Reactive oxygen species (ROS) mediates the
mitochondrial-dependent apoptosis induced by
transforming growth factor ␤ in fetal hepatocytes
BLANCA HERRERA,* ALBERTO. M. ÁLVAREZ,† ARÁNZAZU SÁNCHEZ,*
MARGARITA FERNÁNDEZ,* CÉSAR RONCERO,* MANUEL BENITO,* AND
ISABEL FABREGAT*,1
*Departamento de Bioquı́mica y Biologı́a Molecular, Instituto de Bioquı́mica, Centro Mixto CSIC/
UCM and †Centro de Citometrı́a de Flujo y Microscopı́a Confocal, Facultad de Farmacia,
Universidad Complutense de Madrid, 28040 Madrid, Spain
ABSTRACT Treatment of fetal rat hepatocytes with
transforming growth factor beta (TGF-␤) is followed by
apoptotic cell death. Analysis of radical oxygen species
(ROS) content and mitochondrial transmembrane po-
tential (⌬␺m), using specific fluorescent probes in
FACScan and confocal microscopy, showed that TGF-␤
mediates ROS production that precedes the loss of
⌬␺m, the release of cytochrome c, and the activation of
caspase 3. TGF-␤ 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 ⌬␺m collapse and release of cytochrome c.
The presence of radical scavengers blocks the decrease
in bcl-xL levels, ⌬␺m collapse, cytochrome c release,
and activation of caspase 3; in contrast, the presence of
glutathione synthesis inhibitors such as BSO accentu-
ated the effect. The incubation of fetal hepatocytes in
the presence of ter-butyl-hydroperoxide alone pro-
duces a decrease in bcl-xL. These results indicate that
during the apoptosis mediated by TGF-␤ in fetal hepa-
tocytes, ROS may be responsible for the decrease in
bcl-xL mRNA levels that precedes the loss of ⌬␺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) medi-
ates the mitochondrial-dependent apoptosis induced by
transforming growth factor ␤ in fetal hepatocytes.
FASEB J. 15, 741–751 (2001)
Key Words: cytochrome c 䡠 caspases 䡠 Bcl-x
The mechanisms that regulate cell death are essential
for normal development and maintenance of ho-
meostasis. 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 apopto-
sis; second, liver atrophy occurs by apoptosis of liver
cells in the absence of regeneration; and third, patho-
0892-6638/01/0015-0741 © FASEB
physiological processes can trigger the cellular apopto-
tic machinery leading to disease processes (for a review,
see ref 1). Endogenous factors such as transforming
growth factor beta (TGF-␤ 1), activin A, Fas ligand, and
tumor necrosis factor alpha (TNF-␣) may be involved in
the induction of liver apoptosis (2). The TGF-␤ family
comprises a great many structurally related polypeptide
factors, each capable of regulating several cellular
processes, including cell proliferation, lineage determi-
nation, differentiation, motility, adhesion, and death
(3). Numerous observations suggest that TGF-␤ plays
an important biological role mediating hepatocyte apo-
ptosis (1). Furthermore, disruption of the TGF-␤ path-
way at the prereceptor, receptor, or postreceptor levels
occurs in hepatocellular carcinomas and can cause
dysregulation of apoptosis (4). However, the mecha-
nisms by which TGF-␤ induces cell death are still only
partly understood.
In previous works from our group, we have found
that TGF-␤ inhibits growth of fetal hepatocytes, arrest-
ing cells in G1 and down-regulating myc expression (5).
But when used at higher concentrations, TGF-␤ 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-␤ in fetal hepatocytes is blocked by radical scaven-
gers, which decrease the percentage of apoptotic cells
(6). All these results provide evidence for the involve-
ment of an oxidative process necessary for the apopto-
sis induced by TGF-␤ in hepatocytes. It has also been
reported that processing/activation of caspase 3 is
involved in TGF-␤-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 pro-
1
Correspondence: Dpto. de Bioquı́mica y Biologı́a Molecular,
Facultad de Farmacia, Universidad Complutense de Madrid,
28040 Madrid, Spain. E-mail: isabelf@eucmax.sim.ucm.es
741
cesses (oxidative stress and apoptosis), and the synthe-
sis of a new protein might be necessary in the upstream
events responsible for the activation of ROS produc-
tion.
Recent reports have provided evidence that mito-
chondria 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 pro-
caspases, 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 multi-
protein 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 circum-
stances. 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 po-
tential (⌬␺ m), which has been defined as an early stage
of apoptosis (10, 11).
Recently, Rodrigues et al. have demonstrated that
TGF-␤ decreases ⌬␺ m and provokes cytochrome c
release in adult hepatocytes (20). However, a question
that warrants addressing is whether the loss of ⌬␺ 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 gener-
ated only after loss of ⌬␺ 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 permeabili-
zation both in vitro and in vivo (11). In agreement with
this last idea, decreasing superoxide levels blocks the
loss of ⌬␺ m in a model of activated T cell apoptosis
(22) and remarkable elevations of ROS precede me-
gamitochondria 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
742 Vol. 15 March 2001 The FASEB Journal
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-␤ in fetal
hepatocytes and their possible relation to the oxidative
stress generated by this factor.
MATERIALS AND METHODS
Materials
Human recombinant TGF-␤ 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 diac-
etate (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 Biotech-
nology (Santa Cruz, Calif.). Anti-rat albumin polyclonal anti-
body was from Nordic Immunological Laboratories (Tilburg,
The Netherlands). Anti-cytochrome c monoclonal antibody
and the caspase 3 substrate Ac-DEVD-AMC were from Phar-
Mingen (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 (2.5⫻106 cells/fetus) as described
previously (24) and plated on plastic (noncoated) dishes in
arginine-free medium 199, supplemented with ornithine (200
␮M), fetal calf serum (10%), penicillin (120 ␮g/ml), and
streptomycin (100 ␮g/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 cy-
tometry 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 ␮M 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 ␮M
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
HERRERA ET AL.
(Bio-Rad) and Confocal Assistance (Free Software by Todd-
Clark-Brelje).
Measurement of intracellular ROS
For visualization and analysis of intracellular ROS, the oxida-
tion-sensitive probe DCFH-DA was used, as previously de-
scribed (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 ␮M 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 analy-
sis, 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 ␮M 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 ␮g/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 Teflon™ pestle
(Overhead Stirrer, Wheaton Instruments, Millville, N.J.). Hy-
pertonic 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 concentra-
tion of lysates was determined using the Bio-Rad (Hercules,
Calif.) protein assay kit according to the manufacturer’s
specifications. After electrophoresis separation of 50 ␮g pro-
tein/condition in sodium dodecyl sulfate (SDS) -12% poly-
acrylamide, 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 perox-
ide-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, mea-
sured photometrically at 550 nm as described previously (25).
ROS MEDIATES MITOCHONDRIAL-DEPENDENT APOPTOSIS BY TGF-␤
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 ␮l cellular lysates, 325 ␮l assay buffer (20 mM
HEPES pH 7.5, 10% glycerol, 2 mM dithiothreitol), and 20
␮M caspase 3 substrate (Ac-DEVD-AMC). After 2 h incubation
in the dark, enzymatic activity was measured in a Lumines-
cence Spectrophotometer (Perkin Elmer LS-50) (␭excitation,
380 nm; ␭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 spectro-
photometer 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/␮g 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, glutathi-
one 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 centrifu-
gation 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 ␮g/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 anal-
ysis of cytochrome c release section). After protein concen-
tration analysis of cell lysates, by using the Bio-Rad protein
assay kit, 75–100 ␮g protein/condition were separated by
SDS-12% polyacrylamide gel electrophoresis, and transferred
to nitrocellulose membranes. Immunoblot sequential incuba-
tions with primary antibodies (1:1000 dilution) and second-
ary 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 formalde-
hyde, and blotted on GeneScreenTM membranes (NEN Re-
search Products, Dupont, Boston, Mass.). Hybridization con-
ditions 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 (␣-32P)dCTP by
random priming. 18S ribosomal cDNA was a gift from Dr.
Rozengurt (UCLA, Los Angeles, Calif.) and was labeled with
743
Figure 1. Changes in the mitochondrial transmembrane potential (⌬␺m) induced by TGF-␤ in fetal hepatocytes. A) Fetal
hepatocytes incubated for 12 h in the absence (control) or presence of 2 ng/ml TGF-␤ were detached by tripsinization. After
30 min of incubation with 0.1 ␮M 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-␤ and further incubated with PBS and 0.1 ␮M 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 ␮m in both
cases. C) Dose response analysis of CMXRos fluorescence. Fetal hepatocytes incubated for 12 h with different concentrations of
TGF-␤ were treated as described in panel A. Parallel dishes incubated during 15 h with TGF-␤ 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-␤ were treated to analyze 1) ⌬␺m (as described in panel A), 2) ROS production (by measuring cellular fluorescence
intensity after 30 min incubation with 5 ␮M DCFH-DA), and 3) nuclear DNA content (as described in panel C). An experiment
representative of three is shown.

(␣-32P)dCTP by nick translation reaction. Sequential hybrid-
ization with the different probes was performed.
RESULTS
TGF-␤ mediates ROS production, which precedes the
loss of ⌬␺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-␤-induced apoptosis in fetal hepatocytes
could be coincident with changes in the mitochondrial
transmembrane potential (⌬␺m) of these cells. We used
CMXRos as a fluorescent probe to 1) detect changes in
the mitochondrial membrane potential by flow cytom-
etry and 2) visualize mitochondria by confocal laser
microscopy. Cells were incubated in the absence (con-
trol) or presence of apoptotic concentrations of TGF-␤
(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 hepato-
cytes in primary culture (because of their different
degree of differentiation), cells presented variable lev-
els of CMXRos fluorescence, but the decrease in inten-
sity was evident when cells were treated with TGF-␤
either by flow cytometry (Fig. 1A) or confocal micros-
copy (Fig. 1B). To know whether this decrease in ⌬␺m
was related to apoptosis, we analyzed the response to
different concentrations of TGF-␤. We had previously
found that 0.1 ng/ml of this factor is enough to inhibit
growth of fetal hepatocytes without inducing a signifi-
cant 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-␤. A detailed analysis of the time course for ⌬␺m
changes showed that the fluorescence started to de-
crease at 10 h, with a peak at 12 h (Fig. 1D). The
decrease in ⌬␺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-␤ in these cells. For

744 Vol. 15 March 2001 The FASEB Journal HERRERA ET AL.

analysis of intracellular ROS we used the oxidation-
sensitive probe DCFH-DA at 5 ␮M, as we have previ-
ously described (6). As shown in Fig. 1D, and in
agreement with our previous results, DCFH-DA fluores-
cence increased, with a peak around 3–5 h, whereas
CMXRos fluorescence has not changed yet. Thus, ROS
production preceded the loss in ⌬␺m in the mechanism
of apoptosis induced by TGF-␤ in fetal hepatocytes.
There is an apparent reversion of the changes in ⌬␺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-␤, as we previ-
ously described (27).
In view of these results, we decided to study whether
the loss in ⌬␺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-␤ (T),
mitochondria were separated from cytosol and cyto-
chrome 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 enzy-
matic protein located in the inner mitochondrial mem-
brane) presented identical activity in mitochondrial
extracts from control and TGF-␤-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-␤ (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-␤-treated cells presented an increase of 10-fold in
caspase-3 activity. A detailed time course of these re-
sponses (release of cytochrome c and caspase 3 activity)
showed that they started at 8 –10 h, reaching a maxi-
mum between 12–14 h (results not shown), coincident
with the decrease in ⌬␺m. Furthermore, these effects
were observed only at apoptotic concentrations of
TGF-␤, and not at lower concentrations (data not
shown).
TGF-␤ produces a decrease in Bcl-xL levels without
affecting expression and/or translocation to
mitochondria of Bax
We next decided to analyze whether TGF-␤ could
modulate the expression of some of the Bcl-2 family of
pro- and antiapoptotic proteins. For this, after incuba-
tion of the cells for 14 h in the absence (C) or presence
of TGF-␤ (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
ROS MEDIATES MITOCHONDRIAL-DEPENDENT APOPTOSIS BY TGF-␤
Figure 2. Release of cytochrome c and activation of caspase 3
induced by TGF-␤ in fetal hepatocytes. A) Release of cyto-
chrome c from mitochondria to cytosol. After incubation of
cells for 14 h in the absence (C) or presence of 2 ng/ml
TGF-␤ (84), mitochondria were separated from cytosol and
cytochrome c content was analyzed by Western blot, as
described in Materials and Methods. The activity of cyto-
chrome 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 ␮mol
oxidized cytochrome c/min ⫻ 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-␤ (T), cells were
lysed and caspase 3 was assayed using the fluorescent sub-
strate 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/␮g 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.
kDa appeared, which indicated that we were visualizing
Bcl-xL (Fig. 3A). This band considerably decreased in
TGF-␤-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-␤. 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-␤ (Fig. 3A). To completely
exclude a possible role for Bax in the TGF-␤-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-␤-treated cells. In con-
trast and in agreement with the results described above,
mitochondrial Bcl-xL levels considerably decreased in
TGF-␤-treated cells. To further study the possible reg-
ulatory role of TGF-␤ 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
745
 We have previously described that TGF-␤-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-␤
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-␤ (Fig. 4A). A similar result was obtained when we

Figure 3. Effect of TGF-␤ on Bcl-xL and Bax expression in

fetal hepatocytes. A) After 12 h incubation of cells without (C)
or with 2 ng/ml TGF-␤ (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 repre-
sentative of five is shown. B) After 12 h incubation of cells
without (C) or with 2 ng/ml TGF-␤ (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-␤ 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-␤ on the decrease in
Bcl-xL protein levels analyzed by Western blot after incuba-
tion for 12 h of fetal hepatocytes with different concentra-
tions of TGF-␤. 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-␤ on bcl-xL
mRNA levels. 20 ␮g total RNA, extracted from the pooled
cells of two 92 mm diameter dishes, were denatured, sepa-
rated 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 Figure 4. EGF prevents the decrease in Bcl-xL levels and the
cDNA were performed as described in Materials and Meth- mitochondrial collapse induced by TGF-␤ in fetal hepato-
ods. An experiment representative of two is shown. cytes. A) Bcl-xL levels: After 12 h incubation of fetal hepato-
cytes without (C) or with 2 ng/ml TGF-␤ (T), 20 ng/ml EGF
(E), or 2 ng/ml TGF-␤ ⫹ 20 ng/ml EGF (E⫹T), proteins
⌬␺m (no significant changes in Bax expression were were extracted and the levels of Bcl-xL and Bax were analyzed
observed, results not shown). Furthermore, the dose- by Western blot. An experiment representative of three is
dependence of the effect was identical to that one shown. B) CMXRos Fluorescence: fetal hepatocytes were
shown in Fig. 1C for the loss in ⌬␺m and the appearance incubated for 12 h under the same conditions described in
of hypodiploid cells. As a control, we analyzed the panel A. Results are expressed in each case as % with respect
to control in the absence of TGF-␤ and are mean ⫾ se of 6
expression of albumin, which does not reveal any independent experiments. Data were compared among them
change in response to TGF-␤. To know whether TGF-␤ by the Student’s t test, *P ⬍ 0.01. C) Release of cytochrome c
could be modulating bcl-xL mRNA levels, we per- from mitochondria to cytosol. After incubation of cells for
formed a Northern blot analysis. As can be seen in Fig. 14 h under the same conditions described in panel A,
3E, a detailed time course experiment showed that mitochondria were separated from cytosol and cytochrome c
bcl-xL mRNA levels started to decrease 5 h after TGF-␤ content was analyzed by Western blot. An experiment repre-
treatment. Thus, the decrease in bcl-xL mRNA levels sentative of five is shown. D) Caspase 3 activity. After incuba-
tion of cells as described in panel A, cells were lysed and
preceded the decay in Bcl-xL protein. All these results caspase 3 assayed as described in Materials and Methods and
suggest that TGF-␤ is regulating bcl-xL expression in a Fig. 2B. Results are mean ⫾ se of five independent experi-
dose- and time-dependent manner, coincident with its ment with duplicate dishes. Data were compared among them
apoptotic effect on these cells. by the Student’s t test, *P ⬍ 0.05; **P ⬍ 0.01.

746 Vol. 15 March 2001 The FASEB Journal HERRERA ET AL.

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-␤. If added later (when bcl-xL was down-regu-
lated), 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-␤ in fetal hepatocytes and indicate that a straight
correlation exists between Bcl-xL levels and the mito-
chondrial events leading to apoptosis.

Role of ROS in the regulation of Bcl-xL expression,

disruption of ⌬␺m, and cytochrome c release induced
by TGF-␤ in fetal hepatocytes

Since TGF-␤ modulated bcl-xL expression in fetal hepa-

tocytes (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 previ-
ously reported that antioxidants or radical scavengers
prevented the apoptosis induced by TGF-␤ in fetal
hepatocytes (6). One of the most powerful combina-
tions was pyrrolidine carbodithioic acid (PDTC) ⫹
ascorbic acid (ASC) (6). We decided to use these
compounds to analyze the role of ROS in the regula-
tion of Bcl-xL expression by TGF-␤ 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-␤. Identical results were ob-
served when we analyzed bcl-xL mRNA levels (results
not shown). We also used diphenyl iodonium (DPI), a
mitochondrial and microsomal NADPH oxidase inhib-
itor that has been used to inhibit the TGF-␤-induced
H2O2 production (29). DCFH-DA fluorescence de-
creased in DPI-treated hepatocytes to 35– 40% with
respect to untreated cells, in agreement with other
results previously described in adult hepatocytes (30).
TGF-␤-induced increase in peroxide content (DCFH-
DA fluorescence in 6 h TGF-␤-treated cells: 140 –150%
with respect to control in three independent experi-
ments) was not observed whether hepatocytes were
incubated in the presence of DPI (DCFH-DA fluores-
cence in 6 h TGF-␤ ⫹ 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)-sulfoxi-
mine), 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-␤ could be
ruling the decrease in Bcl-xL levels. To further confirm
this hypothesis, we decided to examine whether exog-
Figure 5. Role of ROS in the regulation of Bcl-xL levels by
TGF-␤ in fetal hepatocytes. A—C) Levels of Bcl-xL analyzed by
Western blot with 100 ␮g protein from cells incubated for
12 h without (C) or 2 ng/ml TGF-␤ (T): A) Effect of the
presence of radical scavengers (1 mM ascorbate ⫹ 50 ␮M
PDTC). An experiment representative of three is shown. B)
Effect of the presence of 20 ␮M diphenyl iodonium (DPI), an
inhibitor of the NADPH oxidase complex that inhibits the
TGF-␤-induced H2O2 production. An experiment represen-
tative of three is shown. C) Effect of the presence of an
inhibitor of glutathione synthesis (1 mM BSO). An experi-
ment representative of three is shown. D) Effect of 0.25 mM
ter-butyl-hydroperoxide on Bcl-xL levels analyzed by Western
blot using 100 ␮g protein extracted from cells incubated
during different times with this factor. An experiment repre-
sentative of two is shown.
enous 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.
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-␤. These agents prevented the increase
in peroxide content induced by TGF-␤ in fetal hepato-
cytes and, as expected, completely blocked the decrease
in glutathione content, i.e., the oxidative stress pro-
duced by this cytokine (Fig. 6A). Under these condi-
tions, TGF-␤ did not decrease ⌬␺m (Fig. 6B). Further-
more, the cytochrome c release (Fig. 6C), and the
activation of caspase 3 (Fig. 6D) induced by TGF-␤ were
completely abolished when ASC ⫹ PDTC were present.
Thus, antioxidant conditions prevented the decrease in

ROS MEDIATES MITOCHONDRIAL-DEPENDENT APOPTOSIS BY TGF-␤ 747


Figure 6. Protective effect of radical scavengers on the intra-
cellular events induced by TGF-␤ in fetal hepatocytes. A) Left:
ROS intracellular content analyzed by DCFH-DA fluores-
cence, after 5 h in the absence (C) or presence of TGF-␤ (2
ng/ml) (T). Effect of the presence of 1 mM ascorbate ⫹ 50
␮M PDTC. Right: glutathione intracellular content analyzed
after 8 h in the absence (C) or presence of TGF-␤ (2 ng/ml)
(T). Effect of the presence of 1 mM ascorbate ⫹ 50 ␮M
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 ␮M PDTC were treated for 12 h without
(control) or with TGF-␤ (2 ng/ml). ⌬␺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-␤
(2 ng/ml) (T) as described in Fig. 2A. Effect of the presence
of 1 mM ascorbate ⫹ 50 ␮M PDTC. An experiment represen-
tative of three is shown. D) Caspase 3 activity analyzed after
14 h in the absence (C) or presence of TGF-␤ (2 ng/ml) (T)
as described in Fig. 2B. Effect of the presence of 1 mM
ascorbate ⫹ 50 ␮M PDTC. Results are mean ⫾ se of four
independent experiments. Data were compared to control by
the Student’s t test, *P ⬍ 0.01.
Bcl-xL levels (Fig. 5), mitochondria collapse, and re-
lease of cytochrome c (Fig. 6).
Taken together, we can conclude from these results
748 Vol. 15 March 2001 The FASEB Journal
that in the apoptosis process induced by TGF-␤ in fetal
hepatocytes, ROS production could be responsible for
the decrease in Bcl-xL protein levels and mitochondrial-
mediated apoptosis.
DISCUSSION
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-␤
plays an important biological role mediating hepato-
cyte apoptosis (6, 31, 32). However, the mechanisms by
which this cytokine induces cell death are not com-
pletely understood. In this work we demonstrate the
participation of the mitochondria in the apoptotic
process initiated by TGF-␤ in fetal hepatocytes. Two
pieces of evidence shown here support this idea. First,
this factor induces disruption of the ⌬␺m (Fig. 1). This
event correlates with apoptotic doses of TGF-␤ and
precedes the appearance of hypodiploid cells. Second,
⌬␺m disruption is followed by the release of cyto-
chrome 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 hepato-
cytes (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-␤
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 ⌬␺m, release of cyto-
chrome 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-␤-induced
apoptosis in some cells (35), but not in others (36). In
fact, it has recently been reported that TGF-␤ 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 af-
fected in TGF-␤-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-␤ appears to modulate
bcl-xL mRNA levels (Fig. 3E), which could suggest a
HERRERA ET AL.
regulation on gene transcription and/or mRNA stabil-
ity, but we cannot exclude a possible additional effect at
the translational level and/or protein stability. Al-
though it has recently been reported that TGF-␤ 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 impor-
tant survival signal for TGF-␤-induced apoptosis in fetal
hepatocytes (7), maintains Bcl-xL levels, preventing the
⌬␺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-␤ in fetal hepatocytes.
Various observations indicate that some of the TGF-␤
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– 45). In addi-
tion, the intracellular oxidized state after treatment of
rat hepatocytes with TGF-␤ has been linked to a de-
crease 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-␤-mediated
ROS production in fetal hepatocytes precedes the loss
of ⌬␺m and the release of cytochrome c (Fig. 1D). In
agreement with this model of cell death: 1) the de-
crease in superoxide levels also blocks the loss of ⌬␺ m
in a model of activated T cells apoptosis (22), 2)
remarkable elevations of ROS precede megamitochon-
dria 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-␤ (Fig. 6A), also abolishes the ⌬␺m collapse,
cytochrome c release, and activation of caspase-3 in
TGF-␤-treated hepatocytes (Fig. 6B–D). These results
suggest that ROS and Bcl-xL levels play an essential role
in the mitochondrial-dependent apoptosis elicited by
TGF-␤ 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
ROS MEDIATES MITOCHONDRIAL-DEPENDENT APOPTOSIS BY TGF-␤
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 prom-
ising therapeutic implications. At least three well-de-
fined transcription factors—nuclear factor kappa B,
activator protein-1, and signal transducers and activa-
tors of transcription— have been identified to be regu-
lated by the intracellular redox state (51, 52). Bcl-xL
expression appears to be regulated by the three families
of transcription factors (53–55). Further work will be
necessary to completely understand the molecular
mechanism by which TGF-␤, 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-␤ in fetal
hepatocytes. A summary of the proposed model is
presented in Fig. 7. During the apoptosis mediated by
TGF-␤ in fetal hepatocytes, ROS production (which
precedes the loss of ⌬␺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
Figure 7. Summary of the proposed mechanism for the
apoptosis induced by TGF-␤ in fetal hepatocytes in primary
culture. TGF-␤ 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 ⌬␺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 produc-
tion of ROS (i.e., cycloheximide or DPI) or scavenge them
(i.e., ascorbate and/or PDTC) prevent mitochondrial col-
lapse and cell death.
749
of mitochondrial-dependent apoptosis. Thus, ROS
would play an essential role upstream the mitochon-
dria. Production of ROS would be dependent on
protein synthesis, as described previously (9, 29). Thus,
the mechanism by which TGF-␤ 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, culmi-
nating in cell death.
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 recip-
ients of fellowships from the Ministerio de Educación y
Cultura and Comunidad de Madrid, respectively.
REFERENCES
1. Patel, T., Roberts, L. R., Jones, B. A., and Gores, G. J. (1998)
Dysregulation of apoptosis as a mechanism of liver disease: an
overview. Semin. Liver Dis. 18, 105–114
2. Pitot, H. C. (1998) Hepatocyte death in hepatocarcinogenesis.
Hepatology 28, 1–5
3. Massague, J., and Chen, Y. G. (2000) Controlling TGF⫺␤
signaling. Genes Dev. 14, 627– 644
4. Thorgeirsson, S. S., Teramoto, T., and Factor, V. (1998) Regu-
lation of apoptosis in hepatocellular carcinoma. Semin. Liver Dis.
18, 115–122
5. Sánchez, A., Álvarez, A. M., Benito, M., and Fabregat, I. (1995)
Transforming growth factor beta modulates growth and differ-
entiation of fetal hepatocytes in primary culture J. Cell. Physiol.
165, 398 – 405
6. Sánchez, A., Álvarez, A. M., Benito, M., and 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
7. Fabregat, I., Sánchez, A., Álvarez, A. M., Nakamura, T., and
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
8. Inayat-Hussain, S. H., Couet, C., Cohen, G. M., and 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
9. Sánchez, A., Álvarez, A. M., Benito, M., and Fabregat, I. (1997)
Cycloheximide prevents apoptosis, reactive oxygen species pro-
duction, and glutathione depletion induced by transforming
growth factor beta in fetal rat hepatocytes in primary culture
Hepatology 26, 935–943
10. Green, D. R., and Reed, J. C. (1998) Mitochondria and apopto-
sis. Science 281, 1309 –1312
11. Kroemer, G., and Reed, J. C. (2000) Mitochondrial control of
cell death. Nature Med. 6, 513–519
12. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J.,
Peng, T. I., Jones, D. P., and Wang, X. (1997) Prevention of
apoptosis by Bcl-2: release of cytochrome c from mitochondria
blocked. Science 275, 1129 –1132
13. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer,
D. D. (1997) The release of cytochrome c from mitochondria: a
primary site for Bcl-2 regulation of apoptosis. Science 275,
1132–1136
14. Crompton, M. (1999) The mitochondrial permeability transi-
tion pore and its role in cell death. Biochem. J. 341, 233–249
15. Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Bcl-2 family
proteins regulate the release of apoptogenic cytochrome c by
750 Vol. 15 March 2001 The FASEB Journal
the mitochondrial channel VDAC. Nature (London) 399, 483–
487
16. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ah-
mad, M., Alnemri, E. S., and Wang, X. (1997) Cytochrome
c and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell 91,
479 – 489
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., and Kroemer, G. (1998) Bax and adenine nucleo-
tide translocator cooperate in the mitochondrial control of
apoptosis Science 281, 2027–2031
18. Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J.,
Matsuda, H., and 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
19. Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., Cotter,
T. G., and 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
20. Rodrigues, C. M., Ma, X., Linehan-Stieers, C., Fan, G., Kren,
B. T., and 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
21. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho,
A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., and
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
22. Hildeman, D. A., Mitchell, T., Teague, T. K., Henson, P., Day,
B. J., Kappler, J., and Marrack, P. C. (1999) Reactive oxygen
species regulate activation-induced T cell apoptosis. Immunity
10, 735–744
23. Karbowski, M., Kurono, C., Wozniak, M., Ostrowski, M., Teran-
ishi, M., Nishizawa, Y., Usukura, J., Soji, T., and Wakabayashi, T.
(1999). Free radical-induced megamitochondria formation and
apoptosis. Free Radic. Biol. Med. 26, 396 – 409
24. Roncero, C., Lorenzo, M., Fabregat, I., and Benito, M. (1989)
Rates of lipogenesis in fetal hepatocytes in suspension and in
primary culture: hormonal effects. Biochim. Biophys. Acta 1012,
320 –324
25. Lowerson, S. A., Taylor, L., Briggs, H. L., and Turnbull, D. M.
(1992) Measurement of the activity of individual respiratory
chain complexes in isolated fibroblast mitochondria. Anal.
Biochem. 205, 372–374
26. Chomczynski, P., and Sacchi, N. (1987) Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenol-chloro-
form extraction. Anal. Biochem. 162, 156 –159
27. Sánchez, A., Álvarez, A. M., López-Pedrosa, J. M., Roncero, C.,
Benito, M., and Fabregat, I. (1999) Apoptotic response to TGF-␤
in fetal hepatocytes depends upon their state of differentiation.
Exp. Cell Res. 252, 281–291
28. Stoll, S. W., Benedict, M., Mitra, R., Hiniker, A., Elder, J. T., and
Núñez, G. (1998) EGF receptor signalling inhibits keratinocyte
apoptosis: evidence for mediation by Bcl-xL. Oncogene 16, 1493–
1499
29. Thannickal, V. J., and 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
30. Caraceni, P., Ryu, H. S., van Thiel, D. H., and Borle, A. B. (1995)
Source of oxygen free radicals produced by rat hepatocytes
during postanoxic reoxygenation. Biochim. Biophys. Acta 1268,
249 –254
31. Oberhammer, F. A., Pavelka, M., Sharma, S., Tiefenbacher, R.,
Purchio, A. F., Bursch, W., and Schulte-Hermann, R. (1992)
Induction of apoptosis in cultured hepatocytes and in regress-
ing liver by transforming growth factor beta 1. Proc. Natl. Acad.
Sci. USA 89, 5408 –5412
32. Fan, G., Ma, X., Kren, B. T., and Steer, C. J. (1996) Regulation
of apoptosis-associated genes in the regenerating liver. Oncogene
12, 1909 –1919
33. Adams, J. M., and Cory, S. (1998) The Bcl-2 protein family:
arbiters of cell survival. Science 281, 1322–1326
HERRERA ET AL.
34. Fadeel, B., Zhivotovsky, B., and Orrenius, S. (1999) All along the
watchtower: on the regulation of apoptosis regulators. FASEB J.
13, 1647–1657
35. Teramoto, T., Kiss, A., and 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
36. Saltzman, A., Munro, R., Searfoss, G., Franks, C., Jaye, M., and
Ivashchenko, Y. (1998) Transforming growth factor-␤-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
37. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K.,
Schumacker, P. T., and Thompson, C. B. (1997) Bcl-xL regulates
the membrane potential and volume homeostasis of mitochon-
dria. Cell 91, 627– 637
38. Brenner, C., Cadiou, H., Vieira, H. L., Zamzami, N., Marzo, I.,
Xie, Z., Leber, B., Andrews, D., Duclohier, H., Reed, J. C., and
Kroemer, G. (2000) Bcl-2 and Bax regulate the channel activity
of the mitochondrial adenine nucleotide translocator. Oncogene
19, 329 –336
39. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Marti-
nou, 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., and
Komatsu, Y. (1998) Inhibition by dexamethasone of transform-
ing growth factor beta1-induced apoptosis in rat hepatoma cells:
a possible association with Bcl-xL induction. Hepatology 27,
959 –966
41. Shima, Y., Nakao, K., Nakashima, T., Kawakami, A., Nakata, K.,
Hamasaki, K., Kato, Y., Eguchi, K., and Ishii, N. (1999) Activa-
tion of caspase-8 in transforming growth factor-beta-induced
apoptosis of human hepatoma cells. Hepatology 30, 1215–1222
42. Christensen, J. G., Gonzales, A. J., Cattley, R. C., and Goldswor-
thy, T. L. (1998) Regulation of apoptosis in mouse hepatocytes
and alteration of apoptosis by nongenotoxic carcinogens. Cell
Growth Differ. 9, 815– 825
43. Ohba, M., Shibanuma, M., Kuroki, T., and Nose, K. (1994)
Production of hydrogen peroxide by transforming growth fac-
tor-beta 1 and its involvement in induction of egr-1 in mouse
osteoblastic cells. J. Cell Biol. 126, 1079 –1088
44. Lafon, C., Mathieu, C., Guerrin, M., Pierre, O., Vidal, S., and
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
ROS MEDIATES MITOCHONDRIAL-DEPENDENT APOPTOSIS BY TGF-␤
45. Garcı́a-Trevijano, E. R., Iraburu, M. J., Fontana, L., Domı́nguez-
Rosales, J. A., Auster, A., Covarrubias-Pinedo, A., and Rojkind,
M. (1999) Transforming growth factor ␤1 induces the expres-
sion of ␣ 1(I) procollagen mRNA by a hydrogen peroxide-C/
EBP␤-dependent mechanism in rat hepatic stellate cells. Hepa-
tology 29, 960 –970
46. Kayanoki, Y., Fujii, J., Suzuki, K., Kawata, S., Matsuzawa, Y., and
Taniguchi, N. (1994) Suppression of antioxidative enzyme
expression by transforming growth factor-beta 1 in rat hepato-
cytes. J. Biol. Chem. 269, 15488 –15492
47. Garcı́a-Ruı́z, C., Colell, A., Parı́s, R., and 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 acti-
vation. FASEB J. 14, 847– 858
48. Kowaltowski, A. J., and Vercesi, A. E. (1999) Mitochondrial
damage induced by conditions of oxidative stress. Free Radic.
Biol. Med. 26, 463– 471
49. Byrne, A. M., Lemasters, J. J., and 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
50. Constantini, P., Chernyak, B. V., Petronilli, V., and 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
51. Sen, C. K., and Packer, L. (1996) Antioxidants and redox
regulation of gene transcription. FASEB J. 10, 709 –720
52. Simon, A. R., Rai, U., Fanburg, B. L., and Cochran, B. H. (1998)
Activation of the JAK-STAT pathway by reactive oxygen species.
Am. J. Physiol. 275, C1640 –C1652
53. Chen, C., Edelstein, L. C., and Gelinas, C. (2000) The Rel/NF-
kappa B family directly activates expression of the apoptosis
inhibitor Bcl-x(L). Mol. Cell. Biol. 20, 2687–2695
54. Jacobs-Helber, S. M., Wickrema, A., Birrer, M. J., and Sawyer,
S. T. (1998) AP1 regulation of proliferation and initiation of
apoptosis in erythropoietin-dependent erythroid cells. Mol. Cell.
Biol. 7, 3699 –3707
55. Silva, M., Benito, A., Sanz, C., Prosper, F., Ekhterae, D., Núñez,
G., and Fernandez-Luna, J. L. (1999) Erythropoietin can induce
the expression of bcl-x(L) through Stat5 in erythropoietin-
dependent progenitor lines. J. Biol. Chem. 274, 22165–22169
Received for publication May 22, 2000.
Revised for publication August 14, 2000.
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