Cesar Roncero

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)

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.

Characterization of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4

We have cloned CHS4, a gene that complements the resistance to Calcofluor of the Saccharomyces cerevisiae cal2 mutant. We show that CHS4 is allelic to the previously described SKT5 and CSD4 genes. CHS4 encodes a 696 amino acids protein with no potential transmembrane domain. chs4‐null mutants are resistant to Calcofluor white and exhibit a considerable reduction in cell wall chitin and in chitin synthase III (CSIII) activity. Biochemical characterization of chitin synthase III from these null mutants indicates that the defect is due to a reduced Vmax of the enzyme. This defect can be overcome in vitro by trypsin treatment of the membrane preparations. Chs4p does not act as a transcriptional or translational regulator of CHS3, the gene coding for the catalytic subunit of CSIII activity, and we therefore propose that Chs4p would be an essential component of the CSIII complex, acting as a post‐translational regulator of this activity. In addition to the chitin defect, the chs4 mutant shows a severe defect in mating.

Calcofluor Antifungal Action Depends on Chitin and a Functional High-Osmolarity Glycerol Response (HOG) Pathway: Evidence for a Physiological Role of the Saccharomyces cerevisiae HOG Pathway under Noninducing Conditions

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.

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.

Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae.

Deletion of the 1,3‐β‐D‐glucan synthase gene FKS1 in Saccharomyces cerevisiae induces a compensatory mechanism that is reflected in a significant increase in chitin synthase III (CSIII) activity, leading to high rates of chitin synthesis. Deregulation of CSIII activity is mainly due to the intracellular delocalization of Chs3p and Chs4p, the two main components of the CSIII active complex.

EGF blocks NADPH oxidase activation by TGF-β in fetal rat hepatocytes, impairing oxidative stress, and cell death

Epidermal growth factor (EGF) is a survival signal for transforming growth factor‐beta (TGF‐β)‐induced apoptosis in hepatocytes, phosphatidylinositol 3‐kinase (PI 3‐K) being involved in this effect. Here, we analyze the possible cross talks between EGF and TGF‐β signals to understand how EGF impairs the early pro‐apoptotic events induced by TGF‐β. Data have indicated that neither SMAD nor c‐Jun NH2 Terminal Kinase (JNK) activations are altered by EGF, which clearly interferes with events directly related to the radical oxygen species (ROS) production, impairing oxidative stress, p38 MAP kinase activation, and cell death. Activation of a NADPH‐oxidase‐like system, which is responsible for the early ROS production by TGF‐β, is completely inhibited by EGF, through a PI 3‐K‐dependent mechanism. Activity of RAC1 increases by TGF‐β, but also by EGF, and both act synergistically to get maximum effects. Fetal rat hepatocytes express nox4, in addition to nox1 and nox2, and TGF‐β clearly upregulates nox4. EGF blocks up‐regulation of nox4 by TGF‐β. Interestingly, in the presence of PI 3‐K inhibitors, EGF is not able to counteract the nox4 upregulation by TGF‐β. Taking together these results indicate that impairment of TGF‐β‐induced NADPH oxidase activation by EGF is a RAC1‐independent process and correlates with an inhibition of the mechanisms that address the increase of nox4 mRNA levels by TGF‐β. J. Cell. Physiol. 207: 322–330, 2006.

The RIM101 Pathway Contributes to Yeast Cell Wall Assembly and Its Function Becomes Essential in the Absence of Mitogen-Activated Protein Kinase Slt2p

ABSTRACT The Saccharomyces cerevisiae ynl294cΔ (rim21Δ) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Δ also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Δ and rim101Δ mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Δ, as well as rim101Δ, was synthetic lethal with slt2Δ, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Δ rlm1Δ and rim101Δ rlm1Δ double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.