(Received for publication, June 30, 1995; and in revised form, November 27, 1995)
From the
Transforming growth factor- (TGF-
), a growth regulator
of fetal hepatocytes in primary culture, also regulates death of these
cells. Dose-response analysis showed that the TGF-
concentration
needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed
to inhibit growth in these cells (0.5 ng/ml). In response to TGF-
,
hepatocytes induced DNA fragmentation and the appearance of nuclei with
a DNA content lower than 2C (diploid content), typical of a programmed
cell death model. TGF-
-induced apoptosis in fetal hepatocytes was
preceded by an induction of reactive oxygen species production and a
decrease in the glutathione intracellular content, indicating that this
factor induces oxidative stress in fetal hepatocytes. Studies performed
to analyze levels of c-fos mRNA, a gene whose expression is
modulated by redox state, demonstrated that only high, apoptotic
concentrations of TGF-
(2.5 ng/ml) produced an increase in the
mRNA levels of this gene, the level of induction being similar to that
found when cells were incubated in the presence of tert-butyl
hydroperoxide. Gel mobility shift assays showed that the
c-fos-induced expression was coincident with an increase in
AP-1 activity. Finally, cell death induced by TGF-
in fetal
hepatocytes was partially blocked by radical scavengers, which
decreased the percentage of apoptotic cells, whereas these agents did
not modify the growth-inhibitory effect elicited by TGF-
in these
cells. In summary, the results presented in this paper provide evidence
for the involvement of an oxidative process in the apoptosis elicited
by TGF-
in fetal hepatocytes.
Mechanisms that regulate cell death are essential for normal
development and maintenance of homeostasis. Cell death can be
developmentally controlled, apoptosis being the most common morphologic
expression of such programmed cell death(1) . Lethal cellular
programs that lead to apoptosis may be triggered by a variety of
exogenous and environmental stimuli. Transforming growth factor-
(TGF-
) (
)is one of the best known physiological
inhibitors of epithelial cell proliferation. It is a member of a large
family of structurally related factors that play a critical role during
embryogenesis in mammals, frogs, and flies(2, 3) .
TGF-
is particularly multifunctional, being able to regulate cell
proliferation, differentiation, and morphogenesis, and recently
attention has turned to its possible role in cell death. Localized cell
death by apoptosis was described in Drosophila by mutation in
a gene coding for a TGF-
homolog(4) . TGF-
1 is one of
the genes activated by the initiation of apoptosis during prostate
regression(5) ; it has been found to stimulate apoptotic cell
death in cultured human gastric carcinoma cells (6) and in
endometrial stromal cells(7) ; and, furthermore, it both
inhibits proliferation and increases apoptosis in cultured uterine
epithelial cells(8) , in rat hepatocytes(9) , and in
hepatoma cells(10) .
The mechanisms by which TGF-1
exerts its effect are still only partly understood. Cross-linking
experiments have revealed that most cells contain three types of
receptors (for a review, see (11) ). The type III receptor (or
betaglycan) is a proteoglycan with a short cytoplasmic domain and is
not likely to mediate any of the known biological activities of
TGF-
. The type I (65-70-kDa) and type II (85-110-kDa)
receptors have recently been cloned and identified as transmembrane
serine/threonine (Ser/Thr) kinases, both receptors being essential for
signaling. Recent advances in the characterization of the cell cycle
machinery have stimulated studies aimed at understanding how TGF-
signaling leads to growth arrest in the late G
phase of the
cell cycle(12) . In spite of these findings, the question of
the signaling pathways from the receptor to the nuclei remains to be
elucidated. Some reports have related the action of TGF-
to the
production of hydrogen peroxide(13, 14) . Reactive
oxygen intermediates are, in general, considered to be cytotoxic and
are implicated in the progression of cancer, inflammation, radiation
injury, and aging (for a review see (15) ). Peroxides and
highly reactive free radicals can also trigger cell death, and although
some investigators believe that oxidation is merely another metabolic
disturbance that leads cells to respond to external stimuli, others
propose a more central role for reactive oxygen species in cell death.
In this context, overexpression of bcl-2, a protooncogene that
is unique among cellular genes in its ability to block apoptotic death
in different cell types (16) decreases lipid peroxidation and
can increase resistance to apoptotic killing by hydrogen peroxide,
menadione, and depletion of glutathione(17) . It has recently
been shown that the Bcl-2 oncoprotein functions as a prooxidant and
presumably protects cells from oxidative stress by activating the
cellular antioxidant defenses(18) . To molecularly dissect
growth arrest and apoptosis induced by TGF-
, Selvakumaran et
al.(19) performed a number of elegant studies showing
that elevated expression of Bcl-2 blocked the TGF-
1-induced
apoptotic pathway but did not prevent the growth arrest induced by this
factor in myeloid leukemia cells.
In light of all these
observations, the aim of our work has been to evaluate the possible
implications of reactive oxygen intermediates in the mechanisms by
which TGF- induces apoptotic death in the liver. Our experimental
model uses fetal hepatocytes in primary culture. These cells are able
to carry out both proliferation and differentiation processes
simultaneously. We have shown that some growth factors such as
epidermal growth factor (EGF), hepatocyte growth factor, or
transforming growth factor-
(20, 21) are able to
induce DNA replication in these cells, whereas some hormones (glucagon,
noradrenaline, glucocorticoids), by themselves or in synergism with the
positive growth factors, regulate the expression of some liver-specific
genes(22, 23, 24) . TGF-
inhibits the
EGF and/or hepatocyte growth factor-induced DNA synthesis and modulates
protooncogene and liver-specific gene expression in these
cells(20, 21, 25, 26) . Furthermore,
high concentrations of this factor induce fetal hepatocyte
death(26) . All of these results suggest that primary cultured
fetal hepatocytes are a good model in which to study the possible role
of TGF-
in the regulation of liver apoptosis during fetal life and
the implication of oxygen radicals in its molecular mechanism.
Figure 1:
Effects of TGF- on growth and cell
viability of cultured rat fetal hepatocytes. A, cells were
cultured in the absence or presence of different concentrations of
TGF-
for 48 h. After this time the number of viable cells was
analyzed by staining with crystal violet. Results are expressed as
percentage of absorbance, at 560 nm, with respect to control
(untreated) cells. Values are the mean ± S.E. of three separate
experiments of triplicate dishes; B, fetal hepatocytes were
cultured in the presence of EGF (20 ng/ml) and in the absence or
presence of different concentrations of TGF-
for 48 h. After this
time, DNA synthesis was analyzed by measuring
[
H]thymidine incorporation into acid-precipitable
material over the last 40 h of culture (0.5 µCi/ml, 1 µM thymidine). A representative experiment of four is shown. C, cells were cultured in the presence of 2.5 (
), 5
(
), and 10 (
) ng/ml TGF-
for different times (8, 12,
24, and 48 h), and cell viability was calculated as described for A.
TGF- induced changes in cell morphology (Fig. 2).
8-10 h after the addition of the factor, cells lose cell contacts
and migrate on the plate (Fig. 2B), and 5-10 h
later (12-20 h in the presence of TGF-
), cellular blebbing
and detachment of degenerated hepatocytes were always observed (Fig. 2C).
Figure 2:
Morphological changes induced by TGF-
in rat fetal hepatocytes in primary culture. Light microscopy
photographs of cultured fetal hepatocytes in the absence (A)
or presence of TGF-
(2.5 ng/ml) for about 8 and 16 h (B and C, respectively). TGF-
induces losing in cell
contacts (B, long arrow), and cells migrate on the
plate (B, short arrow). At longer times, cell
blebbing (C, long arrow) and detachment of the
hepatocytes (C, short arrow) is observed.
Magnification: 200
.
To assess if fetal hepatocyte death
involved a process of apoptosis, we first analyzed the normal nuclear
DNA content by flow cytometry, staining nuclei with propidium iodide (Fig. 3A, upper panel). Cells that had been incubated
in the presence of TGF- (2.5 ng/ml) for 12 h showed between 12 and
15% of the nuclei with a DNA content lower than 2C (Fig. 3A, lower panel). Then, in order to know
if these cells showed fragmentation in their DNA, we isolated the
extranuclear DNA from parallel dishes that had been incubated in the
absence or presence of TGF-
(2.5 ng/ml). A representative
experiment is shown in Fig. 3B. DNA from
TGF-
-treated cells appeared cleaved into a ``ladder,''
which is generally associated with the internucleosomal site cleavage
occurring in the apoptotic process. This effect was first observed
after 6-8 h of treatment and continued throughout for the next
12-16 h (20-24 h in the presence of the factor; data not
shown).
Figure 3:
Effect of TGF- on nuclear DNA content
and DNA fragmentation. A, DNA content histograms of untreated
hepatocytes (top panel) and hepatocytes treated with TGF-
(2.5 ng/ml) (bottom panel) as measured by flow cytometry.
Fetal hepatocytes cultured in the absence or presence of TGF-
for
12 h were collected and treated for analysis of DNA content as
described under ``Materials and Methods.'' A representative
experiment of four is shown. B, after an 8-h incubation of
fetal hepatocytes in the absence (C) or in the presence of
TGF-
(T), cells were scraped and subjected to cytoplasmic
DNA extraction. Purified DNA, from parallel 10-cm diameter dishes, was
electrophoresed in a 1.5% agarose gel and visualized by UV fluorescence
after staining with ethidium bromide. A representative experiment of
five is shown.
Figure 4:
TGF- induction of intracellular
reactive oxygen species content. Fetal hepatocytes cultured in the
absence or presence of TGF-
were detached and incubated for
20-30 min with 5 µM of an oxidation-sensitive
fluorescent probe (DCFH-DA), measuring the fluorescence intensity by
flow cytometry. The top panel shows a representative histogram
of cells untreated (shadowed area) or treated for 3 h with 2.5
ng/ml TGF-
(white area). The bottom panel represents a time course analysis of intracellular reactive oxygen
species content. Cells were incubated for different times (1.5, 3, 12,
and 24 h) in the presence of TGF-
(2.5 ng/ml), and the production
of reactive oxygen species was analyzed as described previously. Data
are expressed as a percentage of DCFH fluorescence with respect to
control value and are means ± S.E. from three independent
experiments.
Figure 5:
Effect of TGF- on glutathione
intracellular levels. Left, after incubating fetal hepatocytes
in the absence or presence of 2.5 ng/ml TGF-
, cells were
collected, and cellular glutathione was extracted and determined
spectrophotometrically as described under ``Materials and
Methods.'' The means of triplicate dishes of a representative
experiment are shown (A). The GSSG/(GSH + GSSG) ratio (%)
in TGF-
-treated cells is presented as a function of time in the
presence of the factor. Control cells (in the absence of TGF-
) did
not show changes in this ratio (data not shown). B, in this same
experiment, the glutathione (GSH + GSSG) intracellular
concentration is presented versus time of TGF-
treatment.
In this case, data are calculated, at each time, as percentage with
respect to control (untreated) cells. Right, cells were
incubated for 3 h in the absence (C) or presence of 2.5 ng/ml
TGF-
(TGF) or 0.25 mM TBH. GSH intracellular
concentrations are expressed as nmol/µg protein and are the mean of
duplicate dishes of three independent experiments. Bars represents S.E.
Figure 6:
Effect of TGF- on c-fos mRNA
levels in cultured fetal hepatocytes. A, cells were cultured
in the absence (lane 1) or presence of 0.5 ng/ml TGF-
(lane 2), 2.5 ng/ml TGF-
(lane 3), and 0.25
mM TBH (lane 4). After 3, 8, 24, and 48 h of
treatment, total RNA was isolated, and 20 µg were subjected to
Northern blot analysis using the cDNA probes specific for fos, ras, and
-fetoprotein mRNAs. An 18 S ribosomal probe was used for
RNA normalization. A representative experiment of three is shown. B, the radioactivity visualized in the Northern blots with
c-fos probe was measured by scanning densitometry and
normalized with the 18 S probe. Data are expressed as percentage of
mRNA levels at zero time (before TGF-
treatment) and are the mean
± S.E. of three independent experiments.
, control;
,
2.5 ng/ml TGF-
.
To further characterize the
cell response to oxidative stress, we looked at AP-1 binding activity.
Nuclear extracts from fetal hepatocytes incubated for 24 h, in the
absence or presence of TGF-, were used in a gel mobility shift
assay using a consensus AP-1 site oligonucleotide as probe. After 24 h
of treatment, TGF-
induced AP-1 binding activity in a
dose-dependent manner (Fig. 7). These results are consistent
with the elevation of c-fos mRNA previously seen at apoptotic
concentrations of TGF-
(2.5 ng/ml).
Figure 7:
Effect of TGF- on the specific
binding to the AP-1. A, 2 µg of nuclear extracts from
control or TGF-
-treated (24 h) fetal hepatocytes were incubated
for 20 min with 0.5 ng of
P-labeled doubled-stranded
oligonucleotide used as AP-1 probe (see ``Experimental
Procedures''). After this time, the mixture was electrophoresed
through a 6% polyacrylamide gel. The autoradiograph of a representative
experiment is shown. B, dose-response increase in AP-1
activity. The radioactivity visualized in the gel mobility shift assays
with AP-1 probe was measured by scanning densitometry. Results are mean
± S.E. of three independent
experiments.
Figure 8:
Effect of radical scavengers on
TGF--induced apoptosis. Left, cells were left untreated
or were treated with 2.5 ng/ml TGF-
alone or in the presence of
different radical scavengers: 50 µM PDTC, 1 mM ascorbic acid (ASC), 300 units/ml superoxide dismutase (SOD), 20 mM NAC, and 5 µM NDGA added 15
min after the TGF-
treatment. Following a 16-h treatment period,
cells were stained with crystal violet, and the viability was
quantified as described under ``Materials and Methods.''
Results are expressed as percentage of control and are means ±
S.E. of triplicate determinations from 3-5 experiments. Right, after 12-h incubation of the cells in the absence (C) or presence of 2.5 ng/ml TGF-
(TGFB) and the
combination of 2.5 ng/ml TGF-
+ 50 µM PDTC (PDTC
+ TGFB), nuclear DNA content was analyzed in a flow cytometer by
staining nuclei with propidium iodide. Representative histograms
together with the proportion of nuclei (%) with a DNA content lower
than 2C are shown.
We have evaluated whether the protective effect on cell death
elicited by the antioxidants is coincident with a decrease in the
percentage of apoptotic cells by measuring nuclear DNA content. When
cells were incubated with TGF- but in the presence of ASC,
superoxide dismutase, or PDTC, the percentage of nuclei with a DNA
content lower than 2C decreased from 13-15% (TGF-
-treated
cells) to about 7-8% (control values were 2-3%). Thus, it
is clearly the case that these radical scavengers offer a high degree
of protection for fetal hepatocytes against TGF-
-induced
apoptosis. A representative experiment for PDTC effect is shown in Fig. 8(right panel).
In contrast to these results
the inhibitory effect of TGF- on fetal hepatocyte growth was not
modified in the presence of radical scavengers. Thus, when these cells
were incubated in the presence of EGF (20 ng/ml) and TGF-
(2.5
ng/ml), the EGF-induced [
H]thymidine
incorporation into DNA was completely blocked by TGF-
, regardless
of whether ascorbate or PDTC were present (Fig. 9). Identical
results were observed when 0.5 ng/ml TGF-
was used (data not
shown). These results seem to indicate that the oxidative stress
induced by TGF-
does not preclude its well understood inhibitory
effect on fetal hepatocyte growth.
Figure 9:
Effect of radical scavengers on TGF-
inhibition of fetal hepatocyte growth. Cells were left untreated (NONE) or treated for 24 h with 20 ng/ml EGF or 20 ng/ml EGF
+ 2.5 ng/ml TGF-
(E + T), alone (C) or in the presence of different radical scavengers: 1
mM ascorbic acid (ASC) or 50 µM PDTC
added 15 min after the TGF-
treatment. DNA synthesis was analyzed
as described under ``Materials and Methods'' after incubating
the cells in the presence of [
H]thymidine (0.5
µCi/ml, 1 µM thymidine) for the previous 20 h. Results
are mean ± S.E. of three independent
experiments.
Programmed cell death, or apoptosis, is the process whereby
cells are induced to activate their own death or cell suicide.
Apoptosis occurs in a wide variety of cell types and is required during
the development of many tissues. Failure to negatively regulate
apoptosis is associated with degenerative diseases, and failure to
positively regulate apoptosis is associated with cancer and autoimmune
diseases (1) . In many models of apoptosis, cells are induced
to die as a result of changes in environmental stimuli, such as growth
factors and hormones. TGF- constitutes one of these apoptotic
factors for some types of cells, such as rat hepatocytes (9) and hepatoma cells (10) .
We have recently shown
that TGF- may be a modulator of both growth and differentiation of
fetal hepatocytes in primary culture(26) . The results
presented here demonstrate that TGF-
also regulates death of these
cells. Hepatocytes die in response to the factor ( Fig. 1and Fig. 2), inducing DNA fragmentation and the appearance of nuclei
with a DNA content lower than 2C (Fig. 3), typical of a
programmed cell death model. These last observations contrast with
those of Oberhammer et
al.(9, 41, 42) , who showed that
apoptosis induced by TGF-
, both in adult hepatocytes and in
regressing liver, did not show DNA fragmentation. These differences are
likely due to the method used for DNA fragmentation analysis. In our
studies, only cytosolic, fragmented DNA is isolated, as in total DNA
isolation experiments it was difficult to observe the typical
oligosomal ladder (results not shown). This may be due to the fact that
these primary cultures do not respond syncronically to TGF-
, and
so, at any given time, not more than 12-15% of the cells show
nuclei with a DNA content lower than 2C (Fig. 3). However, we
cannot rule out the possibility of adult and fetal hepatocytes having
different responses to TGF-
in terms of apoptosis. In contrast,
our results agree with those of Fukuda et al.(10) in
hepatoma cells, where TGF-
induced ladder-pattern DNA cleavage.
TGF--induced apoptosis in fetal hepatocytes is preceded by an
enhancement in reactive oxygen species production (Fig. 4), an
increase in the GSSG/(GSH + GSSG) ratio, and a decrease in the
glutathione intracellular content (Fig. 5). Reduced glutathione
(GSH) is used intracellularly to reduce numerous oxidizing compounds,
including reactive oxygen species. Agents that induce oxidative stress
in hepatocytes, such as tert-butyl hydroperoxide, cause
accumulation of GSSG because the capacity of NADP-dependent
GSSG-reductase becomes rate-limiting and the NADPH/NADP ratio decreases (39, 43) . It has been proposed that efflux of GSSG
from the cell then occurs in order to preserve the cellular normal
redox state so that a depletion in the glutathione levels is always
observed(43) . Thus, an increase in the reactive oxygen species
production and a decrease in the glutathione concentrations indicate
that TGF-
induces an oxidative stress in fetal hepatocytes.
Support for this idea also comes from the observations that (i)
H
O
production by TGF-
has already been
found in bovine pulmonary artery endothelial cells (13) and in
mouse osteoblastic cells(14) , (ii) Kayanoki et al.(44) have recently described that TGF-
supresses the
expression of antioxidative enzymes in adult rat hepatocytes, thereby
showing that production of peroxides is increased in these cells, and
(iii) Abdel-Razzak et al.(45) have shown that
TGF-
down-regulates cytochromes P-450 1A1 and 1A2, two genes whose
expression is modulated by oxidative stress (46) in adult
hepatocytes. However, at present it is not clear if TGF-
-induced
peroxide production may cause growth inhibition, apoptosis, or both
things. The results presented in this paper clearly relate TGF-
reactive oxygen species production to fetal hepatocyte cell death.
First, low concentrations of TGF-
(0.5 ng/ml) sufficient to
completely block fetal hepatocyte growth (Fig. 1) do not induce
reactive oxygen intermediate production in these cells (results not
shown). Second, studies performed to analyze c-fos expression,
as a gene modulated by redox state(40, 47) ,
demonstrate that only high, apoptotic concentrations of TGF-
(2.5
ng/ml) produce an increase in its mRNA levels; this induction is
coincident with an increase in the nuclear AP-1 binding activity ( Fig. 6and Fig. 7). Finally, TGF-
-induced cell death
in fetal hepatocytes may be either partially blocked by single radical
scavengers (Fig. 8) or totally blocked by combinations of these.
However, these agents do not preclude the TGF-
growth-inhibitory
effect in these cells (Fig. 9). Antioxidants, such as ascorbic
acid (vitamin C) or PDTC, a thiol compound and radical scavenger, have
proved to be the most potent inhibitors of apoptosis, although the
addition of superoxide dismutase alone to the culture medium also
partially blocks cell death (Fig. 8). These results strongly
suggest that TGF-
may be inducing apoptosis in fetal hepatocytes
through the generation of reactive oxygen intermediates.
N-Acetyl-L-cysteine or NDGA are unable to prevent
apoptosis induced by TGF- in the fetal hepatocytes. The addition
of NAC to cells increases intracellular glutathione levels (48) , but the utilization of NAC by rat hepatocytes is limited
by their rate of uptake and conversion to cysteine(49) .
Moreover, García-Ruiz et al.(50) have recently shown that NAC-treatment does not
result in a significant increment of the mitochondrial pool GSH,
despite significantly increasing the cytosol GSH pool size, so that
this compound is probably unable to counteract a mitochondrial
oxidative stress in hepatocytes. NDGA is a 5-, 12-, and 15-lipoxygenase
inhibitor. Lipoxygenase metabolites have been implicated in TNF
responses such as cytotoxicity, induction of the transcription factor
c-fos, and the mitochondrial superoxide radical scavenging
enzyme, manganous superoxide dismutase (for review, see (51) ).
The inability of NDGA to protect against TGF-
-induced apoptosis in
fetal hepatocytes indicates that the lipoxygenase pathway is unlikely
to be responsible for the oxidative stress induced by TGF-
in
these cells.
Elevated expression of c-fos has previously
been related to programmed cell death(52) . However, the time
course analysis of c-fos mRNA levels shows that its gene
expression increases just when cells stop dying ( Fig. 1and Fig. 6). So, we can speculate that c-fos might be a
survival gene in fetal hepatocytes. Moreover, we have found a
correlation between c-fos mRNA expression and AP-1 activity
after 24 h of TGF- treatment (Fig. 7). These results
support the idea that functional Fos, in its AP1 form, may be involved
in cell response to TGF-
-induced oxidative stress and in cell
survival.
Reactive oxygen intermediates, such as hydrogen peroxides
or oxygen radicals are, in general, cytotoxic. The involvement of
oxygen radicals in inflammation or aging processes is well established.
In recent years, on the basis of the elegant studies by Kane et al.(53) and Hockenbery et al.(17) oxidative stress and radical oxygen intermediates
have been proposed as integral control elements in the cell's
decision to enter apoptosis. Results presented in this paper provide
evidence for the involvement of an oxidative process in the apoptosis
elicited by TGF- in fetal hepatocytes. Further work will be
necessary to completely understand the molecular mechanism by which
high concentrations of TGF-
induce oxygen radical production and
apoptosis, whereas lower concentrations of this factor (enough to
inhibit cell growth) do not.