From the Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom
Received for publication, November 5, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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We have previously shown that
transforming growth factor- Transforming growth factor- TGF- The formation of the Apaf-1-containing apoptosome complex is central to
the activation of the caspase cascade in mitochondrion-mediated cell
death, and we have recently shown that TGF- The mechanism by which TGF- Therefore, we have used DNA microarrays to characterize
time-dependent changes in gene expression during
TGF- Cell Culture--
FaO cells were cultured in Ham's nutrient
mixture F-12 with Glutamax supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin and maintained in a
humidified atmosphere of 5% CO2 at 37 °C. Cells were
seeded at a density of 2 × 104/cm2 on day
0. After 24 h, the medium was changed to low serum Ham's nutrient
mixture F-12 containing 1% fetal calf serum. After another 24 h,
the medium was changed again, and the cells were treated with 0.5 ng/ml
TGF- Annexin V Staining--
Apoptosis was assessed using the annexin
V staining method adapted for adherent cells as previously described
(28). Attached cells were trypsinized (0.5× trypsin/EDTA in
phosphate-buffered saline) and combined with the detached cells,
therefore composing the total cell population, which was then pelleted
(200 × g, 5 min, 4 °C), resuspended in 10 ml of
fresh medium containing 10% fetal calf serum, and incubated for 20 min
at 37 °C. Cells (0.5 × 106) were pelleted and
resuspended in 1 ml of annexin V buffer (10 mM HEPES/NaOH
(pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM
CaCl2). Annexin V (1.5 µl) was added, and the cells were incubated for a further 8 min at room temperature before labeling with
30 µl of propidium iodide (50 µg/ml) for 1 min and subsequent analysis by flow cytometry.
RNA Extraction and Labeling--
Attached cells were removed
from the flasks with a scraper and pelleted (200 × g,
5 min, 4 °C) with the detached cells. Cell pellets were then washed
with ice-cold phosphate-buffered saline, snap-frozen, and stored at
Data processing was carried out using ConvertData Version
3.3.3 2 before importing
files into GeneSpring Version 4.0.4 (Silicon Genetics, Redwood City,
CA) for overall analysis. K-means clustering analysis using
smooth correlation was performed according to the advanced analysis
techniques manual supplied with GeneSpring. Genes were selected as
significantly altered if they showed a consistent change in three
different experiments and the mean value represented a -fold change of
at least ±1.5 (where +1 represents a 100% increase, i.e.
double the control value, and RT-PCR--
Validation of changes in gene expression for nine
selected genes was carried out by RT-PCR. PCR primers for each rat gene were designed using Gene Tool Lite Version
1.0 3 based on sequence data
available from the NCBI Protein
Database.4 cDNA was
prepared from 1 µg of RNA using Superscript RNase H Sequencing--
For practical purposes, only the sequences of
the genes discussed under "Results" were confirmed using an ABI
PRISM® BigDyeTM Terminator Version 3.0 cycle
sequencing kit.
Reagents--
TGF- Global Changes in Gene Expression during
TGF-
To identify potential mediators of TGF-
Experiments with the actinomycin D- and cycloheximide-inhibited FaO
cells showed that both cycloheximide and actinomycin D affected a very
large number of genes that are constitutively expressed (data not
shown). Consequently, it was difficult to identify those genes that
were specifically involved in TGF-
Gene expression profiles of the normalized data were collated from
three independent experiments and revealed that, in all treatments,
~5000 genes (~90% of the total gene array) hybridized successfully
to FaO cDNA. The majority of the genes that were detected showed
little or no alteration in their expression levels. However, after
TGF- Genes Up-regulated by TGF- Cluster A: Extracellular Matrix and Cytoskeleton--
This gene
cluster contains 25 genes, nine of which were significantly induced by
4 h and remained up-regulated throughout the time course (Fig.
2A). This cluster includes a number of genes coding for
structural proteins such as TNNT2
(troponin T),
DSP (desmoplakin),
ARHB (ras homolog gene family, member B),
SDC4 (proteoglycan), and SMTN
(smoothelin), consistent with the
fundamental role played by TGF- Cluster B: Amplification of TGF- Cluster C: TGF- During TGF- Gene Expression Is Significantly Altered in the Presence of
Z-VAD-fmk and TGF- Confirmation of Selected Gene Changes by RT-PCR--
Significant
changes in selected gene expression as detected by the microarrays were
validated by RT-PCR. Due to the large number of gene changes observed,
we selected relevant genes from each of the functionally significant
clusters or groups we had identified by GeneSpring analysis. Thus, from
cluster A (extracellular matrix and cytoskeleton), we selected
CTGF and ARHB for RT-PCR analysis. At 4 h,
both these genes were significantly up-regulated as shown by microarray
analysis (Fig. 2A) and clearly confirmed by RT-PCR (Fig.
5A). Similarly,
GLCLC and SEPP1 from the antioxidant group of
genes were shown to be down-regulated by the microarray analysis (Fig.
3) and confirmed by RT-PCR (Fig. 5A), particularly with
GLCLC, which was markedly down-regulated. The pro-apoptotic gene CASP8 and TANK were shown by the microarray
analysis to be up-regulated at later times (16 h), particularly in the
presence of Z-VAD-fmk; and this was confirmed by RT-PCR (Fig.
5C). However, the RT-PCR results also showed that
TGF- TGF- The apoptotic related gene cluster (cluster B) encodes a number of both
pro- and anti-apoptotic proteins, including XIAP, cIAP2, CASP8, TP53, BAK1,
BAD, and NOTCH4. The up-regulation of CASP8 is in agreement with previous studies showing that
caspase-8 is processed/activated during TGF- Although the main action of IAP proteins is to prevent apoptosis by
inhibiting active caspases (51), XIAP has also been shown to associate
with TGFBR-1 and to potentiate TGF- ATF3 homodimers bind to the ATF/cAMP-responsive element consensus sites
and repress transcription of downstream genes. However, ATF3 also forms
a nonfunctional heterodimer with GADD153, thereby up-regulating gene
transcription (60). GADD153, a small nuclear protein, dimerizes with
other CCAAT/enhancer-binding proteins and is normally expressed at very
low levels, but is markedly up-regulated during endoplasmic reticulum
stress (61) and apoptosis (62). Parallel up-regulation of
ATF3 and GADD153 transcripts has also been
observed during MG132-induced apoptosis in MCF-7 cells (63) and Jurkat
cells.7 These results suggest
that GADD153/ATF3 heterodimers may be formed and that genes normally
repressed by ATF3 would be up-regulated, perhaps facilitating the
necrotic cell death induced by TGF- The stress-response cluster also contains TAB1 and
TANK, which have been implicated in the activation of
NF- The down-regulation of a battery of enzymes that are involved in
protecting the cell against oxidative stress suggests that these genes
are coordinately regulated. Cells treated with agents such as
tert-butylhydroquinone are protected against oxidative stress via the antioxidant-responsive element. Antioxidant-responsive element-like elements have been found in the promoter regions of the
rat NQO1 and human NQO2 (NADPH:quinone
oxidoreductase) genes, rat glutathione synthetase, and other
antioxidant-related enzymes (for review, see Ref. 71; Ref. 72).
Interestingly, a recent microarray analysis of
tert-butylhydroquinone-treated human neuroblastoma cells has
identified 63 genes that are significantly increased, many of which are
involved (including GLCLC, GSR, and GSTM3 (brain glutathione
S-transferase)) in protecting the
cell against oxidative stress (73). In contrast TGF- Another gene that was markedly up-regulated during TGF- The microarray data indicate that TGF-1
(TGF-
1)-induced apoptosis in FaO hepatoma cells is
mediated by cytochrome c release, apoptosome formation, and
caspase activation. Although TGF-
1 acts via the SMAD
signaling pathway to initiate de novo gene transcription,
little is known about the downstream gene targets that are involved in
the regulation of apoptosis. Therefore, in this study, we used in-house
microarrays (~5500 genes) to identify pathway-specific gene
clustering in TGF-
1-treated cells. A total of 142 genes
showed time-dependent changes in expression during
TGF-
1-induced apoptosis. The polycaspase inhibitor
benzyloxycarbonyl-VAD-fluoromethyl ketone, which, on its own, had no
effect on gene transcription, blocked TGF-
1-induced cell
death and significantly altered the expression of 261 genes, including
185 down-regulated genes. Cluster analysis identified up-regulation of
early response genes (0-4 h) encoding for the extracellular matrix and
cytoskeleton, including the pro-apoptotic CTGF gene, and
delayed response genes (8-16 h), including pro-apoptotic genes. A
second delayed response cluster (44 genes) was also observed when
TGF-
1-induced caspase activation was blocked by
benzyloxycarbonyl-VAD-fluoromethyl ketone. This cluster included genes
encoding stress-related proteins (e.g. Jun, ATF3, TAB1, and
TANK), suggesting that their up-regulation may be in response to
secondary necrosis. Finally, we identified an early response set of
nine down-regulated genes that are involved in antioxidant defense. We
propose that the regulation of these genes by TGF-
1
could provide a molecular mechanism for the observed elevation in
reactive oxygen species after TGF-
1 treatment and may
represent the primary mechanism through which TGF-
1
initiates apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
(TGF-
1)1 is an
important cytokine that regulates cell proliferation, differentiation,
matrix accumulation, chemotaxis, and apoptosis in a wide range of cell types (for review, see Ref. 1). TGF-
1 induces apoptotic
cell death in normal and regressing livers (2, 3) and cultured hepatocytes and hepatoma cell lines (2-8). Consequently,
TGF-
1 can cause liver disease by disrupting the normal
homeostasis between cell proliferation and apoptotic cell death (9,
10). The importance of TGF-
1 in controlling liver
homeostasis is demonstrated by the extensive apoptosis and fibrosis
observed in the livers of transgenic mice overexpressing
TGF-
1 (11). Disruption of the TGF-
1
pathway and disregulation of apoptosis have been implicated in
hepatocellular carcinoma (12, 13).
1 induces its many biological effects by binding to
specific receptors on the plasma membrane, initiating a
serine/threonine kinase-catalyzed signaling pathway. Although all the
subsequent downstream effects in the apoptotic pathway have not been
delineated, it is clear that caspase activation is an essential
component, as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
(Z-VAD-fmk), a polycaspase inhibitor, not only blocks activation of
caspase-3 and caspase-7, but also abrogates apoptotic cell death (6, 7). In addition, studies in hepatoma cell lines have shown that
caspase-2, -8, and -9 are also processed and activated during TGF-
1-induced cell death (14, 15). Activation of
effector caspases (caspase-3 and caspase-7), which kill the cell by
cleaving and inactivating/activating key proteins (for review, see
Refs. 16 and 17), can occur by one of two major pathways involving either stimulation of cell-surface death receptors or perturbation of
mitochondria (18). In both pathways, a caspase cascade is activated by
a two-step mechanism in which initiator (apical) procaspases
(procaspase-8 and procaspase-9) are recruited and activated within
large multiprotein complexes known as the death-inducing signaling
complex and the apoptosome, respectively (for review, see Ref. 19). In
the case of mitochondrion-mediated cell death, the release of
cytochrome c is a common response to many apoptotic stimuli
(20, 21), initiating the ATP/dATP-dependent oligomerization of Apaf-1 to form the apoptosome. This large multiprotein complex recruits and facilitates autoprocessing of caspase-9 to form an Apaf-1·caspase-9 holoenzyme complex, which then recruits and
processes the effector caspases (22-27).
1-induced
apoptosis in FaO hepatoma cells induces cytochrome c release
and assembly of the ~700-kDa apoptosome complex, which then activates
the effector caspases (caspase-3 and caspase-7) (28). Cytochrome
c release during TGF-
1-induced apoptosis has
subsequently been confirmed in fetal hepatocytes (29). Furthermore,
overexpression of Bcl-xL (30), an anti-apoptotic Bcl-2
family member, protects against TGF-
1-induced cytochrome
c release and apoptosis in prostate epithelial cells. Thus,
although TGF-
1-induced apoptosis is a receptor-mediated
phenomenon, it does not involve death-inducing signaling complex
formation and direct primary activation of caspase-8 (28), but
acts by triggering the mitochondrial caspase activation pathway.
1 initiates cytochrome
c release is as yet unknown. TGF-
1 induces
its other varied biological effects by acting through specific
transmembrane type I and II serine/threonine kinase receptors (TGFBR-1
and TGFBR-2). The cytokine binds to TGFBR-2, which then phosphorylates
and activates the TGFBR-1 kinase (1, 32), which, in turn,
phosphorylates the receptor-associated Smad2 and Smad3 proteins. These
are then released from the receptor complex and bind to Smad4 to form a
heterotrimeric complex, which translocates to the nucleus (33, 34).
SMAD complexes interact directly or indirectly with
TGF-
1-responsive promoter sequences and, in combination
with other transcription factors, regulate the transcription of
specific genes (1, 35). This mechanism has been well established in a
variety of TGF-
1 signaling paradigms, and recent studies
have shown that overexpression of dominant-negative Smad2 and Smad3 and
also the inhibitor Smad7 not only prevents SMAD-mediated signal
transduction, but also abrogates the apoptotic effects of
TGF-
1 (36, 37). Thus, TGF-
1-induced
apoptosis appears to require changes in the expression levels of key
proteins. In support of this, earlier studies have shown that
TGF-
1-induced apoptosis in adult and fetal rat
hepatocytes is blocked by cycloheximide (7, 38). Furthermore, we have shown that TGF-
1-induced apoptosis in FaO hepatoma cells
requires both de novo transcription and translation, as it
is blocked by actinomycin D and cycloheximide (see Fig. 1) (39). As
TGF-
1-induced apoptosis occurs via transcriptional
activation, some of the resultant and downstream changes in gene
expression must be involved in the release of cytochrome c
and induction of apoptosis.
1-induced apoptosis. Our results have identified
distinct clusters of both up- and down-regulated genes that may act in
a coordinated manner to initiate and amplify the apoptotic program. At
early times, a cluster of genes involved in protection against reactive
oxygen species (ROS) was down-regulated, allowing an increase in ROS,
which would be predicted to induce cytochrome c release and
subsequently apoptosome formation. A second cluster of pro-apoptotic
genes was up-regulated at later times, thereby amplifying the apoptotic
response. Rather surprisingly, in the presence of TGF-
1,
the polycaspase inhibitor Z-VAD-fmk up-regulated a number of genes
involved in stress responses, suggesting that the expression of some
TGF-
1-regulated genes is caspase-dependent.
In conclusion, our studies show that TGF-
1 induces
apoptosis by time-dependent changes in the expression of a
number of critical genes, which then act in a concerted manner to
initiate and propagate apoptotic cell death.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (2 µg/ml stock in 4 mM HCl
containing 1 mg/ml bovine serum albumin) or an HCl control. In some
experiments, cells were pretreated with 50 µM Z-VAD-fmk
(200 mM stock in Me2SO), 0.1 µg/ml
cycloheximide (0.1 mg/ml stock in phosphate-buffered saline), or 250 nM actinomycin D 30 min before TGF-
1 administration.
80 °C. Array construction, RNA extraction and labeling,
hybridization, and analysis of fluorescence were all carried out as
previously described (40), except that the optimal hybridization time
was found to be 48 h.
1 represents a 50% decrease,
i.e. half the control value). This method of -fold change
analysis gives equal emphasis to underexpressed and overexpressed genes
while avoiding the loss of detail that occurs when logarithms are used.
Using this formula [+/
(ext(abs(log(ratio))))
1] to calculate the change in gene expression means that the y
axis can be numbered from positive to negative infinity. The
gene designations used in the figures and tables are those approved by
the HUGO Gene Nomenclature Committee, although in some cases, the
alternative, more usual designation (published) is also shown.
reverse transcriptase (Invitrogen). After an initial 3-min denaturation at 94 °C, specific genes were amplified for 30-40 cycles of
94 °C for 30 s, 55-66 °C for 30 s, and 72 °C for
30 s and a final 5-min extension at 72 °C. PCR products were
separated by electrophoresis on a 2% agarose gel and visualized by
ethidium bromide staining. The following sense and antisense primers,
respectively, were used: FAT10,
5'-ATGGCTTCCTGCGTCTGTGT-3' and 5'-GCTTCTCATCACCCCACTCC-3'; CTGF, 5'-GTGTGAAGACCTACCGGGCTAAGT-3' and
5'-AAGCTATAATGTCCCTC-CCCTGTC-3'; jun,
5'-GGTGGGTGGGGGCTTACAAA-3' and 5'-GGCTGTCCCTCTCCCCTTGC-3'; glyceraldehyde-3-phosphate dehydrogenase, 5'-CGGCAAGTTCAACGGCACAG-3' and 5'-TGCCAGTGAGCTTCCCGTTC-3'; ARHB,
5'-TCCGCAAGAAGCTGGTGGTG-3' and 5'-CTGGGCCGTCTCGAAAACCT-3';
GCLC, 5'-TGTCCCAAGGCTCGCCACTG-3' and
5'-GCGATGCAGCACTCAAAGCC-3'; SEPP1,
5'-TGGGCATGAGCATCTTGGGA-3' and 5'-GGCTGGCTTCTGTGGGGCTT-3';
TANK, 5'-ACGCGAGCAACAGGAACAGC-3' and
5'-CCACAGGCGGAAACTTGACA-3'; ATF3, 5'-GCCATCGTCCCCTGCCTCTC-3' and 5'-CTTCAGGGTTTGGGGTGG-3'; and CASP8,
5'-CGAAGAACTGGCTGCCCTCA-3' and 5'-TCCTCCCGTGCTTTGCTGAA-3'.
1 was purchased from R&D
Systems (Oxford, UK). All cell culture reagents were obtained from
Invitrogen (Paisley, Scotland). The caspase inhibitor Z-VAD-fmk was
purchased from Enzyme Systems Products (Dublin, CA). Annexin V was
purchased from Bender Medsystems Diagnostics GmbH (Vienna, Austria).
All other chemicals were purchased from Sigma (Poole, Dorset, UK).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-induced Apoptosis--
Previous studies have
shown that TGF-
1-induced apoptosis requires de
novo protein synthesis (7, 38) and involves receptor-associated SMAD proteins (36), indicating that TGF-
1-induced
apoptosis requires transcriptional and translational alterations in the expression of key proteins. Therefore, we reasoned that
TGF-
1 should induce a set of early response genes that
would initiate apoptotic cell death and that other genes might be
activated later in response to the cellular changes brought about by
the cell death program. To characterize these changes, we investigated gene expression throughout the time course of
TGF-
1-induced apoptotic cell death and also examined the
effect of transcriptional (actinomycin D) and translational
(cycloheximide) inhibitors on both apoptosis and gene expression. In
addition, we also used the polycaspase inhibitor Z-VAD-fmk to block
caspase activation and thereby elucidate any potential gene changes
caused by activation of the execution phase of the cell death program.
Both actinomycin D and cycloheximide significantly inhibited
TGF-
1-induced apoptosis in FaO cells at 4 and 8 h
after treatment (Fig. 1), although by
16 h, the degree of inhibition was less marked.
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Fig. 1.
Time course of inhibition of
TGF- 1-induced apoptosis in FaO
hepatoma cells. Cells were treated with 0.5 ng/ml
TGF-
1, 50 µM Z-VAD-fmk
(ZVAD.FMK) plus TGF-
1, 0.1 µg/ml
cycloheximide (CHX) plus TGF-
1, 250 nM actinomycin D (act D) plus
TGF-
1, or vehicle control; harvested at the indicated
times; and analyzed for apoptotic cell death using annexin V/propidium
iodide staining as described under "Experimental Procedures."
Results are means ± S.E. from three separate experiments.
Apoptotic cell death is shown in control cells and in cells treated
with TGF-
1, 50 µM Z-VAD-fmk plus
TGF-
1, 0.1 µg/ml cycloheximide plus
TGF-
1, and 250 nM actinomycin D plus
TGF-
1.
1-induced
apoptosis, gene expression profiles were analyzed from the various
treatments using in-house human DNA microarrays containing 5548-5784
cDNA clones and included as many IMAGE clones for apoptotic genes
as were available at the time of the experiments. These arrays have been successfully used to detect the changes in gene expression in
human cell lines after exposure to
various apoptotic stimuli.5 Homologene
software6 was used to
determine, for several key genes, the degree of identity between
curated/calculated human and rat orthologs. In the main, the
observed homology between rat and human clones was >80%. Using cross-species arrays, it is possible to get false negatives, but not
false positives; and in our experiments, only a small number of the
genes on the arrays exhibited no hybridization (see below), thereby
validating our approach.
1-induced apoptosis.
We therefore decided to focus on the time-dependent changes
in gene expression that occur during TGF-
1-induced
apoptosis, with a view to correlating these changes with the
corresponding apoptosis-related biochemical changes that we have
detailed in a previous study (28). In this study, we showed that, in
the first 4 h after TGF-
1 treatment, there was
little or no caspase processing and only a small increase in annexin
V-positive cells (Fig. 1 in this study and Figs. 1-3 in Ref. 28).
After 8 h, caspase processing and activity were initiated and
continued to increase to a maximum between 16 and 24 h. We
therefore examined the gene expression changes at 4, 8, and 16 h
after exposure to TGF-
1, when ~12, ~25, and ~40%
of the cells were apoptotic, respectively (Fig. 1). A second set of
samples that had been co-treated with TGF-
1 and
Z-VAD-fmk was also analyzed. Apoptosis was completely inhibited by
Z-VAD-fmk at 4 and 8 h and was still extensively inhibited
(70-80%) at 16 h.
1 treatment, 142 genes were significantly changed
(>1.5-fold), with 32 genes up-regulated and 110 genes down-regulated.
Unexpectedly, pretreatment with Z-VAD-fmk, which abrogates apoptosis by
blocking downstream caspases, resulted in an additional 44 up-regulated
and 75 down-regulated genes, giving a total of 261 genes with
significantly altered gene expression. These changes were not caused by
Z-VAD-fmk itself, as cells treated with Z-VAD-fmk alone showed no
significant alterations in gene expression at any of the time points.
1 Cluster into Discrete
Functional Groups--
To further investigate the alterations in gene
expression induced by TGF-
1, the 76 up-regulated genes
were analyzed using a clustering algorithm that groups genes according
to their time-dependent expression profiles. This analysis
assumes that co-regulated genes will cluster together and therefore
either have similar functions or are involved in the same biochemical
pathway/phenomenon. A smooth correlation analysis technique using
K-means clustering, as described under "Experimental
Procedures," was used to identify co-regulated genes. Three clusters were
identified (Fig. 2 and Table I). The
TGF-
1-induced changes in gene expression in clusters A
(extracellular matrix and cytoskeleton) and B (apoptosis) were largely
unaffected by co-treatment with TGF-
1 and Z-VAD-fmk, whereas cluster C (stress response) was detected (except for
FAT10) only in the presence of both Z-VAD-fmk and
TGF-
1.
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Fig. 2.
Expression profiles of
TGF- 1-up-regulated gene
clusters. Cells were treated with 0.5 ng/ml TGF-
1,
50 µM Z-VAD-fmk (ZVAD.FMK) plus
TGF-
1, or vehicle control and harvested at the indicated
times, and gene expression was analyzed on DNA microarrays as described
under "Experimental Procedures." Gene expression in
TGF-
1-treated cells versus control cells and
in 50 µM Z-VAD-fmk/TGF-
1-treated cells
versus control cells is shown in A and
B, respectively. Changes in gene expression were quantified
by comparing mRNA intensity in control and
TGF-
1-treated FaO hepatoma cells and analyzed by
GeneSpring software. Genes were selected as significantly altered if
they showed a consistent (i.e. always up or down) change in
three different experiments and the mean value showed a -fold change of
at least ±1.5 (where +1 represents a 100% increase, i.e.
double the control value, and
1 represents a 50% decrease,
i.e. half the control value). The 76 significantly
up-regulated genes were analyzed by GeneSpring and sorted into three
groups using K-means clustering by smooth correlation
as described under "Experimental Procedures." The clusters were
then classified on the basis of the function of the majority of genes.
A, extracellular matrix (ECM) and cytoskeleton.
In addition to the five labeled genes, this cluster contains
ERF, ANXA13, KIAA0824,
SDC4, CITED1, CYR61, C4A,
HREV107, ODC1, BTG2, DSP,
CES1, GPX3, ABCB1, CD5,
NSF
(N-ethylmaleimide-sensitive
factor), S100A6, IREB2,
fos, and DUSP6. B, apoptosis.
Functional details and changes in expression of the proteins encoded by
the genes in this cluster are listed in Table I and Supplemental Table
I, respectively. C, stress response. In addition to the
seven labeled genes, this cluster contains TARBP1,
TSC22, CGR19, CSNK1G2,
BRF1, DCTN4, COL14A1,
TWEAK, SAT, IGFBP5, AES,
NR5A2, CAMP, ID3, APPBP1,
SFRS8, DDIT3/GADD153, and
CASP8AP2/FLASH.
Apoptosis-related genes
1 in regulating
cytoskeletal proteins and in extracellular matrix remodeling (41).
Significantly, this cluster also contains CTGF
(connective tissue growth
factor), which has been shown to induce apoptosis and to
activate caspase-3 in human aortic smooth muscle cells (42).
CTGF belongs to a family of immediate-early growth-response
genes (43), including NOV, ELM1, COP1,
WISP3, and CYR61, which were also found in
cluster A.
1-induced Apoptosis
by Up-regulation of Apoptosis-related Genes--
This apoptotic
cluster contains 26 genes, none of which reached a significant level of
expression until 8 h, but thereafter gradually increased with time
(Fig. 2B and Table I). This cluster contains anti-apoptotic
genes XIAP and cIAP2 and pro-apoptotic genes
CASP8, TP53, BAK1, BAD, and
NOTCH4. Up-regulation of these genes was delayed, and they
are therefore unlikely to play a role in the initial events that
initiate the apoptotic cascade. However, up-regulation of these genes
was generally unaffected by Z-VAD-fmk, suggesting that the
TGF-
1-induced increase in these pro-apoptotic genes
serves to maintain and amplify the apoptotic process.
1 Induces a Stress Response in the
Absence of Caspase Activation--
This cluster contains 25 genes
that, in the main, were up-regulated only in the presence of Z-VAD-fmk.
It contains genes that have been implicated in the activation of
NF-
B, viz. TWEAK, TAB1 (TGF-
1-activated
kinase-binding protein-1), and TANK
(TRAF-associated NF-
B activator) (Fig. 2C).
Several other stress-related transcription factors were also
up-regulated, including ATF3, a member of the CCAAT/enhancer-binding
protein family (44), and Jun, the major form of the AP-1 transcription
factor. Together, these gene changes suggest that the cells may undergo
a stress response that may be important in the caspase-independent cell
death induced by TGF-
1 at later time points (28).
Interestingly, this cluster also contains FAT10, which,
although it was up-regulated in the TGF-
1-alone
treatment, exhibited an even greater increase in the presence of
Z-VAD-fmk. FAT10 is a ubiquitin-like protein that forms covalent
conjugates and induces apoptosis (45).
1-induced Apoptosis, Genes Encoding
Proteins Involved in Antioxidant Defense Are Down-regulated--
Of
the 185 significantly down-regulated genes, including a number of
oncogenes and cell division genes, a substantial proportion (44 genes)
were metabolic enzymes, suggesting that the cells may undergo an
adaptive response and switch off nonessential functions. However, the
most interesting genes that were observed to be down-regulated are
those known to be involved in antioxidant
pathways (Fig. 3 and Table II).
Importantly, these nine genes include both the first, GLCLC
(glutamate-cysteine ligase
catalytic subunit (
-glutamylcysteine synthetase)), and
second, glutathione synthetase, enzymes of the glutathione synthesis
pathway, and all were down-regulated independently of Z-VAD-fmk.
Notably, GLCLC was already down-regulated by 4 h, indicating that this may be a primary transcriptional response. The
down-regulation of antioxidant defense mechanisms may facilitate TGF-
1-induced apoptosis, which is known to involve the
generation of ROS (38).
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Fig. 3.
Expression profiles of antioxidant genes
down-regulated by TGF- 1. The
185 significantly down-regulated genes include nine genes encoding
enzymes and proteins involved in antioxidant defense. These
changes were also observed in the presence of Z-VAD-fmk
(ZVAD.FMK) plus TGF-
1, indicating that they
are caspase-independent.
Antioxidant genes down-regulated by TGF-1
1 (see also Fig. 3) within
4 h. Gene names, IMAGE numbers, and a summary of functions are
indicated.
1--
Although the initial aim of
these experiments was to identify upstream mediators of
TGF-
1-induced apoptosis, the significant changes in gene
expression observed in the presence of Z-VAD-fmk pretreatment were very
intriguing. Therefore, we analyzed the data further to identify genes
whose expression during TGF-
1-induced apoptosis was
significantly changed (p < 0.05) by Z-VAD-fmk
treatment. This analysis showed that 24 genes were up-regulated
in the presence of Z-VAD-fmk (Fig. 4 and
Table III); and interestingly, 17 of
these genes are members of the stress-response cluster shown in Fig. 2C. These results imply that cellular stress responses are
enhanced when the caspase-dependent apoptotic pathway is
blocked. The time course of these changes in stress-related genes shows
that they occurred late, and this correlates with the delayed necrosis
that we have previously observed in FaO hepatoma cells treated with TGF-
1 (28). These results indicate that
TGF-
1 can potentially alter the expression of an
additional set of genes, except that these changes in gene expression
are suppressed by the action of active caspases. Consequently,
inhibition of the caspase cascade by Z-VAD-fmk then allows the
appropriate genes to be up- or down-regulated.
View larger version (35K):
[in a new window]
Fig. 4.
Statistically significant alterations in gene
expression in the presence of Z-VAD-fmk. The data were analyzed by
K-means clustering for any genes whose expression was
statistically different using a non-parametric test (p < 0.05) when co-treated with Z-VAD-fmk (ZVAD-FMK). This
analysis produced a group of 25 genes, 24 of which were up-regulated
only in the presence of Z-VAD-fmk. The genes and functions of the
proteins they encode are listed in Table III and Supplemental Table
II.
Genes whose expression is altered by Z-VAD-fmk treatment
1 alone produced marked up-regulation of these two
genes. Up-regulation of FAT10, which was clustered in the
stress group (Fig. 2C), was confirmed by RT-PCR and was
clearly a response to TGF-
1 treatment, which was markedly enhanced in the presence of Z-VAD-fmk. jun
expression as measured by RT-PCR was up-regulated in all the treatments
(Fig. 5B), whereas in the microarray results, it was only
significantly up-regulated in the
TGF-
1/Z-VAD-fmk-treated cells (Figs. 2C and 4). However, the changes in ATF3 expression as measured by
RT-PCR (Fig. 5B) correlated exactly with the microarray
results (Figs. 2C and 4).
View larger version (48K):
[in a new window]
Fig. 5.
Verification of selected gene
expression changes by RT-PCR. RT-PCR was carried out on treated
and control samples as described under "Experimental Procedures."
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression
was used as a loading control. In A, the expression of
selected genes is shown at each time point, with and without Z-VAD-fmk.
In those experiments in which Z-VAD-fmk was shown to affect the
TGF- 1 response (B), additional
Z-VAD-fmk-alone samples were also run for comparison. In C,
the expression of two apoptotic genes is shown for the 16-h time point.
Casp-8, caspase-8.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 initiates many of its biological effects,
including apoptosis, by signaling through SMAD proteins and
TGF-
1-responsive promoter sequences that regulate the
expression of a variety of proteins (36, 46). In this study, we used
DNA microarrays to study the changes in gene expression during
TGF-
1-induced apoptosis. We have shown that there was a
time-dependent and coordinated expression of discrete
clusters of genes. Interestingly, many genes were down-regulated by
TGF-
1, demonstrating that transcriptional repression of
specific genes is involved in the initiation of TGF-
1-induced apoptotic cell death. The 32 transcripts specifically up-regulated by TGF-
1 clustered
into immediate-early responses (up-regulated by 4 h) (Fig.
2A) and delayed responses (up-regulated by 8 h or
later) (Fig. 2B). The immediate-early response genes encode
many structural proteins, consistent with the fundamental role of
TGF-
1 in regulating cytoskeletal proteins and in
extracellular matrix remodeling. Only a few specific genes (~12) are
known to have SMAD-responsive elements. However, a recent microarray
study in dermal fibroblasts has identified more SMAD-responsive genes (47). These included five related genes encoding for collagen, TIMP-1 (tissue inhibitor of metalloproteinase-1), an
irreversible inhibitor of collagenases, ARHB
(rhoB), and DSP (desmoplakin I). The latter two
genes were also up-regulated in the extracellular matrix and
cytoskeleton cluster in FaO cells (Fig. 2A). This cluster also includes CTGF, which promotes fibroblast proliferation
and extracellular matrix formation and has been implicated in liver fibrosis (48) and TGF-
1-induced apoptosis in MCF-7 and
human aortic smooth muscle cells (49, 50). The CTGF gene
contains a TGF-
1-responsive element and is strongly
up-regulated in MCF-7 cells (which do not normally express
CTGF mRNA) during TGF-
1-induced apoptosis
(49). Overexpression of CTGF induces apoptosis, which is
abrogated by CTGF antisense oligonucleotides (49). Thus, the
up-regulation of CTGF we observed in FaO cells (Fig. 5)
could amplify the apoptotic effects of TGF-
1 and/or
alternatively induce apoptosis in surrounding cells.
1-induced
apoptosis (15, 28) and would augment apoptotic cell death. However, the
significance of other gene changes is more difficult to assess, as some
of the proteins (e.g. XIAP) could act as inhibitors of
apoptosis, whereas BAK1 and BAD would be expected to amplify the
apoptotic response.
1-induced signaling (52). XIAP binds to TAB1, an activator of TAK1
(TGF-
1-associated kinase-1), a mitogen-activated protein kinase
kinase kinase (53). Interestingly, TAB1 was up-regulated in
the presence of Z-VAD-fmk (Fig. 2C and Table III) and is a
member of the stress-response gene cluster, which also contains
jun, ATF3, and GADD153. The microarray
analysis showed that Jun expression was markedly up-regulated at later
time points only in TGF-
1/Z-VAD-fmk-treated cells. In contrast, the RT-PCR data showed that TGF-
1 alone also
induced the up-regulation of jun, which agrees with previous
studies showing that TGF-
1 induces an immediate
up-regulation of AP-1 component genes (for review, see Ref. 54). During
TGF-
1-induced apoptosis, SMAD proteins bind directly to
the Jun family of AP-1 transcription factors (36). AP-1 proteins are
homo- and heterodimers composed of bZIP (basic region
leucine zipper) DNA-binding proteins belonging to the Jun
(c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), JDP1 and
JDP2 (Jun dimerization partner),
and closely related ATF (activating
transcription factor; ATF2, LRF1/ATF3, and
B-ATF) families (55, 56). Many different apoptosis-inducing agents have
been shown to activate jun and fos expression,
including DNA-damaging agents (57), oxidant injury (58), and natural products such as flavonoids and isothiocyanates (59).
1 after prolonged
exposure (28).
B. TAK1 and its activator TAB1 have been shown to activate I
B
kinase, thus stimulating NF-
B activation (64). Similarly, TANK can
stimulate activation by forming a signaling complex containing TANK,
TRAF2, and TBK1, a novel I
B kinase-related kinase (65). Together,
these data suggest that the cells may undergo an NF-
B-related stress
response in the presence of Z-VAD-fmk. Interestingly, 9 of the 185 down-regulated genes encode proteins involved in antioxidant defense,
particularly the glutathione redox cycle, which is the major defense
system against ROS by normal aerobic mitochondrial metabolism. In the absence of antioxidants, ROS would increase to toxic levels; and significantly, ROS have previously been implicated in
TGF-
1-induced apoptosis in fetal hepatocytes (29, 38,
66). After TGF-
1 treatment, there is an early increase
in ROS and a decrease in glutathione levels (66), which precede a
decrease in
and the release of cytochrome c (29).
Significantly, the TGF-
1-inducible transcription factor
TIEG1 (TGF-
1-inducible
early response gene-1) also
induces apoptosis via an increase in ROS and loss of mitochondrial
(67). TGF-
1-induced oxidative stress and
apoptosis can be blocked in part by antioxidant treatment (66, 67) and
accentuated by inhibitors of glutathione synthesis (29). Glutathione is synthesized in a two-step reaction from glutamate and cysteine to form
L-
-glutamylcysteine, which is then conjugated with
glycine to produce reduced glutathione (for review, see Ref. 68). Our data show that two key enzymes (GLCLC and glutathione synthetase) involved in glutathione synthesis and CTH (cystathionine
-lyase), the terminal enzyme involved in synthesizing cysteine from methionine, are down-regulated at a very early stage in
TGF-
1-mediated cell death (Fig. 3 and Table II). This
would lead to a decrease in the levels of glutathione and an increase
in ROS. In support of this conclusion, a recent study has shown that
TGF-
1-induced glutathione depletion in alveolar
epithelial cells is due to down-regulation of GLCLC
(
-glutamylcysteine synthetase) (69). This would lead to a decrease
in glutathione levels and a subsequent increase in ROS, resulting in
cytochrome c release and apoptosis. Consistent with this
hypothesis, a recent study has shown that superoxide can directly
trigger cytochrome c release and apoptosis in HepG2 cells
via interaction with voltage-dependent anion channel (VDAC) independently of pro-apoptotic Bcl-2 proteins (70).
1
appears to act in the opposite manner to
tert-butylhydroquinone and leads to down-regulation of many
of these genes, perhaps by the induction of an as yet unidentified
antioxidant-responsive element suppressor protein. Significantly, the
time-dependent suppression of other antioxidant enzymes
(viz. manganese-superoxide dismutase, copper/zinc-superoxide dismutase, and catalase) has also been demonstrated in primary hepatocytes following exposure to TGF-
1 (74).
1
and TGF-
1/Z-VAD-fmk treatment was FAT10,
which encodes a ubiquitin-like protein that is synergistically induced
by interferon-
and tumor necrosis factor-
and which has been
shown to induce apoptosis in a caspase-dependent manner
(45). There is increasing evidence that TGF-
1 signaling
is regulated by proteasomal degradation of component members of the
pathway. For example, TIEG1 is rapidly induced by
TGF-
1 and serves to down-regulate the negative feedback inhibition of the inhibitory protein Smad7, which is also induced by
TGF-
1 (75). TIEG1 interacts with SIAH1
(seven in absentia homlogue-1), a ubiquitin-protein isopeptide ligase, and is
targeted for destruction by the proteasome (76). Other studies have
shown that activated Smad2 is polyubiquitinated and degraded by
the proteasome, thereby terminating the signaling pathway (31). Inhibition of proteasomal degradation would be predicted to prolong TGF-
1 signaling, whereas in contrast, we have found that
proteasome inhibitors (MG132 and lactacystin) abrogated
TGF-
1-induced
apoptosis.8 This suggests
that the involvement of the proteasome in TGF-
1-induced apoptosis implicates some other as yet identified target and perhaps that FAT10 targets an anti-apoptotic molecule for proteasomal degradation.
1 induces
apoptosis by specifically down-regulating a set of genes that encode
enzymes involved in protecting the cell against ROS. These antioxidant enzymes are rapidly turned over, and down-regulation would lead to an
increase in ROS, cytochrome c release, and activation of caspases via the apoptosome complex. The later up-regulation of apoptogenic proteins such as caspase-8, BAD, and BAK1 could
serve to amplify the apoptotic response. In addition, the early
up-regulation of CTGF may trigger apoptosis in neighboring cells,
thus providing an additional pathway of amplification (Fig.
6). In conclusion, our study has shown
that TGF-
1-induced apoptosis involves the coordinated
induction and repression of specific gene clusters that initiate and
propagate the process of cell death.
View larger version (42K):
[in a new window]
Fig. 6.
Schematic representation of the possible
mechanism of TGF- 1-induced
apoptosis. The scheme shows some of the possible pathways involved
in TGF-
1-induced apoptosis based on the known
biochemical events and the possible effects of up-regulation (
) or
down-regulation (
) of target genes that have been identified in this
study. Thus, in this scheme, the activation of the
TGF-
1 receptor leads to SMAD-mediated transcriptional
changes in key metabolic proteins. The down-regulation of
GSH and GLRX and its subsequent effects on ROS,
coupled with up-regulation of BAK1 and BAD,
induce cytochrome c release, apoptosome formation, and
activation of the caspase cascade. The up-regulation of CTGF
could also lead to amplification of the apoptotic response by
activating the CTGF receptor and inducing apoptosis by this
pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the Microarray Group at the Medical Research Council Toxicology Unit for providing the arrays used in this study. We are also indebted to David Judah for helpful discussions on microarray data analysis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by European Union Grant QLG1-1999-00739.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental
Tables I and II.
Present address: Children's Brain Tumour Research Centre, Inst.
of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
§ Present address: AVENTIS PHARMA, Centre de Recherches de Paris, 94403 Vitry sur Seine, France.
¶ To whom correspondence should be addressed: MRC Toxicology Unit, Hodgkin Bldg., University of Leicester, P. O. Box 138, Lancaster Rd., Leicester LE1 9HN, UK. Tel.: 44-116-252-5547; Fax: 44-116-252-5616; E-mail: kc5@le.ac.uk.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211300200
2 Available at www.le.ac.uk/cmht/twg1/array-fp.html.
3 Available at www.DoubleTwist.com/.
4 Available at www.ncbi.nlm.nih.gov/.
5 B. Coyle, manuscript in preparation.
6 Available at www.ncbi.nlm.nih.gov/HomoloGene/.
7 B. Coyle, unpublished data.
8 C. Freathy and K. Cain, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-1, transforming growth factor-
1;
Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
ROS, reactive oxygen species;
RT, reverse transcription.
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