From the Experimental Immunology Branch, National
Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, ¶ Genetics Institute, Cambridge,
Massachusetts 02140, and
Division of Cellular Biochemistry,
The Netherlands Cancer Institute,
1066 CX Amsterdam, The Netherlands
Received for publication, May 30, 2000, and in revised form, October 5, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nodal, a member of the transforming growth factor
Members of the transforming growth factor Signaling by TGF- The nodal signaling pathway awaits characterization at the biochemical
level. However, mutational studies in the mouse and zebrafish and
ectopic expression studies in Xenopus and zebrafish suggest
that the nodal and activin signaling pathways may share receptors and
Smads. Targeted mutations in the mouse Smad2 gene (5-8) and the
activin type IB receptor gene (9) and combined mutations of the activin
type IIA and IIB receptor genes (10) show gastrulation phenotypes
resembling the mouse nodal mutant (11, 12). Nodal and activin have
similar mesoderm-inducing capacity in Xenopus embryo
explants or in whole embryos (13), whereas dominant negative forms of
Smad2 (14) or activin receptors (15-17) disrupt endogenous mesoderm
formation. One difference between nodal and activin signaling has been
described in zebrafish. There is genetic evidence that the
extracellular epidermal growth factor-Cripto, FRL1, Cryptic (EGF-CFC)
protein one-eyed pinhead is required for signaling by zebrafish
nodal-related factors but not for activin signaling (18). Whether nodal
signaling in the mouse also depends on the function of the related
EGF-CFC proteins cripto (19) and cryptic (20) has not been established.
However, targeted mutations in both genes result in embryonic lethal
phenotypes that are suggestive; cripto null embryos exhibit
gastrulation defects (21, 22), and the absence of cryptic leads to
left-right defects (23, 24).
Earlier studies on nodal function relied on overexpression from
transfected or injected expression vectors. Here we describe the
production of recombinant nodal protein. The availability of purified
nodal protein allows functional characterization of this
developmentally important pathway not previously possible. We describe
a quantitative assessment of nodal signaling activity in P19 mouse
embryonal carcinoma cells, a model system for the early mouse embryo
(25). We have determined that P19 cells are competent to respond to
nodal signaling and have exploited this capacity and the availability
of recombinant nodal protein to investigate the intracellular pathway
by which the nodal signal is transduced. In this study we provide
direct evidence that nodal signals through activin-TGF- Recombinant Nodal Protein--
The mature region of human nodal
protein was expressed in Escherichia coli and refolded
essentially as described (26). Nodal dimer was isolated by ion exchange
chromatography on S-Sepharose Fast Flow (Amersham Pharmacia Biotech)
using a gradient from 0.05 to 1.0 M NaCl in 25 mM NaAc, 30% isopropanol (pH 4.0), followed by reversed
phase high-performance liquid chromatography over a 2.0 × 0.46-cm C4 column (Supelco, Bellefonte, PA) with an acetonitrile gradient in 0.1% trifluoroacetic acid. Protein purity was assessed by
SDS-polyacrylamide gel electrophoresis, and concentration was determined by amino acid analysis. Protein activity was assessed using
the Xenopus animal cap assay. Briefly, animal caps isolated from stage 8-9 Xenopus embryos were incubated overnight at
16 °C with nodal protein in 50 mM NaCl, 1 mM
KCl, 0.5 mM MgSO4, 1 mM
CaCl2, 2.5 mM HEPES (pH 7.6), 0.1% bovine
serum albumin.
Reagents--
Activin A, TGF- Cell Culture--
P19 cells were obtained from American Type
Culture Collection and were grown as monolayer cultures in high-glucose
Dulbecco's modified Eagle's medium (DMEM) without sodium pyruvate
(Life Technologies) supplemented with 7.5% fetal bovine serum (FBS,
triple 0.1-µm filtered; Hyclone, Logan, UT). Serum was pretreated
with dextran-coated charcoal as described (29). Cells were passaged at
confluency (approximately every third day) at a ratio of 1:4. HepG2
human hepatoma cells were grown as monolayer cultures in low-glucose Dulbecco's minimal essential medium (Life Technologies) supplemented with 10% FBS. Cultures were passaged at confluency (approximately twice weekly) at a ratio of 1:10 using trypsin-EDTA.
Plasmids--
The luciferase reporter plasmids
p(SBE)4 and p(CAGA)12 were described earlier
(30, 31). The pAR3-lux and pTlx2-lux reporter plasmids were described
earlier (32, 33). The pGL3 basic and pGL3 promoter vectors were
obtained from Promega. The pCDNA3 expression vector was obtained
from Invitrogen (Carlsbad, CA). The Transcriptional Response Assay--
The day before transfection,
P19 cells were plated in 24-well plates at 50,000 cells/well in 0.5 ml
of DMEM containing 7.5% FBS and were typically 50-60% confluent at
the time of transfection. Plasmid DNA (typically 0.5 µg of total
including 0.25 µg of luciferase reporter and 5 ng of
pRSV- Luciferase and Immunoblot Analysis--
P19 cells were plated in six-well
plates (2 × 105 cells/well) in 2 ml of media on day
0. On day 2, media were removed and replaced with DMEM containing 0.1%
bovine serum albumin for 3 h. Nodal, activin, or BMP was then
added in 0.1% bovine serum albumin for varying times. Cells were
washed with ice-cold phosphate-buffered saline, and 200 µl of 1×
gel-loading dye was added to each well. Cell lysates were collected,
sonicated for 15 s, and heated for 3 min at 95 °C. Samples were
fractionated by SDS-polyacrylamide gel electrophoresis using a 10-20%
gradient acrylamide gel. Transfer to the polyvinylidene difluoride
membrane and processing were as described previously (34, 35). Briefly,
membranes were incubated in TNT 20 (20 mM Tris-HCl (pH
7.6), 137 mM NaCl, 0.1% v/v Tween 20) with 5% w/v nonfat
dried milk for 1 h, washed in TNT 20 (3-5 min), and incubated
with primary antibody (1:1000) in TNT 20 containing 1% milk. This was
done for 1 h at room temperature for non-phospho-specific
antibodies and overnight at 4 °C for phospho-specific antibodies.
After further washing in TNT 20, membranes were incubated for 1 h
with horseradish peroxidase-linked anti-IgG secondary antibody (Pierce;
1:5000), and immunoreactive proteins were detected using SuperSignal
chemiluminescent substrate (Pierce).
P19 Embryonal Carcinoma Cells Are Responsive to Recombinant Nodal
Protein--
Unpublished work from our laboratory has shown that P19
embryonal carcinoma cells stably transfected with a nodal expression vector can differentiate into mesoderm without chemical
induction.2 These results
suggested that P19 cells are responsive to nodal and may provide an
appropriate model system to study nodal function in the early mouse
embryo. To confirm these results and establish a reporter assay to
dissect the nodal signaling pathway, we carried out transient
transfection of undifferentiated P19 cells using the
p(SBE)4 luciferase reporter. p(SBE)4 has been
shown to be a general reporter for the TGF-
To analyze nodal signaling, we used recombinant human nodal protein,
produced in bacteria and refolded in vitro. Nodal protein activity was assayed using Xenopus embryo explants. Animal
caps treated with nodal protein (at 5 µg/ml) or activin (at 2.5 or 25 ng/ml) underwent elongations characteristic of mesoderm formation (data
not shown), confirming the bioactivity of the recombinant protein
preparation. We then evaluated p(SBE)4 activation by
recombinant nodal treatment of P19 cells. As shown in Fig.
1B, nodal induced p(SBE)4 reporter activity in a
dose-dependent manner with a 14-fold increase at 4 µg/ml.
These studies confirmed that P19 cells are competent to respond to
nodal and provide an experimental system for defining the intracellular
signaling pathway. In further studies, we used 2 µg/ml nodal, because
this amount still gave a significant level of induction within the
range of activation induced by the other ligands.
Nodal Signaling Requires EGF-CFC Function--
The specific
activity of recombinant nodal protein was low compared with the other
ligands tested, perhaps because of a significant amount of inactive
protein in the high-performance liquid chromatography-purified fraction. The requirement of using such large amounts of nodal protein
raised the question of specificity: is the signaling truly representative of endogenous nodal signaling, or is it caused by
low-affinity binding to nonphysiological receptors? To address this
question, we analyzed nodal signaling in HepG2 cells. As shown in Fig.
2A, activin, BMP4, and TGF-
The nonresponsiveness of HepG2 cells to nodal treatment indicates these
cells lack a component specific to the nodal signaling pathway. Based
on results in zebrafish suggesting that nodal signaling requires
EGF-CFC function (18) and unpublished data showing that HepG2 cells
lack expression of full-length
cripto,3 we asked whether
transfection of a cripto expression vector could rescue nodal signaling
in HepG2 cells. As shown in Fig. 2B, nodal treatment of
HepG2 cells expressing cripto led to a significant increase in
p(SBE)4 reporter activity (4.92 ± 0.95-fold) compared with controls. This result provides further support that the
recombinant protein is acting physiologically and suggests that EGF-CFC
activity is a general requirement for vertebrate nodal signaling.
Nodal Induces a TGF- Nodal Signaling Is Mediated by Smad3--
The response of the
activin response element to activin and TGF- Nodal Signaling Is Reduced by a Dominant Negative Smad2--
To
determine the involvement of Smad2 in nodal signaling, we first used a
dominant negative approach. Previous work has shown that C-terminal
serines in Smad2 are phosphorylated by the TGF- Nodal Signaling Results in Phosphorylation of Smad2--
As a
second approach to determine Smad2 involvement in nodal signaling, we
examined Smad2 phosphorylation after nodal treatment of P19 cells.
These studies used antisera specific for the C-terminal phosphorylated
form of Smad2. These antibodies do not recognize the unphosphorylated
(nonactivated) forms of these Smads. P19 cells were treated with nodal
(2 µg/ml), activin (20 ng/ml), or BMP4 (40 ng/ml) for various times,
and cell lysates were analyzed by Western blotting. As shown in Fig.
6A, nodal activated Smad2 within 15 min, and phosphorylation was found for at least 4 h before returning to basal levels by 24 h. Activin treatment also led to Smad2 phosphorylation in P19 cells. However, peak levels for
activin stimulation were seen at 1 h with a return to basal level
by 4 h.
To confirm that nodal signaling does not go through BMP R-Smads, we
used antisera specific for the C-terminal phosphorylated forms of Smads
1, 5, and 8. As shown in Fig. 6B, BMP4 treatment led to
rapid phosphorylation of these BMP R-Smads, which returned to basal
levels by 24 h. However, treatment with nodal had no effect. These
results provide direct evidence that nodal signals through Smad2 and
conclusively show that BMP R-Smads are not used.
We have obtained several lines of evidence indicating that nodal
activity in P19 cells is regulated through the activin-TGF- The involvement of Smad2 in nodal signaling in P19 cells suggests that
a similar pathway exists in the intact embryo. Several different
targeted mutations of Smad2 in the mouse germ line have been reported
(5-8). The studies by Nomura and Li (7) and Weinstein et
al. (6) have described gastrulation defects similar to those of
the nodal null mutant, which lacks mesoderm formation (11, 12, 42). The
similarity of these phenotypes is consistent with nodal signaling using
Smad2 in the gastrulating embryo. Nomura and Li (7) also reported that
embryos heterozygous for both the Smad2 and nodal mutations have
phenotypic defects in left-right axis development and anterior
patterning. Heyer et al. (8) reported similar phenotypes in
Smad2 mutant embryos rescued through gastrulation. These latter
phenotypes are similar to that produced by a hypomorphic nodal
mutation,4 indicating that
nodal signaling uses Smad2 in developmental processes other than
mesoderm formation. However, the Smad2 knockouts reported by Waldrip
et al. (5) and Heyer et al. (8) showed transient mesoderm formation, complicating the conclusion that nodal uses Smad2
in the induction of mesoderm in the embryo. Our conclusive demonstration that nodal signals through Smad2 in P19 cells will aid in
the interpretation of these different Smad2 knockout phenotypes.
We have shown that nodal signaling also can use Smad3 in P19 cells.
However, targeted mutation of Smad3 in the mouse does not lead to early
developmental defects (43-45), suggesting that Smad3 is not involved
in nodal signaling in the embryo or that Smad2 can compensate for the
loss of Smad3. Interestingly, embryos heterozygous for both Smad2 and
Smad3 can display left-right
abnormalities.5 This finding
suggests that nodal signaling may indeed involve both Smad2 and Smad3
in the embryo.
Although we show here that nodal uses an activin-like intracellular
signaling pathway, we have also found some major differences between
nodal and activin signaling. We have found that human HepG2 cells are
not responsive to human nodal protein unless transfected with an
expression vector encoding the extracellular EGF-CFC protein cripto.
Previous genetic studies demonstrated a requirement for the zebrafish
EGF-CFC protein one-eyed pinhead in nodal-related signaling in the
zebrafish but not for activin signaling (18). A recent study showed
that injection of a mouse nodal expression vector into
Xenopus embryos resulted in induction of a luciferase reporter carrying an enhancer element from the nodal gene, but at a
significantly lower level than that obtained with an activin expression
vector. However, co-injection of nodal and cripto expression vectors
led to similarly high levels as with the activin expression vector
(46). Together these data suggest that there is a general requirement
for EGF-CFC protein function in vertebrate nodal signaling.
Another difference between activin and nodal signaling is in the
persistence of Smad2 phosphorylation occurring in response to these two
factors. We found that during continuous activin treatment of P19 cells
the levels of activated Smad2 peaked at 1 h and then declined.
This is similar to what was found for Smad2 activated by continuous
TGF- In conclusion, we have shown that nodal signaling in P19 cells is
mediated intracellularly through the TGF- (TGF-
) superfamily, is implicated in many events critical to the
early vertebrate embryo, including mesoderm formation, anterior
patterning, and left-right axis specification. Here we define the
intracellular signaling pathway induced by recombinant nodal protein
treatment of P19 embryonal carcinoma cells. Nodal signaling activates
pAR3-Lux, a luciferase reporter previously shown to respond
specifically to activin and TGF-
. However, nodal is unable to induce
pTlx2-Lux, a reporter specifically responsive to bone morphogenetic
proteins. We also demonstrate that nodal induces
p(CAGA)12, a reporter previously shown to be
specifically activated by Smad3. Expression of a dominant negative
Smad2 significantly reduces the level of luciferase reporter activity
induced by nodal treatment. Finally, we show that nodal signaling
rapidly leads to the phosphorylation of Smad2. These results provide
the first direct biochemical evidence that nodal signaling is mediated
by both activin-TGF-
pathway Smads, Smad2 and Smad3. We also show
here that the extracellular cripto protein is required for nodal
signaling, making it distinct from activin or TGF-
signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 superfamily of
intercellular signaling factors regulate cell fate and behavior during
development and in the adult (1). The three major subgroups based on
sequence similarity are the TGF-
s, activins and inhibins, and bone
morphogenetic proteins (BMPs; Ref. 1). Nodal and related factors form a
separate subgroup and are implicated in many events critical to the
early vertebrate embryo, including mesoderm formation, anterior
patterning, and left-right axis specification (2).
and related ligands uses two types of receptors,
type I and type II transmembrane serine-threonine kinases. Ligand
binding results in the formation of heteromeric receptor complexes, in
which type II receptors phosphorylate type I receptors (1, 3).
Downstream signal transduction events are mediated by the intracellular
Smad proteins. One class, the receptor-regulated Smads (R-Smads), are
directly phosphorylated by activated type I receptors on a C-terminal
SSXS motif. Upon phosphorylation, R-Smads form complexes
with the co-Smad, Smad4 and then translocate to the nucleus and
regulate transcription of target genes. Biochemical and biological
studies have established that the R-Smads used by TGF-
and activin
signaling, Smad2 and Smad3, are distinct from those used by BMP
signaling, Smad1, Smad5, and Smad8 (1, 3, 4).
pathway-specific Smads but not BMP pathway-specific Smads. In addition,
we show that nodal activity depends on the expression of cripto,
suggesting a general requirement for EGF-CFC function in vertebrate
nodal signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, BMP4, and BMP6 were from R & D systems (Minneapolis, MN). Tissue culture materials and transfection
reagents were purchased from Life Technologies. The gradient gel and
polyvinylidene difluoride membrane were obtained from Novex (San Diego,
CA). Antibodies PS1 (specific to the C-terminal phosphorylated form of
Smad1, Smad5, and Smad8) and PS2 (specific to the C-terminal phosphorylated form of Smad2) were described earlier (27, 28). Additional antibodies to the phosphorylated forms of Smad1 and Smad2,
as well as antibodies to the unphosphorylated forms of Smad1, Smad2,
and Smad3, were from Upstate Biotechnology (Waltham, MA). A Dual light
reporter gene assay kit was purchased from Tropix (Bedford, MA).
-galactosidase reporter
pRSV-
-galactosidase was provided by P. Yen (National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health). A HindIII-NotI fragment containing the full-length cripto cDNA (a gift from M. Shen) was subcloned into the pCDNA3 vector for expression.
-galactosidase) was mixed with 2 µl of LipofectAMINE Plus
reagent (Life Technologies) in 25 µl of DMEM and incubated at room
temperature. After 15 min, 25 µl of media containing 1.5 µl of
LipofectAMINE was added and incubated for another 30 min. Cells were
washed once with DMEM (without FBS), and 200 µl of DMEM was added to
each well. 50 µl of the transfection mixture was added to each well,
and after 2 h, another 250 µl of DMEM containing 15% FBS was
added to each well and incubated overnight (14-18 h). The next day,
cells were washed once with DMEM, and fresh media were added. After
overnight incubation, media were replaced with serum-free DMEM
containing 0.1% bovine serum albumin with or without factors (activin,
BMPs, TGF-
, or nodal) as indicated. The cells were harvested for
luciferase assay after 16-20 h of incubation. Similar starting cell
numbers and transfection conditions were used for HepG2 cells.
Transfections were carried out with 0.5 µg of total DNA (0.25 µg of
reporter and 0.25 µg of cripto expression vector). In control
reactions, 0.25 µg of pCDNA3.0 was used instead of cripto. HepG2
cells were treated with the same concentrations of ligands as described
for P19 cells.
-Galactosidase Assay--
Luciferase and
-galactosidase activity were measured using the Dual light
chemiluminescent reporter gene assay kit (Tropix; Applied Biosystems,
Foster City, CA) as specified by the manufacturer, in an EG&G
Berthold Lumat LB 9507 luminometer. Briefly, cells were washed once
with phosphate-buffered saline. After addition of 120 µl of lysis
buffer, cells were scraped and centrifuged (4 °C, 12,000 × g, 5 min). 10 µl of the supernatant was added to 25 µl
of buffer A, followed by 100 µl of buffer B. After a delay of 2 s, the luciferase signal was measured for 5 s.
-Galactosidase activity was measured 60 min after measuring luciferase activity. All
assays were performed in duplicate. Luciferase activity was normalized
to
-galactosidase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily in HepG2
cells, responding to TGF-
itself, activin, and BMP2 (30). Therefore,
it was a good candidate reporter to confirm nodal signaling in P19
cells without knowing any details of the pathway. We first determined that p(SBE)4 is induced by TGF-
, activin, and BMP2 in
P19 cells. As shown in Fig.
1A, activin (20 ng/ml), BMP4
(40 ng/ml), and TGF-
1 (1 ng/ml) increased p(SBE)4
reporter activity by 9 ± 1.8-, 4 ± 2.3-, and 3 ± 0.4-fold (mean ± S.D.), respectively. These results established
that p(SBE)4 is a general reporter for both TGF-
-activin
and BMP pathway-specific R-Smad activation in P19 cells, as in HepG2
cells.
View larger version (26K):
[in a new window]
Fig. 1.
Recombinant nodal protein activates the
p(SBE)4 reporter in P19 cells. Luciferase activity in
the absence of any factor is taken as 100%. A, treatment
with activin (20 ng/ml), BMP4 (40 ng/ml), or TGF- (1 ng/ml) leads to
a significant increase in p(SBE)4 activity in P19 cells.
The mean + S.D. for six experiments is plotted. B, treatment
of P19 cells with increasing concentrations of nodal (0.25-4 µg/ml)
leads to an increase in p(SBE)4 activity. The mean + S.D.
of two experiments is plotted.
induced p(SBE)4 activity in transfected HepG2 cells by
8.5 ± 1.4-, 8.8 ± 1.4-, and 5.8 ± 0.1-fold,
respectively. However, we saw no activation of p(SBE)4 by
nodal at 2 µg/ml, the same amount that gave a significant level of
induction in P19 cells. This result indicated that our preparation of
recombinant nodal protein, even at this concentration, does not
cross-react with the TGF-
, activin, and BMP receptors clearly present on HepG2 cells.
View larger version (26K):
[in a new window]
Fig. 2.
Nodal activates p(SBE)4 in HepG2
cells only when Cripto is coexpressed. A, treatment of
HepG2 cells with nodal has no effect on p(SBE)4 activity,
whereas activin (20 ng/ml), BMP4 (40 ng/ml), or TGF- (1 ng/ml) leads
to a significant increase. The mean + S.D. for three separate
experiments is shown. B, HepG2 cells transfected with a
Cripto expression vector show a significant increase in
p(SBE)4 activity when treated with nodal (2 µg/ml). The
mean + S.D. for three separate experiments is shown.
and Activin Response but Not a BMP
Response--
To determine whether intracellular nodal signals are
mediated through the TGF-
-activin or BMP pathway, or both, we used
two reporters that differentiate between these pathways. pAR3-lux contains an activin response element from the Xenopus
Mix.2 gene and is activin and TGF-
inducible in
Xenopus embryos (36) and in HepG2 cells (32). The pTlx2-lux
reporter contains a BMP-responsive element and is BMP2, BMP7, and Smad1
inducible in P19 cells (33, 37). pTlx2-lux has also been shown to be
induced by constitutively active forms of the BMP-specific type I
receptors activin receptor-like kinase 2 and activin
receptor-like kinase 6 but not by a constitutively active activin
receptor-like kinase 4 receptor, an activin-specific type I receptor
(37). We transiently transfected P19 cells with pAR3-lux and pTlx2-lux
reporters and treated them with nodal (2 µg/ml), activin (20 ng/ml),
BMP4 (40 ng/ml), and BMP6 (40 ng/ml). As shown in Fig.
3A, activin treatment of cells
transiently transfected with pAR3-lux resulted in a 4.8 ± 0.9-fold induction of luciferase activity, consistent with previous
reports (37). When cells were treated with nodal, pAR3-lux activation
increased by 3 ± 0.4-fold. Neither BMP4 nor BMP6 had any
significant effect on the activity of pAR3-lux. As shown in Fig.
3B, BMP4 and BMP6 induced pTlx2-lux by 1.8 ± 0.12- and
2.2 ± 0.26-fold, respectively. As expected, we did not see any
induction by activin. Nodal also had no detectable effect on the Tlx2
promoter. Taken together, our results suggest that nodal uses only the
TGF-
-activin signaling pathway in P19 cells.
View larger version (29K):
[in a new window]
Fig. 3.
Nodal activates pAR3-lux but not pTlx-2-lux
in P19 cells. Luciferase activity in the absence of any factor is
taken as 100%. A, the pAR3-lux reporter responds to activin
(20 ng/ml) and nodal (2 µg/ml) but not to BMP4 or BMP6 (each 40 ng/ml). B, pTlx-2-lux only responds to BMPs and not to
either activin or nodal. The mean + S.D. for three experiments is
plotted.
treatment was
previously shown to be mediated by either Smad2 or Smad3 (38-40). To
determine whether nodal signals through Smad2 or Smad3, or both, in P19
cells, we first used the p(CAGA)12 luciferase reporter.
This reporter is activated only in response to activin and TGF-
signaling and only through Smad3 and not Smad2 in HepG2 and Mv1Lu cells
(31). As shown in Fig. 4, activin and
TGF-
induced p(CAGA)12 by 9.4 ± 3.1- and 6.5 ± 3.1-fold respectively in P19 cells, whereas BMP4 had no significant
effect. Treatment with nodal led to a 6.2 ± 1.5-fold increase in
p(CAGA)12 activity. These results indicate that Smad3 can
mediate nodal signaling in P19 cells.
View larger version (31K):
[in a new window]
Fig. 4.
Nodal activates the Smad3-specific luciferase
reporter p(CAGA)12 in P19 cells. Luciferase activity
in the absence of any factor is taken as 100%. Activin (20 ng/ml),
nodal (2 µg/ml), and TGF- 1 (1 ng/ml) lead to a significant
increase in p(CAGA)12 reporter activity, whereas BMP4 (40 ng/ml) does not. The mean + S.D. for five experiments is plotted.
receptor, and
mutating these serines to alanines prevents Smad2 phosphorylation and
its nuclear accumulation (41). These mutant forms of Smad2 can still
associate with the receptor, however, and can act as dominant
negatives. We used the dominant negative Smad2 (3S-A) to test whether
it affected nodal-mediated p(SBE)4 activation. P19 cells
were transfected with p(SBE)4 either alone or together with
Smad2 (3S-A), and luciferase activity was measured after
treatment with nodal or activin. The results are shown in Fig.
5A. In the absence of Smad2
(3S-A), p(SBE)4 activity was increased 6- and 9-fold by
nodal and activin, respectively, which was taken as 100% activity. In
the presence of Smad2 (3S-A), we observed only 3.96- and 6.75-fold
induction by nodal and activin, respectively, a 34 and 25% reduction.
We repeated these experiments using the activin- and TGF-
-specific
reporter pAR3-lux. As shown in Fig. 5B, expression of the
dominant negative Smad2 led to a 40% reduction of nodal-induced
pAR3-lux activity. These results indicate that nodal also can use Smad2
for intracellular signaling in P19 cells.
View larger version (32K):
[in a new window]
Fig. 5.
Nodal signaling is decreased in P19 cells
expressing dominant negative Smad2. Cells were cotransfected with
0.2 µg p(SBE)4 or pAR3-lux and 0.01 µg of Smad2 (3S-A).
The level of luciferase activity after either nodal or activin
treatment in the absence of Smad2 (3S-A) is taken as 100%.
A, in Smad2 (3S-A)-transfected cells treated with nodal (2 µg/ml) or activin (20 ng/ml), there is a significant reduction of
p(SBE)4 activity. The mean + S.D. for three experiments is
shown. B, cells expressing Smad2 (3S-A) treated with nodal
(2 µg/ml) or activin (20 ng/ml) show a similar reduction of pAR3-lux
activity.
View larger version (92K):
[in a new window]
Fig. 6.
Nodal signaling leads to phosphorylation of
Smad2 but not BMP R-Smads in P19 cells. A, immunoblots
of nodal- and activin-treated samples. Top panel, samples
analyzed with the PS2 antibody specific to phosphorylated Smad2. Both
ligands cause rapid phosphorylation of Smad2 but with different
kinetics. Bottom panel, same samples analyzed with
anti-Smad2 antibody. B, immunoblots of BMP4- and
nodal-treated samples. Top panel, samples analyzed with the
PS1 antibody specific to phosphorylated Smads 1, 5, and 8. BMP4
treatment results in rapid phosphorylation, whereas nodal has no
effect. Bottom panel, same samples analyzed with anti-Smad1
antibody. The results shown are representative of three separate
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pathway
mediated by Smad2 and Smad3 and not through BMP-regulated Smads. First,
nodal induces pAR3-lux, which was previously shown to be specifically
responsive to TGF-
and activin (37). Nodal does not activate
pTlx2-lux, which has been shown to be strictly BMP inducible (33, 37).
Second, nodal activates the p(CAGA)12 reporter, which was
previously shown to be activated specifically by Smad3 (31). Third,
nodal activation of either p(SBE)4 or pAR3-lux is reduced
significantly by expression of a dominant negative form of Smad2.
Fourth, nodal treatment of P19 cells rapidly induces the
phosphorylation of Smad2 but not BMP R-Smads.
treatment of HaCaT cells (47). However, Smad2 phosphorylation
induced by continuous nodal treatment of P19 cells was sustained for
much longer. This difference may be attributable to differences at the
level of receptor-mediated phosphorylation of Smad2 or at the level of
Smad2 turnover. Lo and Massagué (47) showed that TGF-
activation of Smad2 and translocation to the nucleus leads to
multiubiquitination of Smad2 and subsequent degradation by the
proteasome. Smad2 activated by nodal may persist longer, because it may
be ubiquitinated with different kinetics than Smad2 activated by
TGF-
or activin. Alternatively, the receptor complex brought
together by nodal may be more stable than that formed by TGF-
or
activin receptor binding, perhaps through the action of extracellular
EGF-CFC proteins. Increased receptor complex stability might result in
more sustained activation of Smad2.
-activin pathway, providing
important biochemical evidence that nodal signaling in the embryo uses
Smad2 and Smad3.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mark deCaestecar for the Smad2 dominant negative construct, Jeff Wrana for the pAR3-lux and pTlx2-lux reporters, Michael Shen for the cripto cDNA, Ester Piek for many useful suggestions, and Anita Roberts and Linda A. Lowe for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
§ Present address: Dept. of Embryology, Moscow State University, Moscow 119899, Russia.
** Supported by the Dutch Organization for Scientific Research (Project 809.67.021).
To whom correspondence should be addressed: Experimental
Immunology Branch, NCI, National Institutes of Health, Bldg. 10, Rm.
4B-36, 10 Center Dr., Bethesda, MD 20892-1360. Tel.: 301-435-6476; Fax:
301-496-0887; E-mail: mkuehn@mail.nih.gov.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M004649200
2 V. Novoselov and M. R. Kuehn, unpublished data.
3 D. Salomon, personal communication.
4 L. A. Lowe, S. Yamada, and M. R. Kuehn, submitted for publication.
5 M. Weinstein and C. X. Deng, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; R-Smad, receptor-regulated Smad; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SBE, Smad binding element; EGF-CFC, epidermal growth factor-Cripto, FRL1, Cryptic.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Massagué, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve] |
2. | Schier, A. F., and Shen, M. M. (2000) Nature 403, 385-389[CrossRef][Medline] [Order article via Infotrieve] |
3. | Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737-740[Medline] [Order article via Infotrieve] |
4. | Wrana, J. L. (2000) Cell 100, 189-192[Medline] [Order article via Infotrieve] |
5. | Waldrip, W. R., Bikoff, E. K., Hoodless, P. A., Wrana, J. L., and Robertson, E. J. (1998) Cell 92, 797-808[Medline] [Order article via Infotrieve] |
6. |
Weinstein, M.,
Yang, X.,
Li, C.,
Xu, X.,
Gotay, J.,
and Deng, C. X.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9378-9383 |
7. | Nomura, M., and Li, E. (1998) Nature 393, 786-790[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Heyer, J.,
Escalante-Alcalde, D.,
Lia, M.,
Boettinger, E.,
Edelmann, W.,
Stewart, C. L.,
and Kucherlapati, R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12595-12600 |
9. |
Gu, Z.,
Nomura, M.,
Simpson, B. B.,
Lei, H.,
Feijen, A.,
van den Eijnden-van Raaij, J.,
Donahoe, P. K.,
and Li, E.
(1998)
Genes Dev.
12,
844-857 |
10. | Song, J., Oh, S. P., Schrewe, H., Nomura, M., Lei, H., Okano, M., Gridley, T., and Li, E. (1999) Dev. Biol. 213, 157-169[CrossRef][Medline] [Order article via Infotrieve] |
11. | Iannaccone, P. M., Zhou, X., Khokha, M., Boucher, D., and Kuehn, M. R. (1992) Dev. Dyn. 194, 198-208[Medline] [Order article via Infotrieve] |
12. |
Conlon, F. L.,
Lyons, K. M.,
Takaesu, N.,
Barth, K. S.,
Kispert, A.,
Herrmann, B.,
and Robertson, E. J.
(1994)
Development
120,
1919-1928 |
13. |
Jones, C. M.,
Kuehn, M. R.,
Hogan, B. L.,
Smith, J. C.,
and Wright, C. V.
(1995)
Development
121,
3651-3662 |
14. | Hoodless, P. A., Tsukazaki, T., Nishimatsu, S., Attisano, L., Wrana, J. L., and Thomsen, G. H. (1999) Dev. Biol. 207, 364-379[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hemmati-Brivanlou, A., and Melton, D. A. (1992) Nature 359, 609-614[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Chang, C.,
Wilson, P. A.,
Mathews, L. S.,
and Hemmati-Brivanlou, A.
(1997)
Development
124,
827-837 |
17. | Dyson, S., and Gurdon, J. B. (1997) Curr. Biol. 7, 81-84[Medline] [Order article via Infotrieve] |
18. | Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S., and Schier, A. F. (1999) Cell 97, 121-132[Medline] [Order article via Infotrieve] |
19. |
Dono, R.,
Scalera, L.,
Pacifico, F.,
Acampora, D.,
Persico, M. G.,
and Simeone, A.
(1993)
Development
118,
1157-1168 |
20. |
Shen, M. M.,
Wang, H.,
and Leder, P.
(1997)
Development
124,
429-442 |
21. | Ding, J., Yang, L., Yan, Y. T., Chen, A., Desai, N., Wynshaw-Boris, A., and Shen, M. M. (1998) Nature 395, 702-707[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Xu, C.,
Liguori, G.,
Persico, M. G.,
and Adamson, E. D.
(1999)
Development
126,
483-494 |
23. |
Yan, Y. T.,
Gritsman, K.,
Ding, J.,
Burdine, R. D.,
Corrales, J. D.,
Price, S. M.,
Talbot, W. S.,
Schier, A. F.,
and Shen, M. M.
(1999)
Genes Dev.
13,
2527-2537 |
24. | Gaio, U., Schweickert, A., Fischer, A., Garratt, A. N., Muller, T., Ozcelik, C., Lankes, W., Strehle, M., Britsch, S., Blum, M., and Birchmeier, C. (1999) Curr. Biol. 9, 1339-1342[CrossRef][Medline] [Order article via Infotrieve] |
25. | McBurney, M. W. (1993) Int. J. Dev. Biol. 37, 135-140[Medline] [Order article via Infotrieve] |
26. | Schlunegger, M. P., Cerletti, N., Cox, D. A., McMaster, G. K., Schmitz, A., and Grutter, M. G. (1992) FEBS Lett. 303, 91-93[CrossRef][Medline] [Order article via Infotrieve] |
27. | Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby, S., Engstrom, U., Heldin, C.-H., Funa, K., and ten Dijke, P. (1998) FEBS Lett. 434, 83-87[CrossRef][Medline] [Order article via Infotrieve] |
28. | Piek, E., Westermark, U., Kastemar, M., Heldin, C.-H., van Zoelen, E. J., Nister, M., and ten Dijke, P. (1999) Int. J. Cancer 80, 756-763[CrossRef][Medline] [Order article via Infotrieve] |
29. | Germain, P., and Harbrioux, G. (1993) Anticancer Res. 13, 1581-1585[Medline] [Order article via Infotrieve] |
30. |
Jonk, L. J.,
Itoh, S.,
Heldin, C.-H.,
ten Dijke, P.,
and Kruijer, W.
(1998)
J. Biol. Chem.
273,
21145-21152 |
31. |
Dennler, S.,
Itoh, S.,
Vivien, D.,
ten Dijke, P.,
Huet, S.,
and Gauthier, J. M.
(1998)
EMBO J.
17,
3091-3100 |
32. | Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., Richardson, M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D. (1997) Cell 89, 1165-1173[Medline] [Order article via Infotrieve] |
33. |
Tang, S. J.,
Hoodless, P. A.,
Lu, Z.,
Breitman, M. L.,
McInnes, R. R.,
Wrana, J. L.,
and Buchwald, M.
(1998)
Development
125,
1877-1887 |
34. |
Kumar, A.,
Middleton, A.,
Chambers, T. C.,
and Mehta, K. D.
(1998)
J. Biol. Chem.
273,
15742-15748 |
35. | Kumar, A., Chambers, T. C., Cloud-Heflin, B. A., and Mehta, K. D. (1997) J. Lipid Res. 38, 2240-2248[Abstract] |
36. | Chen, X., Rubock, M. J., and Whitman, M. (1996) Nature 383, 691-696[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Macias-Silva, M.,
Hoodless, P. A.,
Tang, S. J.,
Buchwald, M.,
and Wrana, J. L.
(1998)
J. Biol. Chem.
273,
25628-25636 |
38. | Weisberg, E., Winnier, G. E., Chen, X., Farnsworth, C. L., Hogan, B. L., and Whitman, M. (1998) Mech. Dev. 79, 17-27[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Yeo, C. Y.,
Chen, X.,
and Whitman, M.
(1999)
J. Biol. Chem.
274,
26584-26590 |
40. |
Yagi, K.,
Goto, D.,
Hamamoto, T.,
Takenoshita, S.,
Kato, M.,
and Miyazono, K.
(1999)
J. Biol. Chem.
274,
703-709 |
41. | 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] |
42. | Pfendler, K. C., Yoon, J. W., Taborn, G. U., Kuehn, M. R., and Iannaccone, P. M. (2000) Genesis 28, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
43. | Zhu, Y., Richardson, J. A., Parada, L. F., and Graff, J. M. (1998) Cell 94, 703-714[Medline] [Order article via Infotrieve] |
44. |
Yang, X.,
Letterio, J. J.,
Lechleider, R. J.,
Chen, L.,
Hayman, R.,
Gu, H.,
Roberts, A. B.,
and Deng, C.
(1999)
EMBO J.
18,
1280-1291 |
45. |
Datto, M. B.,
Frederick, J. P.,
Pan, L.,
Borton, A. J.,
Zhuang, Y.,
and Wang, X. F.
(1999)
Mol. Cell. Biol.
19,
2495-2504 |
46. | Saijoh, Y., Adachi, H., Sakuma, R., Yeo, C. Y., Yashiro, K., Watanabe, M., Hashiguchi, H., Mochida, K., Ohishi, S., Kawabata, M., Miyazono, K., Whitman, M., and Hamada, H. (2000) Mol. Cell 5, 35-47[Medline] [Order article via Infotrieve] |
47. | Lo, R. S., and Massagué, J. (1999) Nat. Cell Biol. 1, 472-478[CrossRef][Medline] [Order article via Infotrieve] |