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 (Δψ _m ), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF‐ß mediates ROS production that precedes the loss of Δψ _m , the release of cytochrome c, and the activation of caspase 3. TGF‐ß induces a decrease in the protein and mRNA levels of bcl‐x _L , 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‐x _L , preventing Δψ _m collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl‐x _L levels, Δψ _m 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‐x _L . These results indicate that during the apoptosis mediated by TGF‐ß in fetal hepatocytes, ROS may be responsible for the decrease in bcl‐x _L mRNA levels that precedes the loss of Δψ _m , 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)

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^−/−/p19^ARF−/−, Stat3^Δhc/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.

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 β1 (TGF-β1) is followed by apoptotic cell death. TGF-β1 mediates radical oxygen species (ROS) production that precedes bcl-x_L 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-β1 activates the caspase cascade and whether or not caspase activation precedes the oxidative stress induced by this factor. Our results show that TGF-β1 activates at least caspase-3, -8, and -9 in rat fetal hepatocytes, which are not required for ROS production, glutathione depletion, bcl-x_L down-regulation, and initial cytochrome c release. However, caspase activation mediates cleavage of Bid and Bcl-x_L 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 (ΔΨm) may be a consequence of caspase activity rather than the mechanism by which TGF-β 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-β in fetal hepatocytes inevitably leads to death, with or without caspases. (Hepatology 2001;34:548-556.)

The TGF-β (transforming growth factor-β) 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 α-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-α 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-β in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-β, 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 TβR-I (TGF-β 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-β up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TβR-I-dependent manner. TGF-β mediates activation of the nuclear factor-κB pathway, which is inhibited by DPI and is required for up-regulation of TGF-α and HB-EGF. In contrast, EGFR activation is not required for TGF-β-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-β in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-β-treated cells.