimers, rather than with intrinsically different sites. The asymmetric release of H2A.H2B dimers would be caused by a change in the binding site of one dimer following the release of the other. This behavior might be related to the structural dynamics of nucleosomes.Resumen del trabajo presentado al European Society of Cardiology (ESC) Congress, celebrado en Barcelona (Espana) del 26 al 30 de agosto de 2017.Resumen del poster presentado al 49th European Association for the Study of Diabetes Annual Meeting, celebrado en Barcelona (Espana) del 23 al 27 de septiembre de 2013.-- et al.Trabajo doctoral realizado por Da Rebeca Lapresa Ruiz de Gauna, para optar al grado de doctor por la Universidad de Salamanca.Rationale: Several animal models have been developed to study acute lung injury (ALI); however the majority of these studies are focused on different mechanisms within the acute phase. These models do not allow studying the mechanisms in the later phases or testing any possible long-term treatment. The aim of this study was to develop an experimental ALI model simulating bronchial aspiration of gastric contents with bacterial superinfection with alveolar epithelial damage persisting over time. Methods: Sprague-Dawley rats (200-250g) were anesthetized with isofluorane. ALI was induced by intratracheal instillation of HCl (1 µl/g, 0.1 mol/L pH=1.4) followed by instillation of LPS from Escherichia coli O55:B5 (0, 10, 20, 30 or 40µg/g b.w.) two hours later. Control rats were treated with intratracheal instillations of saline. After 72h, the animals were sacrificed and bronchoalveolar lavage fluid (BALF) was sampled for further analysis of total protein concentration by bicinchoninic acid method. Results: At 72 h, rats suffered a significant loss of weight proportional to the administered dose of LPS (5.6% with 10µg/g b.w, 12.6% with 20µg/g b.w, 14.2% with 30µg/g b.w and 17.7% with 40µg/g b.w). Control rats gained in weight at 72h. LPS at 10, 20, 30 and 40µg/g b.w induced a 1.7, 2.5, 2.9 and 3.4 fold increase in total protein concentration in BAL fluid, respectively, reflecting a substantial increase proportional to the LPS dose. Conclusion: The degree of weight loss and the increase of total protein concentration in BAL fluid in the current model may reflect disease severity and progression. This model would be useful in future for new therapeutical options. Grant acknowledgements: FIS-PI12/02548 and Fundacio Parc Tauli.Resumen del trabajo presentado al European Respiratory Society (ERS) International Congress, celebrado en Paris (Francia) del 15 al 19 de septiembre de 2018.Resumen del trabajo presentado a las 5as Jornadas de Formacion del CIBERES celebradas en Bunyola (Mallorca) del 18 al 19 de octubre de 2012.Resumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Resumen del trabajo presentado al XIII Congreso de la Sociedad Espanola del Dolor, celebrado en Pamplona del 2 al 4 de junio de 2016.This work was supported by grants FIS-01/1048 and FIS-02/1199 from the Fondo de Investigacion Sanitaria and grant SA-087/01 from Junta de Castilla y Leon.Resumen del poster presentado al Joint Meeting of the American Physiological Society and the Physiological Society, celebrado en Dublin (Irlanda) del 29 al 31 de julio de 2016.Trabajo presentado al 5th International Conference on Phospholipase A2 Mediated Signaling in Translational Medicine celebrado en New Orleans (US) del 20 al 21 de mayo de 2013.Tesis Doctoral presentada por Rebeca Torres Merino para optar al grado de Doctora por la Universidad de Valladolid, Facultad de Medicina: Dpto. de Bioquimica y Biologia Molecular y Fisiologia.Poster presentado al Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), celebrado en Seattle, Washington (US) del 1 al 5 de mayo de 2016.Resumen del trabajo presentado al 63rd Annual Meeting Biophysical Society, celebrado en Baltimore, Maryland (USA) del 2 al 6 de marzo de 2019.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Resumen del poster presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.-- Tambien presentado al 15o Congreso Nacional de la Sociedad Espanola de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013.Resumen del trabajo presentado al XXXVI Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Madrid del 4 al 6 de septiembre de 2013.Resumen del trabajo presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.Resumen del poster presentado al XXVIII Congreso Nacional de la Sociedad Espanola de diabetes, celebrado en Bilbao del 20 al 22 de abril de 2016.SAF2016-77703-C2-2-R of the Ministerio de Economia y Competitividad, Spain and the European Regional Development Fund (ERDF); AGAUR 2017-SGR106 and the CERCA Programme of the Generalitat de Catalunya; C. Sanfeliu belong to Group 05 of CIBER Epidemiologia y Salud Publica (CIBERESP) of the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain

Survival and apoptosis: a dysregulated balance in liver cancer

Background/Aims: Dysregulation of the balance between proliferation and cell death represents a protumorigenic principle in human hepatocarcinogenesis. This article aims to provide a review of the current findings about how physiological hepatocyte apoptosis is regulated and whether or not its dysregulation might contribute to the progression towards a hepatocellular carcinoma (HCC) process.

Upregulation of the NADPH oxidase NOX4 by TGF-beta in hepatocytes is required for its pro-apoptotic activity

BACKGROUND/AIMS The transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes through an oxidative stress process. Here, we have analyzed the role of different NADPH oxidase isoforms in the intracellular signalling induced by TGF-beta in hepatocytes, to later explore whether this mechanism is altered in liver tumor cells. METHODS Primary cultures of rat and human hepatocytes, HepG2 and Hep3B cells were used in in vitro studies to analyze the TGF-beta response. RESULTS TGF-beta-induced apoptosis in rat hepatocytes does not require Rac-dependent NADPH oxidases. TGF-beta upregulates the Rac-independent Nox4, which correlates with its pro-apoptotic activity. Regulation of Nox4 occurs at the transcriptional level and is counteracted by intracellular survival signals. siRNA targeted knock-down of Nox4 attenuates NADPH oxidase activity, caspase activation and cell death in rat hepatocytes. NOX4 upregulation by TGF-beta is also observed in human hepatocytes, coincident with apoptosis. In human hepatocellular carcinoma (HCC) cell lines, NOX4 upregulation by TGF-beta is only observed in cells that are sensitive to its cytotoxic effect, such as Hep3B cells. siRNA targeted knock-down of NOX4 in these cells impairs TGF-beta-induced apoptosis. CONCLUSIONS Upregulation of NOX4 by TGF-beta is required for its pro-apoptotic activity in hepatocytes. Impairment of this TGF-beta-induced response might confer apoptosis resistance in HCC cells.

Involvement of EGF receptor and c-Src in the survival signals induced by TGF-beta1 in hepatocytes.

Transforming growth factor beta1 (TGF-β1) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. Although solid data established the role of TGF-βs as suppressor factors in tumorigenic processes, in the context of an advanced stage of disease, TGF-βs could also play a pro-oncogenic role. We have previously shown that TGF-β1 induces both pro- and antiapoptotic signals in foetal rat hepatocytes. In this work, we have focused on its antiapoptotic mechanism. We show that TGF-β1 activates the epidermal growth factor receptor (EGFR) and phosphorylates c-Src. EGFR is required for Akt activation. Blocking EGFR signalling amplifies the apoptotic response to TGF-β1. TGF-β1 induced a rapid activation of the tumour necrosis factor-α-converting enzyme (TACE/ADAM (a disintegrin and metalloprotease) 17). Inhibitors of TACE considerably attenuated Akt activation, which suggests that TGF-β1 activates EGF signalling in hepatocytes by promoting shedding of EGF-like ligands. The activation of c-Src by TGF-β1 is EGFR dependent and is required for full Akt phosphorylation and cell survival. Inhibition of EGFR does not block the epithelial–mesenchymal transition (EMT) induced by TGF-β1 in hepatocytes, which indicates that activation of EGFR plays an essential role in impairing apoptosis, but it is dispensable for the EMT process.

Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion.

The mechanisms for in vivo production of bone marrow–derived hepatocytes (BMDHs) remain largely unclear. We investigated whether granulocyte colony–stimulating factor (G‐CSF)–mediated mobilization of hematopoietic cells increases the phenomenon. Recurrent liver injury in mice expressing green fluorescent protein (EGFP) in all hematopoietic‐derived cells was produced by 3 months of carbon tetrachloride (CCL4) injections. Histologically, there were necrotic foci with histiocyte‐rich infiltrates, but little oval cell proliferation. Subsequently, some animals were mobilized with G‐CSF for 1, 2, or 3 weeks. Animals were sacrificed 1 month after growth factor treatment. BMDH percentages were lower than previously reported, though G‐CSF mobilization significantly augmented BMDH production in injured livers. BMDHs originating from in vivo fusion were evaluated by transplanting female EGFP+ cells into male mice. Binucleated, EGFP+ hepatocytes with one Y chromosome, indicating fusion, were identified. In conclusion, (1) mobilization of hematopoietic cells increases BMDH production and (2) as with the FAH‐null model, the first model demonstrating hematopoietic/hepatocyte fusion, recurring CCl4‐induced injury has macrophage‐rich infiltrates, a blunted oval cell response, and a predominantly in vivo fusion process for circulating cell engraftment into the liver. These findings open the possibility of using hematopoietic growth factors to treat nonhematopoietic degenerative diseases. (HEPATOLOGY 2006;43:108–116.)

Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-β–Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Activation of NADPH oxidase by transforming growth factor-β in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-κB-dependent mechanism

The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.