FASEB J. Pierce now sold as Thermo Scientific
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by HERRERA, B.
Right arrow Articles by FABREGAT, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by HERRERA, B.
Right arrow Articles by FABREGAT, I.
(The FASEB Journal. 2001;15:741-751.)
© 2001 FASEB

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

BLANCA HERRERA*, ALBERTO. M. ÁLVAREZ{dagger}, ARÁNZAZU SÁNCHEZ*, MARGARITA FERNÁNDEZ*, CÉSAR RONCERO*, MANUEL BENITO* and ISABEL FABREGAT*1

* Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM and
{dagger} Centro de Citometría de Flujo y Microscopía Confocal, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain

1Correspondence: Dpto. de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. E-mail: isabelf{at}eucmax.sim.ucm.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({Delta}{psi}m), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF-ß mediates ROS production that precedes the loss of {Delta}{psi}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-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 {Delta}{psi}m collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl-xL levels, {Delta}{psi}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-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 {Delta}{psi}m, the release of cytochrome c, and the activation of caspase 3, culminating in cell death.—Herrera, B., Alvarez, A. M., Sánchez, A., Fernández, 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.


Key Words: cytochrome c • caspases • Bcl-x


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MECHANISMS THAT regulate cell death are essential for normal development and maintenance of homeostasis. Several concepts have recently emerged with respect to the role of apoptosis in liver physiology and pathology. First, liver hyperplasia during development or regeneration may be aided by inhibition of apoptosis; second, liver atrophy occurs by apoptosis of liver cells in the absence of regeneration; and third, pathophysiological processes can trigger the cellular apoptotic machinery leading to disease processes (for a review, see ref 1 ). Endogenous factors such as transforming growth factor beta (TGF-ß 1), activin A, Fas ligand, and tumor necrosis factor alpha (TNF-{alpha}) may be involved in the induction of liver apoptosis (2) . The TGF-ß family comprises a great many structurally related polypeptide factors, each capable of regulating several cellular processes, including cell proliferation, lineage determination, differentiation, motility, adhesion, and death (3) . Numerous observations suggest that TGF-ß plays an important biological role mediating hepatocyte apoptosis (1) . Furthermore, disruption of the TGF-ß pathway at the prereceptor, receptor, or postreceptor levels occurs in hepatocellular carcinomas and can cause dysregulation of apoptosis (4) . However, the mechanisms by which TGF-ß induces cell death are still only partly understood.

In previous works from our group, we have found that TGF-ß inhibits growth of fetal hepatocytes, arresting cells in G1 and down-regulating myc expression (5) . But when used at higher concentrations, TGF-ß also induces fetal hepatocyte apoptosis (6 , 7) , a process that is preceded by an induction of reactive oxygen species (ROS) and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes (6) . Cell death induced by TGF-ß in fetal hepatocytes is blocked by radical scavengers, which decrease the percentage of apoptotic cells (6) . All these results provide evidence for the involvement of an oxidative process necessary for the apoptosis induced by TGF-ß in hepatocytes. It has also been reported that processing/activation of caspase 3 is involved in TGF-ß-induced apoptosis in rat hepatocytes (8) . Pretreatment of hepatocytes with cycloheximide blocks both oxidative stress (9) and caspase-3 activation (8) and, consequently, the apoptotic process. These last findings confirm that a link exists between both processes (oxidative stress and apoptosis), and the synthesis of a new protein might be necessary in the upstream events responsible for the activation of ROS production.

Recent reports have provided evidence that mitochondria are deeply involved in the regulation of cell death (10 , 11) . Mitochondria manifest signs of outer membrane and/or inner membrane permeabilization when exposed to a variety of proapoptotic second messengers. Thus, cytochrome c, which is normally confined in the mitochondrial intermembrane space, is found in the cytosol of cells undergoing apoptosis (12 , 13) . Furthermore, other proteins such as certain procaspases, adenylate kinase 2, and apoptosis-inducing factor (AIF) are also released from mitochondria in response to some apoptotic stimuli (11) . Mitochondrial membrane permeabilization involves a dynamic multiprotein complex formed in the contact site between the inner and outer mitochondrial membranes (14) . This process precedes nuclear apoptosis and is inhibited by the presence of Bcl-2 on these organelles (12 , 13 , 15) . Cytosolic cytochrome c forms a complex with Apaf-1 and procaspase-9, resulting in activation of caspase-9, which then processes and activates other caspases, such as caspase 3, to orchestrate the biochemical execution of programmed cell death (16) . Proapoptotic Bcl-2 family proteins, including Bax, Bak, and Bid, which bear resemblance to channel-forming bacterial toxins, induce the mitochondrial membrane permeabilization and cytochrome c release (15 , 17 , 18) . Accordingly, translocation of Bax from the cytosol (where it is a monomer) to mitochondrial membranes (where it forms a dimer or higher order oligomers) has been reported in a wide array of apoptosis-inducing circumstances. In contrast, Bcl-xL, an antiapoptotic member of the Bcl-2 family, is capable of preventing cytochrome c release while also significantly inhibiting cell death (15 , 19) . Cytochrome c release is frequently coincident with a disruption of the mitochondrial transmembrane potential ({Delta}{psi} m), which has been defined as an early stage of apoptosis (10 , 11) .

Recently, Rodrigues et al. have demonstrated that TGF-ß decreases {Delta}{psi} m and provokes cytochrome c release in adult hepatocytes (20) . However, a question that warrants addressing is whether the loss of {Delta}{psi} m is responsible for the increase in ROS or the other way around. In other cell systems and with other apoptotic stimuli, some reports have shown that ROS are generated only after loss of {Delta}{psi} m (11 , 21) . However, the mitochondrial membrane permeabilization pore has been shown to be sensitive to the redox state and ROS can also induce mitochondrial membrane permeabilization both in vitro and in vivo (11) . In agreement with this last idea, decreasing superoxide levels blocks the loss of {Delta}{psi} m in a model of activated T cell apoptosis (22) and remarkable elevations of ROS precede megamitochondria formation in a model of hepatocyte cell death (23) . Indeed, it has been postulated that ROS may play a dual role in apoptosis, either as activators of permeability transition or a consequence of this transition, depending on the death stimulus (10) .

The aim of this work therefore was to study the implication of the mitochondria and the bcl-2 family members in the apoptosis induced by TGF-ß in fetal hepatocytes and their possible relation to the oxidative stress generated by this factor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Human recombinant TGF-ß was from Calbiochem (La Jolla, Calif.) and human recombinant EGF was kindly provided by Serono Laboratories (Madrid, Spain). Collagenase was from Roche (Barcelona, Spain). Fetal and neonatal calf serum and culture media were from Imperial Laboratories (Hampshire, U.K.). Radiochemicals were from ICN (Irvine, Calif.). The fluorescent probes 2',7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) and chloromethyl-X-rosamine (CMXRos) were from Molecular Probes (Eugene, Oreg.). Anti-Bcl-x and anti-Bax polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-rat albumin polyclonal antibody was from Nordic Immunological Laboratories (Tilburg, The Netherlands). Anti-cytochrome c monoclonal antibody and the caspase 3 substrate Ac-DEVD-AMC were from PharMingen (San Diego, Calif.). Other reagents were from Sigma Chemical Co. (St. Louis, Mo.) or Boehringer (Mannheim, Germany).

Cell isolation and culture
Hepatocytes from 20-day-old fetal Wistar rats were isolated by collagenase disruption (2.5x106 cells/fetus) as described previously (24) and plated on plastic (noncoated) dishes in arginine-free medium 199, supplemented with ornithine (200 µM), fetal calf serum (10%), penicillin (120 µg/ml), and streptomycin (100 µg/ml). Cells were incubated in 5% CO2, at 37°C for 4 h, allowing cell attachment to plates. Media was changed at that time and replaced by one of the same composition except that 10% fetal calf serum was changed by 2% newborn bovine serum. After 18–20 h, the medium was again replaced for one of identical composition but in the absence of serum. Two hours later, cells were ready for all the experiments described below.

Analysis of mitochondrial transmembrane potential
The fluorescent probe CMXRos was used to analyze the mitochondrial transmembrane potential by either flow cytometry or confocal microscopy. For flow cytometry, fetal hepatocytes were incubated in the presence or absence of the different factors; at different times, cells were detached by trypsinization and resuspended in phosphate-buffered saline (PBS). The cellular fluorescence intensity was measured after 30 min incubation of the cells with 0.1 µM CMXRos. A FACScan flow cytometer (Becton-Dickinson, San José, Calif.) was used. For each analysis, 10,000 events were recorded. For confocal microscopy analysis, after incubation in the absence or presence of the different factors, cells were washed twice with PBS and media were replaced by PBS and 0.1 µM CMXRos. Thirty minutes later the cellular fluorescence was detected by using an MRC-1024 laser confocal microscopy (Bio-Rad, Hempstead, England). Digital image analysis from cellular fluorescence was done with Lasersharp Software (Bio-Rad) and Confocal Assistance (Free Software by Todd-Clark-Brelje).

Measurement of intracellular ROS
For visualization and analysis of intracellular ROS, the oxidation-sensitive probe DCFH-DA was used, as previously described (9) . To analyze the net intracellular generation of ROS by flow cytometry, cells were detached by trypsinization after incubation in the absence or presence of the different factors. The cellular fluorescence intensity was measured after 30 min incubation with 5 µM DCFH-DA, by using the same flow cytometer described above. Propidium iodide (0.005%) was used to detect dead cells. For each analysis, 10,000 events were recorded. For confocal microscopy analysis, after incubation of cells in the absence or presence of the different factors, they were washed twice with PBS and the cellular fluorescence intensity was visualized after 30 min of incubation with 5 µM DCFH-DA by using the same confocal microscopy described above.

Analysis of nuclear DNA content by flow cytometry
The ploidy determination of hepatocytes was estimated by flow cytometry DNA analysis. Cells were detached from dishes by addition of 0.25% trypsin-0.02% EDTA, fixed in methanol (-20°C) for 1 min, and treated with RNase (10 µg/ml) for 30 min at 37°C. The DNA content per cell was then evaluated in a FACScan flow cytometer (Becton-Dickinson) after staining cells with propidium iodide (0.05 mg/ml) for 15 min at room temperature in the dark. For the computer analysis, only signals from single cells were considered (10,000 cells/assay).

Analysis of cytochrome c release
Attached cells were scraped off in isotonic isolation buffer (1 mM EDTA; 10 mM HEPES, 250 mM sucrose, pH 7.6), collected by centrifugation at 2,500 g for 5 min at 4°C and resuspended in hypotonic isolation buffer (1 mM EDTA, 10 mM HEPES, 50 mM sucrose, pH 7.6). Cells were incubated at 37°C for 5 min and homogenized under a TeflonTM pestle (Overhead Stirrer, Wheaton Instruments, Millville, N.J.). Hypertonic isolation buffer (1 mM EDTA, 10 mM HEPES, 450 mM sucrose, pH 7.6) was added to balance the buffer’s tonicity. Samples were centrifuged at 2000 g for 5 min at 4°C. Supernatants were recovered and centrifuged again at 10,000 g for 10 min. The pellet contained the mitochondrial fraction resuspended in isotonic isolation buffer and supernatant contained the cytosolic protein extract. Protein concentration of lysates was determined using the Bio-Rad (Hercules, Calif.) protein assay kit according to the manufacturer’s specifications. After electrophoresis separation of 50 µg protein/condition in sodium dodecyl sulfate (SDS) -12% polyacrylamide, gels were transferred by semidry transfer (Bio-Rad Labs, Richmond, Calif.) to nitrocellulose membranes (Schleicher and Schuell, Keene, N.Y.). Immunoblots were blocked in TTBS (10 mM Tris/HCl, 150 mM NaCl, pH 7.5, 0.05% Tween 20) containing 5% non-fat dried milk and incubated overnight with the primary antibody (monoclonal anti-cytochrome c diluted 1:1000 in TTBS 0.5% non-fat dried milk). After washing, membranes were incubated with peroxide-conjugated anti-mouse immunoglobulin (1:5000 in TTBS 0.5% non-fat dried milk) for 2 h and the blot was developed with the ECL system (Amersham, Buckinghamshire, U.K.). Mitochondrial contamination of the cytosolic protein extracts was determined by analysis of cytochrome c oxidase, measured photometrically at 550 nm as described previously (25) .

Analysis of caspase 3 activity
Cells were scraped off in PBS, collected by centrifugation at 2,500 g for 5 min, and lysed at 4°C in 5 mM Tris/HCl, pH 8.0, 20 mM EDTA, 0.5% Triton X-100. Lysates were clarified by centrifugation at 13,000 g for 10 min. Reaction mixture contained 25 µl cellular lysates, 325 µl assay buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM dithiothreitol), and 20 µM caspase 3 substrate (Ac-DEVD-AMC). After 2 h incubation in the dark, enzymatic activity was measured in a Luminescence Spectrophotometer (Perkin Elmer LS-50) ({lambda}excitation, 380 nm; {lambda}emission, 440 nm). We define a unit of caspase 3 activity as the amount of active enzyme necessary to produce an increase in 1 arbitrary unit in the luminescence spectrophotometer after 2 h incubation with the reaction mixture. Protein concentration of cell lysates was determined using the Bio-Rad protein assay kit and final expression of the results is presented as units of caspase 3 activity/µg protein.

Glutathione determination
Fetal hepatocytes were washed twice with PBS, scraped off, and pelleted at 4°C. Cellular glutathione was extracted in a buffer containing 0.2% Triton X-100, 2.5% sulfosalicylic acid. After centrifugation at 15,000 g for 15 min at 4°C, the supernatant was used for the determination of total (GSH +GSSG) glutathione, using the method of Griffith modified as described previously (9) . Using GSH as standard, glutathione content is initially expressed as nmol/106 cells for each condition and represented in the figures as percentage with respect to control cells.

Western blot analysis of Bcl-xL and Bax
To detect Bcl-x, and Bax protein levels, supernatant cells were collected by centrifugation at 2000 g for 5 min at 4°C; attached cells were scraped off in PBS, pelleted by centrifugation at 4000 g, for 10 min, at 4°C and resuspended in a lysis buffer (25 mM HEPES; 2,5 mM EDTA; 0.1% Triton X-100, 1 mM PMSF, 5 µg/ml leupeptin). Samples were sonicated for 30 s at 1,5 mA and lysates were clarified by centrifugation at 13,000 g for 10 min. When Bax and Bcl-xL levels were analyzed in cytosol and mitochondrial extracts, the method to isolate mitochondria was the same described above (in analysis of cytochrome c release section). After protein concentration analysis of cell lysates, by using the Bio-Rad protein assay kit, 75–100 µg protein/condition were separated by SDS-12% polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Immunoblot sequential incubations with primary antibodies (1:1000 dilution) and secondary antibodies (1:5000 dilution) were performed as described above. Blots were developed with the ECL system.

RNA Isolation and Northern blot analysis
For each assay, total RNA was extracted from the pooled cells of two 92 mm diameter dishes, as described by Chomczynski and Sacchi (26) . Twenty milligrams RNA per condition were denatured in 50% formamide, 2.2 M formaldehyde, 20 mM MOPS, pH 7.0, 6% glycerol at 65°C for 15 min, separated by size on gels containing 0.9% agarose and 0.66M formaldehyde, and blotted on GeneScreenTM membranes (NEN Research Products, Dupont, Boston, Mass.). Hybridization conditions were previously described (6) . Bcl-xL cDNA (a 745 bp fragment Flag-Bclx in pcDNA3) was kindly provided by Dr. Núñez (Ann Arbor, Mich.) and labeled with ({alpha}-32P)dCTP by random priming. 18S ribosomal cDNA was a gift from Dr. Rozengurt (UCLA, Los Angeles, Calif.) and was labeled with ({alpha}-32P)dCTP by nick translation reaction. Sequential hybridization with the different probes was performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGF-ß mediates ROS production, which precedes the loss of {Delta}{psi}m, release of cytochrome c, and activation of caspase-3 in fetal hepatocytes in primary culture
The first purpose of this study was to determine whether TGF-ß-induced apoptosis in fetal hepatocytes could be coincident with changes in the mitochondrial transmembrane potential ({Delta}{psi}m) of these cells. We used CMXRos as a fluorescent probe to 1) detect changes in the mitochondrial membrane potential by flow cytometry and 2) visualize mitochondria by confocal laser microscopy. Cells were incubated in the absence (control) or presence of apoptotic concentrations of TGF-ß (2 ng/ml) and treated as described in Materials and Methods. Figure 1A , B shows the results after 12 h of treatment. Due to the heterogeneity of fetal hepatocytes in primary culture (because of their different degree of differentiation), cells presented variable levels of CMXRos fluorescence, but the decrease in intensity was evident when cells were treated with TGF-ß either by flow cytometry (Fig. 1A ) or confocal microscopy (Fig. 1B ). To know whether this decrease in {Delta}{psi}m was related to apoptosis, we analyzed the response to different concentrations of TGF-ß. We had previously found that 0.1 ng/ml of this factor is enough to inhibit growth of fetal hepatocytes without inducing a significant death (5) . However, death is observed from 0.5 to 0.75 ng/ml, with a maximum around 2 ng/ml (6) . As shown in Fig. 1C , CMXRos fluorescence decreased according to the apoptotic dose response. Thus, a greater loss of mitochondrial membrane potential was coincident with a higher proportion of hypodiploid cells when analyzed in dishes treated during 15 h with TGF-ß. A detailed analysis of the time course for {Delta}{psi}m changes showed that the fluorescence started to decrease at 10 h, with a peak at 12 h (Fig. 1D ). The decrease in {Delta}{psi}m was followed by the appearance of hypodiploid cells, which reached the maximum at 15 h. An interesting point was to compare this time course with the ROS production by TGF-ß in these cells. For analysis of intracellular ROS we used the oxidation-sensitive probe DCFH-DA at 5 µM, as we have previously described (6) . As shown in Fig. 1D , and in agreement with our previous results, DCFH-DA fluorescence increased, with a peak around 3–5 h, whereas CMXRos fluorescence has not changed yet. Thus, ROS production preceded the loss in {Delta}{psi}m in the mechanism of apoptosis induced by TGF-ß in fetal hepatocytes. There is an apparent reversion of the changes in {Delta}{psi}m, ROS production and percent of hypodiploid cells after 18–24 h treatment. This could be because at this time the population of cells is enriched in fetal hepatocytes with a lower degree of differentiation that are able to survive to the apoptotic effect of TGF-ß, as we previously described (27) .



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Changes in the mitochondrial transmembrane potential ({Delta}{psi}m) induced by TGF-ß in fetal hepatocytes. A) Fetal hepatocytes incubated for 12 h in the absence (control) or presence of 2 ng/ml TGF-ß were detached by tripsinization. After 30 min of incubation with 0.1 µM CMXRos, the intracellular fluorescence intensity was measured in a FACScan flow cytometer. An experiment representative of more than 10 is shown. B) Confocal microscopy images of fetal hepatocytes treated without or with 2 ng/ml TGF-ß and further incubated with PBS and 0.1 µM CMXRos for 30 min. Cellular fluorescence intensity was visualized by using a MRC-1024 laser confocal microscopy. An experiment representative of three is shown. Bar is 25 µm in both cases. C) Dose response analysis of CMXRos fluorescence. Fetal hepatocytes incubated for 12 h with different concentrations of TGF-ß were treated as described in panel A. Parallel dishes incubated during 15 h with TGF-ß were processed for analysis of nuclear DNA content by flow cytometry as described in Materials and Methods to calculate the percentage of hypodiploid (apoptotic) cells. An experiment representative of three is shown. D) Fetal hepatocytes incubated for different times with 2 ng/ml TGF-ß were treated to analyze 1) {Delta}{psi}m (as described in panel A), 2) ROS production (by measuring cellular fluorescence intensity after 30 min incubation with 5 µM DCFH-DA), and 3) nuclear DNA content (as described in panel C). An experiment representative of three is shown.

In view of these results, we decided to study whether the loss in {Delta}{psi}m could be coincident with the release of cytochrome c. After incubation of the cells for 14 h in the absence (C) or presence of 2 ng/ml of TGF-ß (T), mitochondria were separated from cytosol and cytochrome c content was analyzed by Western blot as described in Materials and Methods. As shown in Fig. 2A , cytochrome c content decreased considerably in the mitochondria, whereas it appeared in the cytosol. In contrast, the levels of albumin (an abundant protein in fetal liver used as a control) did not suffer any change (results not shown). Cytochrome c oxidase (an enzymatic protein located in the inner mitochondrial membrane) presented identical activity in mitochondrial extracts from control and TGF-ß-treated cells (Fig. 2A ), being absent in cytosolic extracts. To corroborate the functionality of cytochrome c in the cytosol, we assayed caspase 3 activity in cell extracts. After incubation of the cells for 14 h in the absence (C) or presence of 2 ng/ml of TGF-ß (T), cells were scraped, lysed, and protein was extracted as described in Materials and Methods. Caspase 3 activity was assayed using the fluorescent substrate Ac-DEVD-AMC. Results are shown in Fig. 2B . TGF-ß-treated cells presented an increase of 10-fold in caspase-3 activity. A detailed time course of these responses (release of cytochrome c and caspase 3 activity) showed that they started at 8–10 h, reaching a maximum between 12–14 h (results not shown), coincident with the decrease in {Delta}{psi}m. Furthermore, these effects were observed only at apoptotic concentrations of TGF-ß, and not at lower concentrations (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Release of cytochrome c and activation of caspase 3 induced by TGF-ß in fetal hepatocytes. A) Release of cytochrome c from mitochondria to cytosol. After incubation of cells for 14 h in the absence (C) or presence of 2 ng/ml TGF-ß (84), mitochondria were separated from cytosol and cytochrome c content was analyzed by Western blot, as described in Materials and Methods. The activity of cytochrome c oxidase, an enzymatic protein located in the mitochondrial membrane, was also analyzed to demonstrate the specificity of the effect and it is expressed as µmol oxidized cytochrome c/min x mg protein. An experiment representative of five is shown, (U.D., undetectable). B) Caspase 3 activity. After incubation of cells for 14 h in the absence (C) or presence of 2 ng/ml TGF-ß (T), cells were lysed and caspase 3 was assayed using the fluorescent substrate Ac-DEVD-AMC, as described in Materials and Methods. A unit is defined as the amount of active enzyme necessary to produce an increase in 1 arbitrary unit in the luminescence spectrophotometer after 2 h incubation with the reaction mixture. Results are expressed as units/µg protein and are mean ± SE of five independent experiment with duplicate dishes. Data were compared among them by the Student’s t test, *P < 0.01.

TGF-ß produces a decrease in Bcl-xL levels without affecting expression and/or translocation to mitochondria of Bax
We next decided to analyze whether TGF-ß could modulate the expression of some of the Bcl-2 family of pro- and antiapoptotic proteins. For this, after incubation of the cells for 14 h in the absence (C) or presence of TGF-ß (T), we extracted proteins and analyzed the expression of the Bcl-2 family proteins by Western blot. We could not find any Bcl-2 expression in our cells, but Bcl-x was expressed. A major band corresponding to 28 kDa appeared, which indicated that we were visualizing Bcl-xL (Fig. 3A ). This band considerably decreased in TGF-ß-treated cells (Fig. 3A ). In contrast, we could not observe any band at 21 kDa (Bcl-xS, the proapoptotic form) in either the absence or presence of TGF-ß. With respect to the proapoptotic forms of the family, we have analyzed Bax expression. Bax exhibited an apparent molecular mass of 21 kDa that did not suffer any change in response to TGF-ß (Fig. 3A ). To completely exclude a possible role for Bax in the TGF-ß-induced apoptosis in fetal hepatocytes, we next investigated the effect of this agent on translocation of the proapoptotic Bax protein from cytosol to mitochondria membrane. Results are presented in Fig. 3B . Western blot analysis of mitochondrial proteins revealed no change in Bax levels between control and TGF-ß-treated cells. In contrast and in agreement with the results described above, mitochondrial Bcl-xL levels considerably decreased in TGF-ß-treated cells. To further study the possible regulatory role of TGF-ß on Bcl-xL expression, we analyzed the time course and the dose response; results are presented in Fig. 3C , D , respectively. As shown, the decrease in Bcl-xL levels was coincident with the loss in {Delta}{psi}m (no significant changes in Bax expression were observed, results not shown). Furthermore, the dose-dependence of the effect was identical to that one shown in Fig. 1C for the loss in {Delta}{psi}m and the appearance of hypodiploid cells. As a control, we analyzed the expression of albumin, which does not reveal any change in response to TGF-ß. To know whether TGF-ß could be modulating bcl-xL mRNA levels, we performed a Northern blot analysis. As can be seen in Fig. 3E , a detailed time course experiment showed that bcl-xL mRNA levels started to decrease 5 h after TGF-ß treatment. Thus, the decrease in bcl-xL mRNA levels preceded the decay in Bcl-xL protein. All these results suggest that TGF-ß is regulating bcl-xL expression in a dose- and time-dependent manner, coincident with its apoptotic effect on these cells.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 3. Effect of TGF-ß on Bcl-xL and Bax expression in fetal hepatocytes. A) After 12 h incubation of cells without (C) or with 2 ng/ml TGF-ß (T), proteins were extracted and the levels of Bcl-xL and Bax were analyzed by Western blot, as described in Materials and Methods. An experiment representative of five is shown. B) After 12 h incubation of cells without (C) or with 2 ng/ml TGF-ß (T), mitochondria were separated from cytosol and the levels of Bcl-xL and Bax were analyzed by Western blot, as described in Materials and Methods. An experiment representative of three is shown. C) Time course analysis of the effect of 2 ng/ml TGF-ß on Bcl-xL protein levels analyzed by Western blot. Albumin content is analyzed as control. An experiment representative of three is shown. D) Dose response effect of TGF-ß on the decrease in Bcl-xL protein levels analyzed by Western blot after incubation for 12 h of fetal hepatocytes with different concentrations of TGF-ß. An experiment representative of three is shown. Albumin content is analyzed as control. E) Time course analysis of the effect of 2 ng/ml TGF-ß on bcl-xL mRNA levels. 20 µg total RNA, extracted from the pooled cells of two 92 mm diameter dishes, were denatured, separated by size on gels containing 0.9% agarose and 0.66M formaldehyde, and blotted on GeneScreenTM membranes. Serial hybridizations with bcl-xL cDNA and 18S ribosomal cDNA were performed as described in Materials and Methods. An experiment representative of two is shown.

We have previously described that TGF-ß-induced apoptosis in fetal hepatocytes may be precluded by EGF (7) . It has been noted that EGF increases bcl-xL mRNA and protein levels in human keratinocytes (28) . We decided to study whether EGF was able to prevent the down-regulation in bcl-xL expression induced by TGF-ß in fetal hepatocytes. Results are shown in Fig. 4 . The presence of 20 ng/ml EGF was able to completely prevent the decrease in Bcl-xL protein levels induced by TGF-ß (Fig. 4A ). A similar result was obtained when we analyzed bcl-xL expression at mRNA levels (results not shown). Under these conditions, the decrease in CMXRos fluorescence and the release of cytochrome c were completely blocked (Fig. 4B , C ) and activation of caspase 3 was attenuated (Fig. 4D ). To prevent these changes and induce survival in fetal hepatocytes, EGF had to be added simultaneously with or up to 6 h after TGF-ß. If added later (when bcl-xL was down-regulated), the survival effect was clearly abolished (results not shown). These results emphasize the important role of Bcl-xL in the apoptotic process induced by TGF-ß in fetal hepatocytes and indicate that a straight correlation exists between Bcl-xL levels and the mitochondrial events leading to apoptosis.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. EGF prevents the decrease in Bcl-xL levels and the mitochondrial collapse induced by TGF-ß in fetal hepatocytes. A) Bcl-xL levels: After 12 h incubation of fetal hepatocytes without (C) or with 2 ng/ml TGF-ß (T), 20 ng/ml EGF (E), or 2 ng/ml TGF-ß + 20 ng/ml EGF (E+T), proteins were extracted and the levels of Bcl-xL and Bax were analyzed by Western blot. An experiment representative of three is shown. B) CMXRos Fluorescence: fetal hepatocytes were incubated for 12 h under the same conditions described in panel A. Results are expressed in each case as % with respect to control in the absence of TGF-ß and are mean ± SE of 6 independent experiments. Data were compared among them by the Student’s t test, *P < 0.01. C) Release of cytochrome c from mitochondria to cytosol. After incubation of cells for 14 h under the same conditions described in panel A, mitochondria were separated from cytosol and cytochrome c content was analyzed by Western blot. An experiment representative of five is shown. D) Caspase 3 activity. After incubation of cells as described in panel A, cells were lysed and caspase 3 assayed as described in Materials and Methods and Fig. 2B . Results are mean ± SE of five independent experiment with duplicate dishes. Data were compared among them by the Student’s t test, *P < 0.05; **P < 0.01.

Role of ROS in the regulation of Bcl-xL expression, disruption of {Delta}{psi}m, and cytochrome c release induced by TGF-ß in fetal hepatocytes
Since TGF-ß modulated bcl-xL expression in fetal hepatocytes (Fig. 4) and the decrease in its mRNA levels were coincident with the increase in ROS production (compare data shown in Fig. 1D with those in Fig. 3E ), we next decided to analyze whether radical scavengers could prevent the decrease in Bcl-xL levels. We previously reported that antioxidants or radical scavengers prevented the apoptosis induced by TGF-ß in fetal hepatocytes (6) . One of the most powerful combinations was pyrrolidine carbodithioic acid (PDTC) + ascorbic acid (ASC) (6) . We decided to use these compounds to analyze the role of ROS in the regulation of Bcl-xL expression by TGF-ß in fetal hepatocytes. Figure 5A shows that the presence of radical scavengers, such as PDTC + ASC prevented the decay in protein levels induced by TGF-ß. Identical results were observed when we analyzed bcl-xL mRNA levels (results not shown). We also used diphenyl iodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor that has been used to inhibit the TGF-ß-induced H2O2 production (29) . DCFH-DA fluorescence decreased in DPI-treated hepatocytes to 35–40% with respect to untreated cells, in agreement with other results previously described in adult hepatocytes (30) . TGF-ß-induced increase in peroxide content (DCFH-DA fluorescence in 6 h TGF-ß-treated cells: 140–150% with respect to control in three independent experiments) was not observed whether hepatocytes were incubated in the presence of DPI (DCFH-DA fluorescence in 6 h TGF-ß + DPI-treated cells: 37–44% with respect to control in three independent experiments). Under these conditions, the decrease in Bcl-xL is not observed (Fig. 5B ). Finally, in the presence of a pro-oxidant such as BSO (DL-buthionine-(S,R)-sulfoximine), an inhibitor of glutathione synthesis that we previously used to decrease intracellular glutathione content in fetal hepatocytes (9) , the decrease in Bcl-xL was more accentuated (Fig. 5C ). These results indicated that the oxidative stress induced by TGF-ß could be ruling the decrease in Bcl-xL levels. To further confirm this hypothesis, we decided to examine whether exogenous peroxide (ter-butyl-hydroperoxide, TBH) would be able to influence Bcl-xL levels. As shown in Fig. 5D , 0.25 mM TBH alone produced a decrease in Bcl-xL as early as 1 h after incubation with the factor without affecting the levels of other proteins, such as albumin, used as control.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 5. Role of ROS in the regulation of Bcl-xL levels by TGF-ß in fetal hepatocytes. A—C) Levels of Bcl-xL analyzed by Western blot with 100 µg protein from cells incubated for 12 h without (C) or 2 ng/ml TGF-ß (T): A) Effect of the presence of radical scavengers (1 mM ascorbate + 50 µM PDTC). An experiment representative of three is shown. B) Effect of the presence of 20 µM diphenyl iodonium (DPI), an inhibitor of the NADPH oxidase complex that inhibits the TGF-ß-induced H2O2 production. An experiment representative of three is shown. C) Effect of the presence of an inhibitor of glutathione synthesis (1 mM BSO). An experiment representative of three is shown. D) Effect of 0.25 mM ter-butyl-hydroperoxide on Bcl-xL levels analyzed by Western blot using 100 µg protein extracted from cells incubated during different times with this factor. An experiment representative of two is shown.

Next, we decided to focus our attention on the effect of antioxidants such as PDTC + ASC on all the other intracellular events related to the apoptotic mechanism induced by TGF-ß. These agents prevented the increase in peroxide content induced by TGF-ß in fetal hepatocytes and, as expected, completely blocked the decrease in glutathione content, i.e., the oxidative stress produced by this cytokine (Fig. 6A ). Under these conditions, TGF-ß did not decrease {Delta}{psi}m (Fig. 6B ). Furthermore, the cytochrome c release (Fig. 6C ), and the activation of caspase 3 (Fig. 6D ) induced by TGF-ß were completely abolished when ASC + PDTC were present. Thus, antioxidant conditions prevented the decrease in Bcl-xL levels (Fig. 5) , mitochondria collapse, and release of cytochrome c (Fig. 6) .



View larger version (33K):
[in this window]
[in a new window]
  		Figure 6. Protective effect of radical scavengers on the intracellular events induced by TGF-ß in fetal hepatocytes. A) Left: ROS intracellular content analyzed by DCFH-DA fluorescence, after 5 h in the absence (C) or presence of TGF-ß (2 ng/ml) (T). Effect of the presence of 1 mM ascorbate + 50 µM PDTC. Right: glutathione intracellular content analyzed after 8 h in the absence (C) or presence of TGF-ß (2 ng/ml) (T). Effect of the presence of 1 mM ascorbate + 50 µM PDTC. In both cases (left and right), results are expressed as % control (untreated cells) and are mean ± SE of three independent experiments with duplicate dishes. Data were compared to their relative control (untreated cells, in the absence of ASC + PDTC) by the Student’s t test: *P < 0.01. B) Cells incubated in the absence (left) or presence (right) of 1 mM ascorbate + 50 µM PDTC were treated for 12 h without (control) or with TGF-ß (2 ng/ml). {Delta}{psi}m was analyzed by CMXRos fluorescence as described in Fig. 1A . An experiment representative of four is shown. C) Cytochrome c release analyzed after 14 h in the absence (C) or presence of TGF-ß (2 ng/ml) (T) as described in Fig. 2A . Effect of the presence of 1 mM ascorbate + 50 µM PDTC. An experiment representative of three is shown. D) Caspase 3 activity analyzed after 14 h in the absence (C) or presence of TGF-ß (2 ng/ml) (T) as described in Fig. 2B . Effect of the presence of 1 mM ascorbate + 50 µM PDTC. Results are mean ± SE of four independent experiments. Data were compared to control by the Student’s t test, *P < 0.01.

Taken together, we can conclude from these results that in the apoptosis process induced by TGF-ß in fetal hepatocytes, ROS production could be responsible for the decrease in Bcl-xL protein levels and mitochondrial-mediated apoptosis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Apoptosis is a programmed process responsible for the ordered removal of superfluous, aged, or damaged cells. Under homeostasis conditions, each mitosis must be compensated by one event of apoptosis. Thus, the control of cell death constitutes one of the key events in biology. Numerous observations suggest that TGF-ß plays an important biological role mediating hepatocyte apoptosis (6 , 31 , 32) . However, the mechanisms by which this cytokine induces cell death are not completely understood. In this work we demonstrate the participation of the mitochondria in the apoptotic process initiated by TGF-ß in fetal hepatocytes. Two pieces of evidence shown here support this idea. First, this factor induces disruption of the {Delta}{psi}m (Fig. 1) . This event correlates with apoptotic doses of TGF-ß and precedes the appearance of hypodiploid cells. Second, {Delta}{psi}m disruption is followed by the release of cytochrome c (Fig. 2) , which is coincident with activation of caspase 3 (Fig. 2) . These results agree with those recently reported by Rodrigues et al. in adult hepatocytes (20) .

The family of Bcl-2-related proteins constitutes one of the biologically most important classes of apoptosis regulatory gene products (33 , 34) . We show that TGF-ß induces in fetal hepatocytes a decrease in the levels of Bcl-xL (Fig. 3) , an antiapoptotic member of the Bcl-2 family capable of preventing cytochrome c release (15 , 19) . Down-regulation of Bcl-xL occurs only at apoptotic doses of this factor (Fig. 3D ), and time course analysis of the process indicates that the decrease in protein levels correlate with changes in {Delta}{psi}m, release of cytochrome c, and activation of caspase 3 (Fig. 3C ). In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family that had been related to TGF-ß-induced apoptosis in some cells (35) , but not in others (36) . In fact, it has recently been reported that TGF-ß does not change Bax expression but induces its translocation from cytosol to mitochondria in adult hepatocytes (20) . Although Bax expression does not appear to be affected in TGF-ß-treated fetal hepatocytes, the ratio Bcl-xL/Bax clearly decreases. The decrease in Bcl-xL would induce cytochrome c release, since this protein may function by regulating the electrical and osmotic homeostasis of mitochondria (37) and in closing the mitochondrial porin channel by binding to it (15) . Moreover, Bax might facilitate cytochrome c release either by interacting with the permeability transition pore complex (38) and/or by forming oligomers, which act as channels that trigger cytochrome c release from mitochondria (39) . TGF-ß appears to modulate bcl-xL mRNA levels (Fig. 3E ), which could suggest a regulation on gene transcription and/or mRNA stability, but we cannot exclude a possible additional effect at the translational level and/or protein stability. Although it has recently been reported that TGF-ß can down-regulate Bcl-xL protein levels in hepatoma cells (40 , 41) or in mouse hepatocytes (42) , our results suggest for the first time a correlation between this effect and changes in the mitochondrial membrane permeability. We also demonstrate that EGF, an important survival signal for TGF-ß-induced apoptosis in fetal hepatocytes (7) , maintains Bcl-xL levels, preventing the {Delta}{psi}m collapse and the release of cytochrome c (Fig. 4) , which indicates the straight correlation between Bcl-xL levels and the mitochondrial events in the apoptotic process induced by TGF-ß in fetal hepatocytes.

Various observations indicate that some of the TGF-ß actions may be mediated by oxidative stress. It has been shown that this cytokine activates an H2O2-generating NADH oxidase (29) and increases ROS intracellular content in different cell types (6 , 29 , 43 44 45) . In addition, the intracellular oxidized state after treatment of rat hepatocytes with TGF-ß has been linked to a decrease in expression of antioxidative enzymes, such as catalase and superoxide dismutase (46) . ROS appear to be involved not only in the apoptosis mediated by this factor (6 , 44) , but also in some of its transcriptional effects (43 , 45) . We show here that TGF-ß-mediated ROS production in fetal hepatocytes precedes the loss of {Delta}{psi}m and the release of cytochrome c (Fig. 1D ). In agreement with this model of cell death: 1) the decrease in superoxide levels also blocks the loss of {Delta}{psi} m in a model of activated T cells apoptosis (22) , 2) remarkable elevations of ROS precede megamitochondria formation in a model of hepatocyte cell death (23) and 3) ROS generation also precedes mitochondrial permeability transition, cytochrome c release, and caspase activation in hepatocytes treated with GD3 ganglioside (47) . Furthermore, results presented in this paper indicate that ROS may be responsible for the decrease in Bcl-xL protein levels (Fig. 5) . First, the presence of radical scavengers (such as ascorbic acid and PDTC) or inhibitors of ROS production, (such as DPI) blocks the decrease in Bcl-xL levels (Fig. 5A , B ); furthermore, the presence of glutathione synthesis inhibitors, such as BSO, accentuated the effect (Fig. 5C ). Second, the incubation of fetal hepatocytes in the presence of ter-butyl-hydroperoxide alone produces a decrease in Bcl-xL as early as 1 h after incubation with the factor, without affecting the levels of other proteins (Fig. 5D ). The presence of radical scavengers, which completely block the oxidative stress generated by TGF-ß (Fig. 6A ), also abolishes the {Delta}{psi}m collapse, cytochrome c release, and activation of caspase-3 in TGF-ß-treated hepatocytes (Fig. 6B , C , D ). These results suggest that ROS and Bcl-xL levels play an essential role in the mitochondrial-dependent apoptosis elicited by TGF-ß in fetal hepatocytes. It has previously been reported that mitochondrial permeability transition may be induced by ROS generating systems such as alloxan, xanthine/xanthine oxidase, 5-aminolevulinic acid, endoperoxide, or ter-butyl-hydroperoxide (23 , 48 , 49) . However, this effect has been associated mainly with oxidation of mitochondrial membrane protein thiols (50) . Here we show that, in addition to this oxidative effect, ROS could also act by regulating the expression of some mitochondrial components such as Bcl-xL. The regulation of gene expression by oxidants, antioxidants, and the redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications. At least three well-defined transcription factors—nuclear factor kappa B, activator protein-1, and signal transducers and activators of transcription—have been identified to be regulated by the intracellular redox state (51 , 52) . Bcl-xL expression appears to be regulated by the three families of transcription factors (53 54 55) . Further work will be necessary to completely understand the molecular mechanism by which TGF-ß, through oxidative stress, regulates Bcl-xL expression in fetal hepatocytes.

Oxidative stress is considered to be an important condition to promote cell death in response to a variety of signals and pathophysiological situations. The results presented in this paper suggest that ROS can mediate the mitochondrial-dependent apoptotic response to a physiological, extracellular factor such as TGF-ß in fetal hepatocytes. A summary of the proposed model is presented in Fig. 7 . During the apoptosis mediated by TGF-ß in fetal hepatocytes, ROS production (which precedes the loss of {Delta}{psi}m, the release of cytochrome c, and the activation of caspase 3) may be responsible for the decrease in Bcl-xL protein levels and the induction of mitochondrial-dependent apoptosis. Thus, ROS would play an essential role upstream the mitochondria. Production of ROS would be dependent on protein synthesis, as described previously (9 , 29) . Thus, the mechanism by which TGF-ß acts inducing apoptosis could include 1) transcriptional induction of redox-related genes, 2) formation of ROS, and 3) loss of bcl-xL (and potentially other survival proteins) expression, cytochrome c release, and caspase 3 activation, culminating in cell death.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. Summary of the proposed mechanism for the apoptosis induced by TGF-ß in fetal hepatocytes in primary culture. TGF-ß would induce the transcription of redox-related genes, which activate the formation of ROS. The oxidative stress would contribute to the down-regulation of bcl-xL expression, the loss of {Delta}{psi}m, the release of cytochrome c and activation of caspase 3. Factors able to prevent bcl-xL down-regulation, such as EGF, and those that block production of ROS (i.e., cycloheximide or DPI) or scavenge them (i.e., ascorbate and/or PDTC) prevent mitochondrial collapse and cell death.


   ACKNOWLEDGMENTS
 
The authors wish to thank Drs. G. Núñez (Ann Arbor, USA) and E. Rozengurt (UCLA, Los Angeles, Calif.) for providing plasmids and A. Vázquez for expert assistance with the flow cytometer. We also thank Drs. J. Gil and A. López-Rivas for helpful discussions. The studies presented in this paper were supported by a grant from the Ministerio de Educación y Cultura (PM97/0052). B.H. and A.S. were recipients of fellowships from the Ministerio de Educación y Cultura and Comunidad de Madrid, respectively.

Received for publication May 22, 2000. Revision received August 14, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Patel, T., Roberts, L. R., Jones, B. A., Gores, G. J. (1998) Dysregulation of apoptosis as a mechanism of liver disease: an overview. Semin. Liver Dis. 18,105-114[Medline]
  2. Pitot, H. C. (1998) Hepatocyte death in hepatocarcinogenesis. Hepatology 28,1-5[Medline]
  3. Massague, J., Chen, Y. G. (2000) Controlling TGF-ß signaling. Genes Dev 14,627-644[Free Full Text]
  4. Thorgeirsson, S. S., Teramoto, T., Factor, V. (1998) Regulation of apoptosis in hepatocellular carcinoma. Semin. Liver Dis. 18,115-122[Medline]
  5. Sánchez, A., Álvarez, A. M., Benito, M., Fabregat, I. (1995) Transforming growth factor beta modulates growth and differentiation of fetal hepatocytes in primary culture J. Cell. Physiol. 165,398-405
  6. Sánchez, A., Álvarez, A. M., Benito, M., Fabregat, I. (1996) Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive oxygen intermediates. J. Biol. Chem. 271,7416-7422[Abstract/Free Full Text]
  7. Fabregat, I., Sánchez, A., Álvarez, A. M., Nakamura, T., Benito, M. (1996) Epidermal growth factor, but not hepatocyte growth factor, suppresses the apoptosis induced by transforming growth factor-beta in fetal hepatocytes in primary culture. FEBS Lett 384,14-18[Medline]
  8. Inayat-Hussain, S. H., Couet, C., Cohen, G. M., Cain, K. (1997) Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes. Hepatology 25,1516-1526[Medline]
  9. Sánchez, A., Álvarez, A. M., Benito, M., Fabregat, I. (1997) Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor beta in fetal rat hepatocytes in primary culture. Hepatology 26,935-943[Medline]
  10. Green, D. R., Reed, J. C. (1998) Mitochondria and apoptosis. Science 281,1309-1312[Abstract/Free Full Text]
  11. Kroemer, G., Reed, J. C. (2000) Mitochondrial control of cell death. Nature Med 6,513-519[Medline]
  12. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., Wang, X. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275,1129-1132[Abstract/Free Full Text]
  13. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275,1132-1136[Abstract/Free Full Text]
  14. Crompton, M. (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341,233-249
  15. Shimizu, S., Narita, M., Tsujimoto, Y. (1999) Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature (London) 399,483-487[Medline]
  16. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., Wang, X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489[Medline]
  17. Marzo, I., Brenner, C., Zamzami, N., Jürgensmeier, J. M., Susin, S. A., Vieira, H. L. A., Prévost, M.-C., Xie, Z., Matsuyama, S., Reed, J. C., Kroemer, G. (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281,2027-2031[Abstract/Free Full Text]
  18. Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., Tsujimoto, Y. (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95,14681-14686[Abstract/Free Full Text]
  19. Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., Cotter, T. G., Green, D. R. (1999) Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274,2225-2233[Abstract/Free Full Text]
  20. Rodrigues, C. M., Ma, X., Linehan-Stieers, C., Fan, G., Kren, B. T., Steer, C. J. (1999) Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 6,842-854[Medline]
  21. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., Kroemer, G. (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182,367-377[Abstract/Free Full Text]
  22. Hildeman, D. A., Mitchell, T., Teague, T. K., Henson, P., Day, B. J., Kappler, J., Marrack, P. C. (1999) Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10,735-744[Medline]
  23. Karbowski, M., Kurono, C., Wozniak, M., Ostrowski, M., Teranishi, M., Nishizawa, Y., Usukura, J., Soji, T., Wakabayashi, T. (1999) Free radical-induced megamitochondria formation and apoptosis. Free Radic. Biol. Med. 26,396-409[Medline]
  24. Roncero, C., Lorenzo, M., Fabregat, I., Benito, M. (1989) Rates of lipogenesis in fetal hepatocytes in suspension and in primary culture: hormonal effects. Biochim. Biophys. Acta 1012,320-324[Medline]
  25. Lowerson, S. A., Taylor, L., Briggs, H. L., Turnbull, D. M. (1992) Measurement of the activity of individual respiratory chain complexes in isolated fibroblast mitochondria. Anal. Biochem. 205,372-374[Medline]
  26. Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156-159[Medline]
  27. Sánchez, A., Álvarez, A. M., López-Pedrosa, J. M., Roncero, C., Benito, M., Fabregat, I. (1999) Apoptotic response to TGF-ß in fetal hepatocytes depends upon their state of differentiation. Exp. Cell Res. 252,281-291[Medline]
  28. Stoll, S. W., Benedict, M., Mitra, R., Hiniker, A., Elder, J. T., Núñez, G. (1998) EGF receptor signalling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-xL. Oncogene 16,1493-1499[Medline]
  29. Thannickal, V. J., Fanburg, B. L. (1996) Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1. J. Biol. Chem. 270,30334-30338[Abstract/Free Full Text]
  30. Caraceni, P., Ryu, H. S., van Thiel, D. H., Borle, A. B. (1995) Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation. Biochim. Biophys. Acta 1268,249-254[Medline]
  31. Oberhammer, F. A., Pavelka, M., Sharma, S., Tiefenbacher, R., Purchio, A. F., Bursch, W., Schulte-Hermann, R. (1992) Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1. Proc. Natl. Acad. Sci. USA 89,5408-5412[Abstract/Free Full Text]
  32. Fan, G., Ma, X., Kren, B. T., Steer, C. J. (1996) Regulation of apoptosis-associated genes in the regenerating liver. Oncogene 12,1909-1919[Medline]
  33. Adams, J. M., Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281,1322-1326[Abstract/Free Full Text]
  34. Fadeel, B., Zhivotovsky, B., Orrenius, S. (1999) All along the watchtower: on the regulation of apoptosis regulators. FASEB J 13,1647-1657[Abstract/Free Full Text]
  35. Teramoto, T., Kiss, A., Thorgeirsson, S. S. (1998) Induction of p53 and Bax during TGF-beta1 initiated apoptosis in rat liver epithelial cells. Biochem. Biophys. Res. Commun. 251,56-60[Medline]
  36. Saltzman, A., Munro, R., Searfoss, G., Franks, C., Jaye, M., Ivashchenko, Y. (1998) Transforming growth factor-ß-mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL down-regulation. Exp. Cell Res. 242,244-254[Medline]
  37. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T., Thompson, C. B. (1997) Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91,627-637[Medline]
  38. Brenner, C., Cadiou, H., Vieira, H. L., Zamzami, N., Marzo, I., Xie, Z., Leber, B., Andrews, D., Duclohier, H., Reed, J. C., Kroemer, G. (2000) Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene 19,329-336[Medline]
  39. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., Martinou, J. C. (2000) Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem. J. 345,271-278
  40. Yamamoto, M., Fukuda, K., Miura, N., Suzuki, R., Kido, T., Komatsu, Y. (1998) Inhibition by dexamethasone of transforming growth factor beta1-induced apoptosis in rat hepatoma cells: a possible association with Bcl-xL induction. Hepatology 27,959-966[Medline]
  41. Shima, Y., Nakao, K., Nakashima, T., Kawakami, A., Nakata, K., Hamasaki, K., Kato, Y., Eguchi, K., Ishii, N. (1999) Activation of caspase-8 in transforming growth factor-beta-induced apoptosis of human hepatoma cells. Hepatology 30,1215-1222[Medline]
  42. Christensen, J. G., Gonzales, A. J., Cattley, R. C., Goldsworthy, T. L. (1998) Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens. Cell Growth Differ 9,815-825[Abstract]
  43. Ohba, M., Shibanuma, M., Kuroki, T., Nose, K. (1994) Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J. Cell Biol. 126,1079-1088[Abstract/Free Full Text]
  44. Lafon, C., Mathieu, C., Guerrin, M., Pierre, O., Vidal, S., Valette, A. (1996) Transforming growth factor beta 1-induced apoptosis in human ovarian carcinoma cells: protection by the antioxidant N-acetylcysteine and bcl-2. Cell Growth Differ 7,1095-1104[Abstract]
  45. García-Trevijano, E. R., Iraburu, M. J., Fontana, L., Domínguez-Rosales, J. A., Auster, A., Covarrubias-Pinedo, A., Rojkind, M. (1999) Transforming growth factor ß1 induces the expression of {alpha} 1(I) procollagen mRNA by a hydrogen peroxide-C/EBPß-dependent mechanism in rat hepatic stellate cells. Hepatology 29,960-970[Medline]
  46. Kayanoki, Y., Fujii, J., Suzuki, K., Kawata, S., Matsuzawa, Y., Taniguchi, N. (1994) Suppression of antioxidative enzyme expression by transforming growth factor-beta 1 in rat hepatocytes. J. Biol. Chem. 269,15488-15492[Abstract/Free Full Text]
  47. García-Ruíz, C., Colell, A., París, R., Fernández-Checa, J. C. (2000) Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation. FASEB J 14,847-858[Abstract/Free Full Text]
  48. Kowaltowski, A. J., Vercesi, A. E. (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radic. Biol. Med. 26,463-471[Medline]
  49. Byrne, A. M., Lemasters, J. J., Nieminen, A.-L (1999) Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butyl hydroperoxide in rat hepatocytes. Hepatology 29,1523-1531[Medline]
  50. Constantini, P., Chernyak, B. V., Petronilli, V., Bernardi, P. (1996) Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271,6746-6751[Abstract/Free Full Text]