From the Department of Medicine, Royal Victoria Hospital, Molecular Endocrinology Laboratory, McGill University, Montreal H3A 1A1, Canada
Received for publication, November 29, 2000, and in revised form, February 12, 2001
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ABSTRACT |
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Activin, a member of the TGF Activin, a member of the
TGF Both Smad3 and Smad4 but not Smad2 can directly bind DNA elements (Smad
binding element) and activate the transcription of the target
genes (12). However, the DNA binding affinity of the Smads is low (13),
and they usually require the presence of other DNA binding proteins to
efficiently interact with the promoters of their responsive target
genes. As a result, the Smad binding elements are often found close to
the DNA binding element of other transcription factors. Among those are
the FAST family members, FAST1 (14) and FAST2 (15), TFE3 (16),
Fos and Jun (17), Sp1 (18), CBP/p300 (19), Evi-1 (20), and ATF2
(21).
The Smad proteins are central elements in the activin receptor
signaling pathway but are not the sole pathway activated by this
receptor complex. Other members of the TGF Activin, its receptors, and the Smads are expressed in myoepithelial
cells as well as in a certain number of human breast cancer cell lines
(26, 27), suggesting a role for this growth factor and its downstream
effectors, the Smads, in mammary cell growth and differentiation.
Several reports have recently implicated TGF In the present study, we investigated the role and mechanism of action
of activin in breast cancer cells. We show here for the first time that
activin strongly inhibits cell growth of the human breast cancer cell
line T47D. In addition, our results indicate that activin induces the
Smad pathway in these cells but also activates the p38 MAPK pathway.
Activation of this pathway further leads to phosphorylation of the
transcription factor ATF2. Furthermore, we show that specific
inhibitors of the p38 MAPK pathway antagonize the activin-mediated cell
growth arrest in T47D cells. Thus, this highlights for the first time
the involvement of this p38 kinase pathway downstream of the activin
receptor signal transduction pathways leading to cell growth arrest.
Cell Culture and Proliferation Assay--
T47D cells were
cultured in Dulbecco's Modified Eagle's Medium (DMEM) in the presence
of 10% fetal calf serum. For proliferation assay, cells were plated in
triplicates in 96-well dishes, at 5000 cells/100 µl in 2% FCS. Cells
were stimulated or not with activin (0.5 nM) and
grown over a 5-day period. Cell proliferation was assessed using direct
cell counting, and the non-radioactive MTT cell proliferation assay for
eukaryotic cells (Cell Titer 96, Promega G 4000). Absorbance was
measured at 570 nm with a reference wavelength at 450 nm, using a
Bio-tek Microplate reader.
Transfection and Reporter Assay--
T47D cells (107
cells) were transfected by electroporation (Bio-Rad Gene Pulser II) in
500 µl of phosphate-buffered saline (240 V and 975 microfarads) with 10 µg of each of the indicated cDNAs.
Following transfection, cells were plated in 6-well dishes in DMEM
(10% FCS) for 24-h recovery. The following day, cells were starved
overnight in DMEM without serum and stimulated or not with activin (0.5 nM) for 16 h. Then, cells were washed once with
phosphate-buffered saline and lysed in 250 µl of lysis buffer (1%
Triton X-100, 15 mM MgSO4, 4 mM
EGTA, 1 mM dithiothreitol, 25 mM glycylglycine)
on ice. The luciferase activity of each sample was measured using 45 µl of cell lysate (EG&G Berthold luminometer) and normalized to the
relative RNase Protection Assay--
RNase protection assay was performed
using the hcc-2 template set and RiboQuant kit from PharMingen (San
Diego, CA) according to the manufacturer's instructions, with minor
modifications. Radiolabeled antisense RNA probes were prepared by
in vitro transcription of the hcc-2 templates with T7 RNA
polymerase in the presence of [ Western Blot Analysis--
T47D cells were plated at
106 cells/ml in 6-well dishes in DMEM (10% FCS). The
following day, cells were starved for an overnight period and
stimulated or not with activin for different periods of time as
indicated. Total cell extracts prepared from these cells were then
separated on a polyacrylamide gel, transferred onto nitrocellulose, and
incubated with the indicated specific antibody overnight at 4 °C
(p38 (New England BioLabs, catalogue no. 9212), phosphop38 (New
England BioLabs, catalogue no. 9210), ATF2 (New England BioLabs,
catalogue no. 9222), phosphoATF2 (Santa Cruz Biotechnologies, catalogue
no. 8398), Smad2/3 (Santa Cruz, catalogue no. 8332), phosphoSmad2
(Upstate Biotechnology Inc., catalogue no. 6829), Smad4 (Santa
Cruz, catalogue no. 7966), ERK1/2 (New England BioLabs, catalogue no.
9102), and phosphoERK1/2 (New England BioLabs, catalogue no. 9101)).
Following incubation, the membranes were washed twice for 10 min in
washing buffer (50 mM Tris-HCl, pH 7.6; 200 mM
NaCl; 0.05% Tween 20) and incubated with a secondary antibody coupled
to peroxidase (from Santa Cruz; at a 1/10,000 dilution) for 1 h at
room temperature. Then, the membranes were washed four times for 15 min
in the washing buffer, and immunoreactivity was normalized by
chemiluminescence (Lumi-Light Plus Western blotting substrate, Roche
Molecular Biochemicals) according to the manufacturer's instructions
and revealed using an Alpha Innotech Fluorochem imaging system
(Packard Canberra).
Activin Inhibits T47D Human Breast Cancer Cell
Growth--
Although activin and its receptors are expressed in a
number of breast cancer cell lines, the role of activin in the
regulation of breast cancer cell growth has not yet been fully
investigated. To analyze the role of activin in regulating growth of
human breast cancer cells, we utilized the human breast cancer cell
line T47D, which endogenously expresses activin-responsive Smad2,
Smad3, and Smad4 (31). Using a cell growth and viability assay (MTT assay), we show that activin treatment of T47D cells leads to a
significant inhibition in their growth, apparent as early as day 2 and
reaching 40% inhibition at day 3 (Fig.
1A). To verify that activin
affects cell growth and not the metabolic rate of the cell, direct cell
counting was also performed. As shown in Fig. 1B, activin
stimulation of T47D cells for 3 days also results in clear cell growth
inhibition. Therefore, activin appears as a potent cell growth
inhibitor for T47D breast cancer cells.
Activin Modulates Cell Cycle Regulators in Breast Cancer
Cells--
TGF Activin Induces Smad2 Phosphorylation in T47D Cells--
To then
analyze the role of the Smad pathway in T47D cells, we first examined
the activation state of Smad2, following activin stimulation. Cells
were starved for an overnight period and treated with 0.5 nM activin for a different period of time, as indicated in
Fig. 3. Total cell lysates were separated
by SDS-polyacrylamide gel electrophoresis, and resolved proteins were
transferred to a nitrocellulose membrane for Western blotting analysis.
The membrane was probed with a specific antibody to phospho-Smad2 that
recognizes the two phosphorylated serine residues in the C-terminal end
of the MH2 domain of Smad2 (SSXS). As shown in Fig.
3, upper panel, activin treatment of T47D cells leads to a
clear phosphorylation of Smad2, as early as 15 min following ligand
stimulation of the cells. The membrane was stripped and reprobed with a
polyclonal antibody that recognizes both Smad2 and Smad3 (Fig. 3,
middle panel) and subsequently with a monoclonal antibody to
Smad4 (Fig. 3, lower panel) and shows equal levels of all
Smads in all samples. These data indicate that the Smad pathway is
functional in T47D cells and is activated in response to activin
stimulation.
Activin Induces 3TPLux and ARE-Lux Promoters in T47D Cells--
To
further examine the activation of the activin receptor/Smad pathway in
T47D cells, we analyzed the ability of activin to induce two activin
receptor/Smad-responsive promoter constructs (3TPLux and ARE-Lux). T47D
cells were transiently co-transfected as shown under "Materials and
Methods" with the promoter construct 3TPLux or ARE-Lux and an
expression vector encoding the co-activator Fast1. As shown in Fig.
4A, activin treatment of T47D
cells led to a 2.6- and 2.7-fold induction of 3TPLux and
ARE-Lux, respectively. Furthermore, T47D cells were also co-transfected
with 3TPLux or ARE-Lux/Fast1 and an expression vector encoding a
constitutively active form of the activin type I receptor (ALK4 T Activin Activates the p38 Kinase Pathway in T47D
Cells--
Recently, the p38 mitogen-activated protein kinase (MAPK)
pathway was shown to regulate gene expression in response to TGF
We then analyzed the activin effects on the phosphorylation of the
transcription factor ATF2, a downstream target of the p38 kinase. Total
cell lysates were analyzed by immunoblot using specific antibodies
directed against the phosphorylated ATF2 (pATF2) or ATF2
(ATF2). As shown in Fig. 5B (upper
panel), there is a time-dependent phosphorylation of
the transcription factor ATF2 following activin treatment of the cells.
The phosphorylation of ATF2 correlates with the activation of the p38
MAPK and shows a maximum phosphorylation at 40 min. The membrane was
stripped and reprobed with an anti-ATF2 antibody and shows an equal
amount of the transcription factor in all lanes (Fig. 5B,
lower panel). Together, these data demonstrate that the p38
MAPK/ATF2 pathway is activated in T47D cells in response to activin.
The p38 Kinase Inhibitor PD169316 Blocks Activin-induced p38 and
ATF2 Phosphorylation--
To further confirm the involvement of the
p38 MAPK pathway downstream of the activin receptor, T47D cells were
treated with a specific p38 kinase inhibitor (PD169316) or
Me2SO as a control. Cells were then stimulated or
not with activin for 30 min, and total cell lysates were analyzed by
Western blotting using different antibodies directed against phosphop38
or p38, phospho-ATF2 or ATF2. As shown in Fig.
6A (upper panel),
in the presence of Me2SO, activin induces phosphorylation
of the kinase p38, confirming the previously seen results (Fig. 5).
However, in the presence of the specific p38 kinase inhibitor PD169316,
this activin effect on p38 phosphorylation is abolished (Fig.
6A, upper panel). The membrane was stripped and
reprobed with an antibody directed against p38 and shows an equal
amount of proteins in all lanes (Fig. 6A, lower
panel). Similarly, as shown in Fig. 6B, upper
panel, activin induces phosphorylation of the transcription factor
ATF2 in the presence of Me2SO, but this activin-induced
effect is abolished in the presence of the p38 kinase inhibitor
PD169316. An equal amount of protein in all lanes was ensured by
stripping and reprobing of the membrane with an anti-ATF2 antibody
(Fig. 6B, lower panel).
The p38 Kinase Inhibitors Antagonize Activin-induced Cell Growth
Arrest in Breast Cancer Cells--
To evaluate the contribution of the
p38 kinase pathway in activin-mediated cell growth inhibition in T47D
cells, we used different p38 kinase-specific inhibitors (SB202190,
SB203580, PD169316) or an inactive analogue (SB202474) and an
MEK1/ERK1/2 inhibitor (PD98059) as controls, in both MTT (Fig.
7A) and direct cell counting assays (Fig. 7B). T47D cells were cultured in DMEM, 2%
serum for 3 days and stimulated or not with 0.5 nM activin
in the presence or absence of the different inhibitors. As shown in
Fig. 7, A and B, after 3 days cell growth is
reduced by 40% in activin-treated cells as compared with untreated
cells, similar to that previously observed in Fig. 1, A and
B. However, in the presence of each the three specific p38
kinase inhibitors (SB202190, SB203580, PD169316), the inhibitory effect
of activin on cell growth is abolished. On the other hand, the activin
effect on cell growth inhibition is maintained in samples treated with
the inactive form of the p38 kinase inhibitor SB202474 or with the
MEK1/ERK1/2 inhibitor PD98059. Our results indicate that p38
kinase-specific inhibitors nearly completely reverse the activin
effect. Because p38 kinase inhibitors could affect TGF
As activin is potent cell growth inhibitor in many different cell
lines, the effect of the p38 kinase inhibitors were also analyzed in
several activin-responsive cell lines such as K562, Chinese hamster
ovary, and MCF7. Interestingly, the activin inhibitors could reverse
the activin effects on cell growth arrest in all cell lines tested
(data not shown). This indicates that the contribution of the p38 MAPK
pathway to activin-mediated cell growth arrest is critical.
Activin Effect on Cell Growth Arrest Is Not Mediated through the
MEK1/ERK1/2 MAPK Pathway--
The absence of effect of the MEK1/ERK1/2
inhibitor PD98059 on activin-mediated cell growth arrest (Fig. 7)
suggests that activin does not modulate the MAPK MEK1/ERK1/2 pathway to
arrest cell growth. The MEK1/ERK1/2 pathway is known to be involved in
cell proliferation in response to various growth factors. To confirm that activin does not modulate or inhibit activation of this pathway in
response to growth factors, T47D cells were starved overnight and
stimulated with EGF (20 ng/ml) for different periods of time in the
absence or presence of 1 nM activin (Fig.
8). Total cell lysates were then analyzed
by Western blotting using an antibody directed against phospho-ERK1/2
( Members of the TGF Abnormalities in the signaling pathways of activin/TGF The p38 MAPK is involved in regulating cellular responses to stress and
cytokines (38-41). p38 kinase is activated and phosphorylated at the
Thr180-Tyr182 site by the two closely
related dual specificity protein kinases MKK3 and MKK6 (42, 43). The
activated p38 kinase has been shown to phosphorylate several
transcription factors such as ATF2 (44), Max (45), and Elk-1 (46) and
indirectly cAMP-response element-binding protein via activation
of Nrf2 (47), STAT1 (48), and MEF-2 (49). The p38 pathway is
activated in response to TGF Signaling by the MAPK family is organized hierarchically in three
different steps. MAPK, such as p38, are phosphorylated by MAPK
kinases (MAPKKS), such as MKK3 and MKK6, in the case of p38. The MAPKKs
are themselves activated and phosphorylated by the MAPKK kinases
(MAPKKKs), such as MLK, TAK, and ASK1 kinases, which act as
MAPKKKs. Finally, the MAPKKKs are regulated by cell surface receptors or other external stimuli (51, 52). It will be interesting to
identify the upstream kinases and other partner proteins involved in
the activin-mediated p38 activation that are acting between the activin
receptor complex and the p38 kinase in the signaling cascade. Recent
reports indicated that TAK1, a member of the MAPKKK family, is
activated by several cytokines, including TGF It was also recently shown that the Müllerian Inhibitory
substance (MIS) represses the growth of breast cancer cells by
regulating the NF Our results indicate that activin strongly represses the cell growth of
T47D breast cancer cells. Further characterization of the downstream
target genes that are modulated in T47D cells in response to activin
will greatly enhance our understanding of its mechanism of action on
cell growth regulation. Our data suggest that at least some of these
targets could be the cyclin-dependent kinase inhibitors.
However, it will remain to be determined whether other cell cycle
regulators as well as apoptosis regulators are also regulated by
activin in these cells. Indeed, identification of the target genes,
involved in the regulation of cell cycle and/or apoptosis, will be
important in shedding light on the activin receptor mechanism of action
in breast cancer cells.
family inhibits
cell growth in various target tissues. Activin interacts with a complex
of two receptors that upon activation phosphorylate specific
intracellular mediators, the Smad proteins. The activated Smads
interact with diverse DNA binding proteins and co-activators of
transcription in a cell-specific manner, thus leading to various
activin biological effects. In this study, we investigated the role and
mechanism of action of activin in the human breast cancer T47D cells.
We found that activin treatment of T47D cells leads to a dramatic decrease in cell growth. Thus activin appears as a potent cell growth
inhibitor of these breast cancer cells. We show that activin induces
the Smad pathway in these cells but also activates the p38-mitogen-activated protein kinase pathway, further leading to
phosphorylation of the transcription factor ATF2. Finally, specific
inhibitors of the p38 kinase (SB202190, SB203580, and PD169316) but not
an inactive analogue (SB202474) or the MEK-1 inhibitor PD98059
completely abolish the activin-mediated cell growth inhibition of T47D
cells. Together, these results define a new role for activin in human
breast cancer T47D cells and highlight a new pathway utilized by this
growth factor in the mediation of its biological effects in cell growth arrest.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 family, regulates
cell growth of various cell types. Activin interacts with a complex of
two receptors (types I and II), both containing an extracellular
domain, a single transmembrane region, and a large intracellular domain that contain a serine/threonine kinase domain. The type II receptor, which is constitutively phosphorylated (1) transphosphorylates the type
I receptor (ALK4) upon ligand stimulation, on serine and threonine
residues (2-4). The activated receptor complex then recruits the two
receptor-regulated Smad2 and Smad3 (5-8). Following binding and
phosphorylation by the activin type I receptor, Smad2 and Smad3 are
released to the cytoplasm where they associate with the common-partner
Smad4 before being translocated to the nucleus (8-11).
superfamily have been
shown to activate different signaling pathways, in addition to the
Smads. TGF
itself can activate a member of the MAPKKK family of
kinases, TAK1 (TGF-activated kinase) (22). TAK1 then activates the
stress-activated kinase p38 and the transcription factor ATF2, a member
of the b-ZIP family of DNA binding proteins (21). In vitro
studies also suggested that the transcription factor ATF2 could
interact with the MH1 domains of two activin responsive Smads, Smad3
and Smad4 (21, 23). Both TGF
and the Müllerian inhibiting
substance (MIS) were also shown to mediate some of their biological
effects through an NF
B-mediated pathway (24, 25). It is
therefore conceivable that activin also utilizes other signaling
pathways to transduce its signals.
family members or their
downstream signaling pathways in the regulation of breast cancer cell
growth. Indeed, Smad4 can restore cell growth arrest in MDA-MB-468
cells, a breast cancer cell line in which the Smad4 gene is deleted
(28). Genetic mutations or loss of expression of the activin and TGF
receptors is also found in human breast cancers (29, 30). Finally,
TGF
and MIS mediates cell growth arrest in breast cancer by reducing
NF
B DNA binding activity (24, 25), and activin itself can modulate cell growth of the breast cancer cells MCF7 (26).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity.
-32P]UTP (PerkinElmer
Life Sciences, Boston, MA). After DNase I digestion, phenol-chloroform
extraction, and ethanol precipitation, the probes were quantified. RNA
samples (5 µg) were dried in a vacuum centrifuge and resuspended in
20 µl of hybridization buffer containing 8 × 105
cpm of radiolabeled probes. Hybridization (overnight at 56 °C), RNase A/T1 digestion (1 h at 30 °C), proteinase K digestion,
phenol-chloroform extraction, ethanol precipitation, and gel resolution
(5% polyacrylamide, 8 M urea sequencing gel) were carried
out according to the instructions contained in the RiboQuant RNase
protection assay kit. A yeast tRNA-only reaction was included as a
negative control to ensure complete RNase digestion. Undigested RNA
probes were also resolved on each gel to ensure their integrity and to
serve as size markers. The cell cycle genes represented in the assays
and the size of corresponding protected probe/mRNA duplexes were as
follows: p130, 400 bp; Rb, 352 bp; p107, 317 bp; p53, 283 bp; p57, 252 bp; p27, 227 bp; p21, 202 bp; p19, 182 bp; p16, 163 bp; p14/p15, 133 bp; L32 riboprotein (L32, used as a housekeeping control), 113 bp; and
glyceraldehyde-3-phosphate dehydrogenase, 96 bp. An exposure was
made of the dried gel onto x-ray film. The positions of the protected
probes were confirmed by plotting on a semi-log graph.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activin induces cell growth arrest of the
breast cancer cell line T47D. T47D cells were grown in 2% FCS
DMEM over a 5-day period in the presence or absence of 0.5 nM activin. Cell proliferation was assessed by
(A) MTT colorimetric assay in triplicates and (B)
direct cell counting. Values are expressed in arbitrary units.
family members regulate cell growth through
different mechanisms. They often mediate cell cycle arrest through
up-regulation of the three cyclin-dependent kinase
inhibitors p15INK4B, p21CIP1WAF1, and p27
(32-34). Because activin exerts a strong effect on cell growth in T47D
(Fig. 1), we analyzed its effects in modulating gene expression levels
of different cyclin-dependent kinase inhibitors as well as
of other cell cycle regulatory genes. For this, we examined the level
of mRNA species of different cell cycle regulators, using a highly
sensitive and specific ribonuclease protection assay. As shown in Fig.
2, T47D cells were stimulated for
different periods of time with activin (0.5 nM). Total RNA
from unstimulated or stimulated cells were extracted and hybridized
with multiple antisense probes for human cell cycle regulators (p15,
p16, p18, p19, p21, p27, as well as for p53, p57, p107 p130, the
retinoblastoma protein (Rb), and the two housekeeping genes L32 and
glyceraldehyde-3-phosphate dehydrogenase). As shown in Fig. 2, a modest
but reproducible ligand-dependent increase in the mRNA
level of p21CIP1/WAF1 was observed. This is consistent with
a microarray analysis of T47D cells treated for 8 h with activin,
which shows a 1.7-fold increase in p21CIP1/WAF1 mRNA
level.2 mRNA levels for
p15INK4B were also consistently increased upon activin
treatment, although at a lower level than p21CIP1/WAF1.
This experiment was repeated three times and showed consistent results.
None of the other cell cycle regulators (p130, Rb, p107, p53, p57, p27,
p19, p18, or p16) or housekeeping genes (L32 or glyceraldehyde-3-phosphate dehydrogenase) mRNA levels showed any significant or reproducible difference in response to activin (Fig. 2).
Our attempts to detect p15INK4B and
p21CIP1/WAF1 protein levels in these cells were
unsuccessful, probably due to the low level of expression of theses two
proteins. This suggests that at least part of the activin effect on
cell growth arrest in T47D cells is mediated through up-regulation of
p15INK4B and p21CIP1/WAF1.
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Fig. 2.
Expression of cell cycle genes in T47D cells
in response to activin. Total RNA (5 µg) obtained from T47D
cells treated for 0, 4, 8, 16, or 24 h with activin (0.5 nM). An RNase protection assay was performed with
radiolabeled probes for the indicated human cell cycle genes and two
housekeeping control genes (L32 and glyceraldehyde-3-phosphate
dehydrogenase), as described under "Materials and Methods." The
positions of the protected probes are shown to the left of
the autoradiography. Yeast tRNA (lane 6) is shown as a
negative control. Undigested probes (lane 7) were used as
size standards.
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Fig. 3.
Activin induces Smad2 phosphorylation in T47D
cells. T47D cells were treated with 0.5 nM activin for
0, 15, 30, and 60 min. Cell lysates were analyzed by Western blot using
a specific antibody to phospho-Smad2 (upper panel). The
membrane was stripped and reprobed with an anti-Smad2/3 antibody
(middle panel) and an anti-Smad4 antibody (lower
panel).
D).
This point mutation replaces threonine 206 by an aspartic acid and
renders the receptor constitutively active even in the absence of
ligand or type II receptor (4). As shown in Fig. 4B, ALK4
T
D mimics activin effects on the activation of the two promoter
constructs, leading to a 3.2- and 2.7-fold induction of 3TPlux
and ARE-Lux/Fast1, respectively. Finally, to confirm the involvement of
the Smad pathway, a dominant negative form of Smad3, which lacks the
MH2 domain (Smad3
C), was transfected in T47D cells together with 3TPLux. Deletion of the C-terminal domain of Smad3 results in the loss
of homo- and heterodimerization with the wild type Smad4 as well as in
its ability to induce a reporter construct (35). Cells were stimulated
or not with activin and, as seen in Fig. 4C, overexpression
of Smad3
C completely blocks activin-induced 3TPLux activity.
Together, these results confirm that the activin receptor/Smad pathway
is functional in breast cancer cells.
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Fig. 4.
Activin induces 3TPLux and ARE-Lux promoters
in T47D cells. A, T47D cells transfected with the
activin receptor/Smad responsive promoter constructs 3TP-Lux or
ARE-Lux/Fast1 reporter constructs were stimulated with activin 16 h. The luciferase activity was normalized to the relative
-galactosidase values. Results represent means and standard
deviations of three independent experiments. B, T47D cells
transfected with the promoter constructs 3TP-Lux or ARE-Lux/Fast1
reporter constructs in the presence or the absence of the
constitutively active form of the activin type I receptor (ALK4
T
D). The luciferase activity was normalized to the
relative
-galactosidase values. Results represent means and standard
deviations of three independent experiments. C, T47D cells
were transfected with the promoter construct 3TP-Lux in the presence or
absence of the truncated form of Smad3
(Smad3
C) and stimulated with activin 16 h. The luciferase activity was normalized to the relative
-galactosidase values. Results represent means and standard
deviations of three independent experiments.
(23). To assess the role of this pathway in activin-mediated cell
growth inhibition of breast cancer cells, T47D cells were starved
overnight and stimulated with 0.5 nM activin for different periods of time as indicated in Fig.
5A. Total cell lysates were analyzed by immunoblot using specific antibodies directed against the
phosphorylated form of the p38 kinase (pp38) and the normal form of p38 (p38). As shown in Fig. 5A,
upper panel, activin treatment of the T47D cells results in
a clear increase in p38 phosphorylation in a time-dependent
manner. Activin effect is maximal at 20-40 min and then decreases to
return to basal level. The membrane was stripped and reprobed with an
antibody directed against p38 and shows an equal amount of the p38
kinase in all lanes (Fig. 5A, lower panel).
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Fig. 5.
Activin induces the p38 kinase pathway in
T47D cells. T47D cells were starved overnight and stimulated with
0.5 nM of activin for 0, 5, 15, 30, 60, and 90 min.
A, total cell lysates were analyzed by immunoblot using
specific antibodies directed against the phosphorylated form of the p38
kinase (pp38) (upper panel). The membrane was
stripped and reprobed with an anti-p38 (p38) antibody
(lower panel). B, similarly, total cell lysates
were analyzed by immunoblot using a specific antibody directed against
the phosphorylated ATF2 (pATF2) (upper panel).
The membrane was stripped and reprobed with an anti-ATF2
(ATF2) antibody (lower panel).
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Fig. 6.
The p38 kinase inhibitor PD169316 blocks
activin-induced p38 and ATF2 phosphorylation. T47D cells were
pretreated with Me2SO or with the specific p38 inhibitor
PD169316 at 10 µM for 1 h before being stimulated
with activin for 30 min. A, total cell lysates were analyzed
by immunoblot using a specific antibody directed against the
phosphorylated form of the p38 kinase (pp38) (upper
panel). The membrane was stripped and reprobed with an anti-p38
(p38) antibody (lower panel). B,
similarly, total cell lysates were analyzed by immunoblot using a
specific antibody directed against the phosphorylated ATF2
(pATF2) (upper panel). The membrane was stripped
and reprobed with an anti-ATF2 (ATF2) antibody (lower
panel).
receptor
activity (36), we examined their effect on activin-induced Smad2
phosphorylation. As shown in Fig. 7C, although
activin-induced p38 phosphorylation is inhibited by pretreatment of the
cells with all three active forms of p38 kinase inhibitors, we observed
no significant inhibitory effect on Smad2 phosphorylation under the
same conditions. Thus it is likely that the antagonistic effect exerted
by the p38 inhibitors on activin-induced cell growth arrest is mediated
through inhibition of the p38 kinase pathway downstream of the activin
receptor.
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Fig. 7.
p38 kinase inhibitors antagonize
activin-induced cell growth arrest in breast cancer cells. T47D
cells were cultured in DMEM, 2% serum for 3 days and stimulated or not
with 0.5 nM activin in the presence or absence of 10 µM of the different p38 kinase-specific inhibitors,
SB202190, SB203580, and PD169316, or the inactive analogue SB202474 and
the MEK1/ERK1/2 inhibitor PD98059 as controls. Cell growth was assessed
by (A) MTT colorimetric assay in triplicates and
(B) direct cell counting. Values represent means and
standard deviations of five separate experiments and are expressed as
percentage of inhibition compared with the control. C, T47D
cells were pretreated with Me2SO or with the p38 inhibitors
(SB202190, PD169316, or SB203580) at 10 µM for 45 min
before being stimulated with activin for 15 min. Total cell lysates
were analyzed by immunoblot using specific antibodies directed against
the phosphorylated form of Smad2 (pSmad2, upper
panel), the phosphorylated form of p38 kinase (pp38,
middle panel), or p38 kinase (p38, lower
panel).
-PERK). As shown in Fig. 8 (upper panel), EGF very
rapidly and transiently induces the phosphorylation of ERK1/2
(p42/p44). However, activin co-stimulation of the cells does not affect
EGF-induced ERK1/2 phosphorylation. The membrane was stripped and
reprobed with an anti-ERK antibody and shows an equal amount of
MAPK in all samples. Together this indicates that the activin effect on
cell growth arrest in T47D cells is not mediated through the
MEK1/ERK1/2 MAPK pathway.
View larger version (25K):
[in a new window]
Fig. 8.
Activin effect on cell growth arrest is not
mediated through the MAPK pathway. T47D cells were starved
overnight and stimulated with EGF (20 ng/ml) for different period of
times in the absence or presence of 1 nM activin. Total
cell lysates were then analyzed by Western blotting using an antibody
directed against phospho-ERK1/2 (upper panel). The membrane
was stripped and reprobed with an anti-ERK1/2 antibody (lower
panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
family of growth factors are important
factors in regulating cell growth inhibition; hence, it is critical to
characterize their intracellular signaling mechanisms. Although it is
known that activin signals through activation of Smad proteins, the
activation of other intracellular signaling pathways and their contribution to activin-mediated cell growth inhibition remain to be
characterized. In this paper we have examined the role of activin in
mediating cell growth inhibition of breast cancer cells. Our results
indicate that activin induces the Smad pathway in T47D cells and
emphasize the involvement of the p38 MAPK pathway in activin-induced
cell growth inhibition of these breast cancer cells.
have been
clearly linked to various cancers, including breast cancer (37). We
analyzed activin effects on the regulation of cell growth of human
breast cancer cells. Using the human breast cancer cell line T47D, we
found that activin has a profound and significant effect on the growth
of these cells. We further investigated how activin triggers its
effects in this cell line. Activin treatment of T47D cells leads to
rapid phosphorylation of the receptor-regulated Smad2. Furthermore,
both activin or the constitutively active form of the activin type I
receptor (ALK4T
D) induce the two promoter constructs 3TPLux and
ARE-Lux, and this effect is completely abolished in the presence of an
overexpressed dominant negative form of Smad3 (Smad3
C). All
together, these results suggest that the activin receptor/Smad pathway
is activated and can regulate the activin response in breast cancer
cells, confirming the central role played by the Smad proteins in the
mediation of the activin response.
in C2C12, Mv1LU, and 293 cells (21,
23). TGF
can induce phosphorylation of both p38 and the
transcription factor ATF2 in these cell lines. In addition, p38 and
ATF2 can contribute to the activation of the synthetic reporter
construct 3TPLux in these cells, but the physiological significance of
this pathway in the mediation of the TGF
effects remains unclear. We
show here that activin induces the p38 kinase pathway in T47D cells leading to phosphorylation of both the p38 kinase and the transcription factor ATF2. Furthermore, we show that the p38/ATF2 pathway is required
to transduce the activin effects on cell growth inhibition. Indeed,
different specific p38 kinase inhibitors, but not their inactive
analogue or the MEK inhibitor can totally reverse the activin effect on
cell growth inhibition. This highlights a new role for the p38 kinase
pathway in the control of cell growth and proliferation downstream of
the activin/TGF
superfamily of growth factors. TGF
family members
often require the presence of parallel or synergistic pathways to the
Smads to carry out their full biological effects and diversity of the
Smad-interacting partners may contribute to signal specificity
(50). In future studies, it will be interesting to examine the
level of interaction between the Smad and the p38 kinase pathways in
response to activin in T47D cells, because in vitro studies
have suggested that the Smads could physically interact with the
transcription factor ATF2 (21, 23).
(22) and bone
morphogenetic protein (53). TAK1 is a potent activator of p38 kinase
(54). It will be interesting, therefore, to determine whether or not
TAK1 also lies downstream of the activin receptor complex signaling cascade.
B pathway (25). The TGF
effect on cell growth
inhibition of breast cancer has also been shown to be associated with a
reduced NF
B activity (24). This suggests that different members of the TGF
superfamily may regulate cell growth by utilizing different signaling pathways in the same target tissues. Interestingly, TAK1 was
also shown to lead to NF
B activation (55), suggesting a potential
role for this factor downstream of the activin receptor.
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ACKNOWLEDGEMENTS |
---|
We are thankful to Wylie Vale and the
National Hormone & Pituitary program and Dr. Parlow for providing
activin, to Dr. J. Massagué for providing the 3TPLux construct,
to Dr. B. Volgestein for the Fast-1 construct, to Dr. Y. Chen for the
Smad3C construct, and to Drs. J. Wrana and L. Attisano for ARE-Lux construct.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Medical Research Council of Canada (Grant 24836) and by the American Concern Foundation for Cancer Research.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.
A Foundation pour la Recherche en Sante du Quebec scholar.
§ A Medical Research Council scholar. To whom correspondence should be addressed: Dept. of Medicine, Royal Victoria Hospital, Molecular Endocrinology Laboratory, 687 Pine Ave. West, McGill University, Montreal H3A 1A1, Canada. Tel.: 514-842-1231 (ext. 4846); Fax: 514-982-0893; E-mail: JJ.Lebrun@MUHC.McGill.CA.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M010768200
2 J.-J. Lebrun, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF, transforming
growth factor
;
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK
kinase;
MAPKKK, MAPK kinase kinase;
TAK1, TGF-activated kinase 1;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
MIS, Müllerian inhibiting substance;
DMEM, Dulbecco's modified
Eagle's medium;
FCS, fetal calf serum;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
L32, L32 riboprotein;
pp38, phosphorylated form of p38 kinase;
EGF, epidermal growth factor;
bp, base pair(s);
ALK, activin receptor-like
kinase;
Fast1, forkhead activin signal transducer-1;
STAT, signal
transducers and activators of transcription.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Mathews, L. S.,
and Vale, W. W.
(1993)
J. Biol. Chem.
268,
19013-19018 |
2. | Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994) Nature 370, 341-347[CrossRef][Medline] [Order article via Infotrieve] |
3. | Willis, S. A., Zimmerman, C. M., Li, L. I., and Mathews, L. S. (1996) Mol. Endocrinol. 10, 367-379[Abstract] |
4. | Attisano, L., Wrana, J. L., Montalvo, E., and Massague, J. (1996) Mol. Cell. Biol. 16, 1066-1073[Abstract] |
5. | Baker, J. C., and Harland, R. M. (1996) Genes Dev. 10, 1880-1889[Abstract] |
6. |
Chen, Y.,
Lebrun, J. J.,
and Vale, W.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12992-12997 |
7. | Zhang, Y., Feng, X., We, R., and Derynck, R. (1996) Nature 383, 168-172[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Lebrun, J. J.,
Takabe, K.,
Chen, Y.,
and Vale, W.
(1999)
Mol. Endocrinol.
13,
15-23 |
9. | Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J. (1996) Nature 383, 832-836[CrossRef][Medline] [Order article via Infotrieve] |
10. | Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224[Medline] [Order article via Infotrieve] |
11. |
Nakao, A.,
Imamura, T.,
Souchelnytskyi, S.,
Kawabata, M.,
Ishisaki, A.,
Oeda, E.,
Tamaki, K.,
Hanai, J.,
Heldin, C. H.,
Miyazono, K.,
and ten Dijke, P.
(1997)
EMBO J.
16,
5353-5362 |
12. | Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B., and Kern, S. E. (1998) Mol. Cell 1, 611-617[Medline] [Order article via Infotrieve] |
13. | Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague, J., and Pavletich, N. P. (1998) Cell 94, 585-594[Medline] [Order article via Infotrieve] |
14. | Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whitman, M. (1997) Nature 389, 85-89[CrossRef][Medline] [Order article via Infotrieve] |
15. | Labbe, E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. (1998) Mol. Cell 2, 109-120[Medline] [Order article via Infotrieve] |
16. |
Hua, X.,
Liu, X.,
Ansari, D. O.,
and Lodish, H. F.
(1998)
Genes Dev.
12,
3084-3095 |
17. | Zhang, Y., Feng, X. H., and Derynck, R. (1998) Nature 394, 909-913[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Moustakas, A.,
and Kardassis, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6733-6738 |
19. |
Nakashima, K.,
Yanagisawa, M.,
Arakawa, H.,
Kimura, N.,
Hisatsune, T.,
Kawabata, M.,
Miyazono, K.,
and Taga, T.
(1999)
Science
284,
479-482 |
20. | Kurokawa, M., Mitani, K., Irie, K., Matsuyama, T., Takahashi, T., Chiba, S., Yazaki, Y., Matsumoto, K., and Hirai, H. (1998) Nature 394, 92-96[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Sano, Y.,
Harada, J.,
Tashiro, S.,
Gotoh-Mandeville, R.,
Maekawa, T.,
and Ishii, S.
(1999)
J. Biol. Chem.
274,
8949-8957 |
22. | Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract] |
23. |
Hanafusa, H.,
Ninomiya-Tsuji, J.,
Masuyama, N.,
Nishita, M.,
Fujisawa, J.,
Shibuya, H.,
Matsumoto, K.,
and Nishida, E.
(1999)
J. Biol. Chem.
274,
27161-27167 |
24. |
Sovak, M. A.,
Arsura, M.,
Zanieski, G.,
Kavanagh, K. T.,
and Sonenshein, G. E.
(1999)
Cell Growth Differ.
10,
537-544 |
25. |
Segev, D. L.,
Ha, T. U.,
Tran, T. T.,
Kenneally, M.,
Harkin, P.,
Jung, M.,
MacLaughlin, D. T.,
Donahoe, P. K.,
and Maheswaran, S.
(2000)
J. Biol. Chem.
275,
28371-28379 |
26. | Liu, Q. Y., Niranjan, B., Gomes, P., Gomm, J. J., Davies, D., Coombes, R. C., and Buluwela, L. (1996) Cancer Res. 56, 1155-1163[Abstract] |
27. | Ying, S. Y., and Zhang, Z. (1996) Breast Cancer Res. Treat. 37, 151-160[Medline] [Order article via Infotrieve] |
28. | de Winter, J. P., Roelen, B. A., ten Dijke, P., van der Burg, B., and van den Eijnden-van Raaij, A. J. (1997) Oncogene 14, 1891-1899[CrossRef][Medline] [Order article via Infotrieve] |
29. | Di Loreto, C., Reis, F. M., Cataldi, P., Zuiani, C., Luisi, S., Beltrami, C. A., and Petraglia, F. (1999) Eur. J. Endocrinol. 141, 190-194[Medline] [Order article via Infotrieve] |
30. | Chen, T., Carter, D., Garrigue-Antar, L., and Reiss, M. (1998) Cancer Res. 58, 4805-4810[Abstract] |
31. | Pouliot, F., and Labrie, C. (1999) Int. J. Cancer 81, 98-103[CrossRef][Medline] [Order article via Infotrieve] |
32. | Hannon, G. J., and Beach, D. (1994) Nature 371, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
33. | Datto, M. B., Li, Y., Panus, J. F., Howe, D. J., Xiong, Y., and Wang, X. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5545-5549[Abstract] |
34. | Zauberman, A., Oren, M., and Zipori, D. (1997) Oncogene 15, 1705-1711[CrossRef][Medline] [Order article via Infotrieve] |
35. | Wu, R. Y., Zhang, Y., Feng, X. H., and Derynck, R. (1997) Mol. Cell. Biol. 17, 2521-2528[Abstract] |
36. | Eyers, P. A., Craxton, M., Morrice, N., Cohen, P., and Goedert, M. (1998) Chem. Biol. 5, 321-328[Medline] [Order article via Infotrieve] |
37. | Massague, J., Blain, S. W., and Lo, R. S. (2000) Cell 103, 295-309[Medline] [Order article via Infotrieve] |
38. | Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037[Medline] [Order article via Infotrieve] |
39. | Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. (1994) Cell 78, 1039-1049[Medline] [Order article via Infotrieve] |
40. | Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., et al.. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve] |
41. | Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811[Medline] [Order article via Infotrieve] |
42. | Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. J. (1996) Mol. Cell. Biol. 16, 1247-1255[Abstract] |
43. | Derijard, B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Science 267, 682-685[Medline] [Order article via Infotrieve] |
44. |
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426 |
45. | Zervos, A. S., Faccio, L., Gatto, J. P., Kyriakis, J. M., and Brent, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10531-10534[Abstract] |
46. | Price, M. A., Cruzalegui, F. H., and Treisman, R. (1996) EMBO J. 15, 6552-6563[Abstract] |
47. |
Alam, J.,
Wicks, C.,
Stewart, D.,
Gong, P.,
Touchard, C.,
Otterbein, S.,
Choi, A. M.,
Burow, M. E.,
and Tou, J.
(2000)
J. Biol. Chem.
275,
27694-27702 |
48. |
Goh, K. C.,
Haque, S. J.,
and Williams, B. R.
(1999)
EMBO J.
18,
5601-5608 |
49. |
Yang, S. H.,
Galanis, A.,
and Sharrocks, A. D.
(1999)
Mol. Cell. Biol.
19,
4028-4038 |
50. | ten Dijke, P., Miyazono, K., and Heldin, C. H. (2000) Trends Biochem. Sci. 25, 64-70[CrossRef][Medline] [Order article via Infotrieve] |
51. | Herlaar, E., and Brown, Z. (1999) Mol. Med. Today 5, 439-447[CrossRef][Medline] [Order article via Infotrieve] |
52. | Ichijo, H. (1999) Oncogene 18, 6087-6093[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Yamaguchi, K.,
Nagai, S.,
Ninomiya-Tsuji, J.,
Nishita, M.,
Tamai, K.,
Irie, K.,
Ueno, N.,
Nishida, E.,
Shibuya, H.,
and Matsumoto, K.
(1999)
EMBO J.
18,
179-187 |
54. |
Moriguchi, T.,
Toyoshima, F.,
Gotoh, Y.,
Iwamatsu, A.,
Irie, K.,
Mori, E.,
Kuroyanagi, N.,
Hagiwara, M.,
Matsumoto, K.,
and Nishida, E.
(1996)
J. Biol. Chem.
271,
26981-26988 |
55. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256[CrossRef][Medline] [Order article via Infotrieve] |