From the Department of Pathology, North Shore-Long Island Jewish Health System and Biomedical Research Center, Manhasset, New York 11030
Received for publication, November 29, 2000, and in revised form, February 15, 2001
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ABSTRACT |
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Transforming growth factor- TGF- In view of its diverse and potent activities, the signaling by TGF- Besides the vast network of positive and negative intracellular
regulatory factors, there are extracellular proteins that, by directly
interacting with TGF- Materials--
The full-length 1.961-kilobase lefty A
(ebaf) cDNA was derived from a human placental
cDNA library (48). The materials used in this study included an
enhanced chemiluminescence system (Roche Molecular Biochemicals),
polyvinylidene difluoride membrane (Bio-Rad), and Kodak Omat film
(Sigma). Unless otherwise indicated, all other chemicals were from
either Sigma or Fisher. The P19 cell line was obtained from American
Type Culture Collection (Manassas, VA). Recombinant TGF- Cells, Transfection, and Protein Preparation--
P19 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (Life Technologies,
Inc.) and 1% antibiotic/antimycotic mixture (Life Technologies, Inc.).
For transfection, cells were seeded into 6-well plates (Falcon,
Franklin lakes, NJ) at a concentration of 1.3 × 104
cells/ml and maintained in a CO2 chamber at 37 °C for
~16 h. When 60% confluent, cells were transfected with cDNA from
the TGF- SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting--
The proteins in the total cell lysates or cell fractions
(15 µg of protein/lane) were resolved on a 12% gel together with a
prestained protein ladder (Life Technologies, Inc.) and were subsequently blotted onto polyvinylidene difluoride membranes in a
Mini-Trans-Blot apparatus (Bio-Rad). The blots were stained using
protein-specific antibodies, followed by incubation with horseradish
peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology).
The specific bands were detected with the chemiluminescence system as
described by the manufacturer. The relative optical densities of the
bands were quantitated by laser scanning densitometry.
Immunoprecipitation--
Immunoprecipitations of the proteins
were performed as described (49). Briefly, for immunoprecipitation,
2-5 µl of specific antibody was added to each milliliter of cell
lysate (~106 cells). The immunoprecipitates were
subjected to SDS-PAGE and blotting, followed by autoradiography.
Immunohistochemical Staining of Smad4--
Smad4 was localized
in cells grown on coverslips. Cells were fixed for 5 min in 10%
buffered Formalin and washed for 5 min with 0.1 M
phosphate-buffered saline (pH 7.4). Cells were incubated at room
temperature with mouse monoclonal anti-Smad4 antibody for 60 min,
followed by a 5-min washing with phosphate-buffered saline. They were
then incubated for 60 min at room temperature with a peroxidase-labeled
anti-mouse secondary antibody. After washing with phosphate-buffered
saline, staining was developed using a
diaminobenzidine/H2O2 mixture.
Luciferase Reporter Assay--
Cells were transfected with 0.5 µg of p21-Lux, pCdc25-Lux, and pCTGF-Lux using Superfect
according to the manufacturer's instructions. Cytokines (TGF- In Vivo Phosphorylation of Smad Proteins--
Cells were grown
in Dulbecco's modified Eagle's medium/nutrient mixture F-12 for
24 h and then washed and incubated for 2 h in Dulbecco's
modified Eagle's medium without glutamine and phosphate. Cells were
incubated for another 2 h in the same medium supplemented with 10 µCi/ml [32P]orthophosphate. After cytokine treatment,
cells were lysed in 1 ml of radioimmune precipitation assay buffer, and
the samples were subjected to immunoprecipitation with Smad2-specific
antibody. The immunoprecipitates were subjected to SDS-PAGE, followed
by blotting and autoradiography.
Lefty Represses the Activation of Reporter Genes by
TGF-
We next tested the effect of lefty on TGF- Lefty Inhibits the Nuclear Localization of R-Smad·Smad4 Complexes
Induced by TGF- Lefty Inhibits the Heterodimerization of R-Smad Proteins with Smad4
Induced by TGF- Lefty Inhibits the Phosphorylation of R-Smad Proteins Induced by
TGF-
Binding of TGF-
To further validate these findings, we assessed the effect of lefty on
gene transcription induced by the constitutively active TGF- Lefty Inhibition of the Phosphorylation of R-Smad Proteins Induced
by TGF-
We then tested whether the effect of lefty on phosphorylation of R-Smad
depends on induction of another protein that blocks the phosphorylation
of R-Smad proteins. To do this, the phosphorylation of R-Smad was
analyzed in P19 cells treated with cycloheximide and transfected with
the constitutively active TGF- Lefty Inhibits BMP Signaling--
Some data suggested that
lefty-1, the mouse homolog of human lefty, may act as an inhibitor of
BMP signaling (41). Therefore, we tested the effect of lefty on BMP
signaling. Signaling by BMP includes phosphorylation of Smad5, which
leads to transcriptional activity of BMP-responsive genes. We tested
the effect of lefty on both BMP-induced phosphorylation of Smad5 and
transcription of the reporter construct Vent2-Lux. P19 cells
were treated with BMP, lefty, or both in the presence of
[32P]orthophosphate to label phosphorylated proteins.
Analysis of phosphorylated Smad5, which was immunoprecipitated with
anti-Smad5 antibody, showed that although BMP led to the
phosphorylation of Smad5, lefty had little or no effect. However, lefty
reduced the BMP-mediated Smad5 phosphorylation by 50% (Fig.
6A). We next assessed the
effect of lefty on transcription of a Vent2-luciferase reporter
construct. P19 cells were transfected with the reporter construct, and
transfected cells were treated with BMP, lefty, or both. Lefty
inhibited the BMP-induced reporter activity in a
dose-dependent fashion (Fig. 6B). Taken
together, these findings show that lefty acts as an inhibitor of BMP
activity by inhibiting the phosphorylation of R-Smad.
TGF- The action of lefty in inhibiting the phosphorylation of R-Smad
proteins is similar to that exerted by anti-Smad proteins. Smad6
interferes with TGF- The interaction of lefty proteins with TGF- It is now well established that tumorigenesis is associated with
development of resistance to TGF- The lefty protein is expressed as a precursor of 42 kDa that is cleaved
at Arg77 and Arg135 to release polypeptides of
34 and 28 kDa, respectively (71). We found the 28-kDa lefty polypeptide
to induce MAPK activity, but the 34-kDa protein appears to be inactive.
Surprisingly, 42-kDa lefty is also capable of inducing MAPK activity,
indicating that the lefty precursor is biologically active (71). It was
recently shown that activated Ras, acting via Erk MAPKs, causes
phosphorylation of R-Smad proteins at specific sites in the region
linking the DNA-binding domain and the transcriptional activation
domain. This phosphorylation inhibits the TGF- In summary, lefty is an inhibitory member of the TGF- (TGF-
) is a
pleiotropic cytokine that regulates growth and differentiation of
diverse types of cells. TGF-
actions are directed by ligand-induced
activation of TGF-
receptors with intrinsic serine/threonine kinase
activity that trigger phosphorylation of receptor-regulated Smad
(R-Smad) protein. Phosphorylated R-Smad proteins bind to Smad4, and the complexes formed move into the nucleus, where they act as components of
a transcriptional complex. Here, we show that TGF-
signaling is
inhibited by lefty, a novel member of the TGF-
superfamily. Lefty
perturbed TGF-
signaling by inhibiting the phosphorylation of Smad2
following activation of the TGF-
receptor. Moreover, lefty inhibited
the events that lie downstream from R-Smad phosphorylation, including
heterodimerization of R-Smad proteins with Smad4 and nuclear
translocation of the R-Smad·Smad4 complex. Lefty repressed TGF-
-induced expression of reporter genes for the p21,
cdc25, and connective tissue growth factor promoters
and of a reporter gene driven by the Smad-binding element. Similarly,
lefty inhibited both BMP-mediated Smad5 phosphorylation and gene
transcription. The action of lefty does not appear to depend on protein
synthesis, including synthesis of inhibitory Smad proteins. Thus, lefty
provides a repressed state of TGF-
- or BMP-responsive genes and
participates in negative modulation of TGF-
and BMP signaling by
inhibition of phosphorylation of R-Smad proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is a potent and
pleiotropic cytokine that regulates cell growth and differentiation,
embryonic patterning, deposition of the extracellular matrix, and
fibrosis (1-7). To exert these functions, TGF-
brings together two
transmembrane serine/threonine kinases, the type I and II receptors.
The assembly and oligomerization of TGF-
receptors lead to
phosphorylation of receptor-regulated Smad (R-Smad) proteins,
heterodimerization of R-Smad proteins with Smad4, and subsequent
nuclear accumulation of these complexes (8-11). Activated Smad
complexes interact with other transcription factors in the nucleus
(12-15), bind to DNA by their N-terminal Mad homology-1 domains
(16-21), and activate transcription of TGF-
-responsive genes
through the C-terminal Mad homology-2 domains (1, 3, 4).
is under tight regulation both by positive and negative feedback
mechanisms. The two principal events in TGF-
signaling, TGF-
receptor and Smad phosphorylation, are controlled by a network of
regulatory proteins, which modulate the magnitude of signals induced by
TGF-
(22). Positive regulators include ligand accessory receptors
and substrate-anchoring factors such as membrane-anchored proteoglycan,
betaglycan, and SARA (Smad anchor for
receptor activation). Betaglycan, also known as
the TGF-
type III receptor, binds TGF-
and increases its affinity
for the signaling receptors (23, 24). By binding to the Mad homology-2
domains of Smad2 and Smad3, SARA facilitates the interaction of these
Smad proteins with TGF-
receptors (25). A second set of factors
participating in the negative regulation of TGF-
signaling includes
the cytosolic growth factor-sequestering protein FKBP-12, the
pseudoreceptor transmembrane protein BAMBI, and a set of inhibitory
Smad proteins that include Smad6 and Smad7. Some of these factors
inhibit the function of TGF-
by interacting with TGF-
receptors
or Smad proteins. By virtue of binding to TGF-
type I
receptor, FKBP-12 prevents its transphosphorylation by the TGF-
type
II receptor (26). BAMBI, on the other hand, blocks TGF-
-mediated
signaling by forming inactive dimers with the type I receptor (27). By binding to receptor-activated Smad1, Smad6 forms a Smad1·Smad6 complex, which appears to be inactive (28), whereas a second inhibitory
Smad protein, Smad7, binds to the activated TGF-
receptor, blocking
the phosphorylation of receptor-regulated Smad (34, 35). Among the
intracellular transcriptional repressors thus far identified that block
TGF-
signaling are two closely related members of the Ski/sno family
of nuclear oncoproteins, SnoN and Ski (29-35). Ski was identified on
the basis of homology to v-Ski, the transforming protein of the
Sloan-Kettering virus. SnoN and c-Ski both inhibit TGF-
signaling by
interfering with Smad function (36). TGF-
-mediated Smad signaling
integrates with and is controlled by other signaling factors such as
those in the Ras/Erk and Jak1/Stat pathways (37, 38). Erk, activated by
growth factors such as epidermal growth factor and hepatocyte
growth factor, induces the phosphorylation of R-Smad proteins in the
linker region and without inhibiting the association of R-Smad proteins
with Smad4 and prevents the nuclear accumulation of Smad proteins (38). Thus, the magnitude of the TGF-
effect is the net outcome of actions
of these positive and negative signals.
, prevent its binding to receptors (22). The
latency-associated peptide noncovalently binds TGF-
, keeping it in a
latent form incapable of binding to betaglycan or its signaling
receptors (23). Some indirect evidence suggests that lefty, a member of
the family of morphogens that is thought to encode a signal for
"leftness," is poised to act as an inhibitor of TGF-
family
members (39-41). Antivin, a lefty-related gene product, acts as a
specific competitive inhibitor for activin during embryo development in
zebrafish (42, 43), and the lefty-related factor Xatv acts as a
feedback inhibitor of nodal signaling in induction of mesoderm and
left/right axis development in Xenopus (44). Overexpression
of Xatv in the marginal zone in Xenopus suppresses mesoderm
formation, causes defects in gastrulation, and inhibits the secondary
axis formation induced by Xnr1 and Xactivin, suggesting that
Xatv acts as a feedback inhibitor of activin signaling (45). Injection
of lefty into mouse blastocysts leads to neurogenesis, a function
attributable to BMP inhibitors such as chordin, noggin, and
follistatin (41, 44-47). These findings suggest (but do not prove)
that lefty is an inhibitory member of the TGF-
superfamily. In this
report, we carried out experiments to determine whether lefty is
capable of blocking TGF-
and BMP signaling and the associated gene
transcriptional activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was
obtained from Sigma, and recombinant BMP-4 was from R&D Systems
(Minneapolis, MN). Recombinant Escherichia coli lefty A,
expressed and refolded from Ser136 to Phe369,
was obtained from Regeneron Pharmaceuticals (Tarrytown, NY). Anti-Smad2/3 (R-Smad proteins) and anti-Smad4-7 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
constitutively active TGF-
type I receptor was a gift from the
laboratory of Dr. J. Massagué (37).
type I receptor and reporter constructs using Superfect transfection reagent (QIAGEN Inc.) or Fugene (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturers' protocol. The amount of protein in the cytosolic, nuclear, and cell
lysates was determined with the Bio-Rad protein assay kit.
and
lefty) were added 24 h after transfection, and luciferase activity
was measured with a luciferase kit (Promega).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
As a first step toward identifying the mode of action of
lefty, we examined its effect on TGF-
-mediated gene transcriptional activity. We chose P19 cells since these cells have an intact TGF-
signaling pathway and have been used in elucidating the role of TGF-
proteins in embryogenesis (50). P19 cells were transfected with the
artificial construct pSBE-Lux. In this construct, the luciferase gene
is under the control of the Smad-binding element, which is in the
promoter of TGF-
-responsive genes and is activated by direct binding
of the TGF-
-induced transcriptional complex (17). The luciferase
activity of cells transfected with the construct was assessed in the
presence of TGF-
and varying amounts of lefty. Lefty inhibited the
activity of the reporter in a dose-dependent fashion (Fig.
1A). The inhibitory activity
of lefty on the reporter could be overridden by increasing the
concentration of TGF-
(Fig. 1B).
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Fig. 1.
Lefty inhibits the transcriptional activity
of TGF- . A-E, P19 cells were
transfected with reporter constructs (pSBE-Lux, p21-Lux, pCdc25-Lux,
and pCTGF-Lux). Twenty-four hours after transfection, cells were
treated with TGF-
and/or lefty for 30 min. Cells were removed
24 h after treatment, and luciferase activity was quantitated and
is expressed in arbitrary units. CTGF, connective tissue
growth factor.
-mediated regulation of
the activity of reporters of the cell cycle factors p21 and Cdc25. The
G1 cell cycle events mediated by TGF-
in untransformed epithelial cells include up-regulation of p21cip1 and reduction
of Cdc25 (51, 52). P19 cells were transfected with p21-Lux and
pCdc25-Lux constructs, and the reporter activity was assessed in the
presence of TGF-
and lefty. TGF-
increased the p21 reporter
activity and decreased the Cdc25 reporter activity (Fig. 1,
C and D). Although lefty did not have any
discernible effect, it opposed reduction of the Cdc25 reporter
activity induced by TGF-
(Fig. 1, C and D).
Whereas the reporter activity was increased by lefty close to 50%, the
activity of the p21 promoter was reduced >4-fold. These findings imply
that lefty inhibits several well known functions of TGF-
that
control cell proliferation in epithelial cells. To determine whether
lefty actions are primarily confined to TGF-
-mediated control of
cell cycle factors or also target other known functions of this
cytokine, we further tested the effect of lefty on TGF-
-mediated
connective tissue growth factor promoter activity. TGF-
leads to
fibrogenesis by activating the transcription of connective tissue
growth factor, a cytokine that induces collagen synthesis by
fibroblasts (53). Lefty significantly reduced the activity of the
reporter induced by 5 (Fig. 1E) and 15 (data not shown)
ng/ml TGF-
. These findings show that lefty is a broad-range
inhibitor of TGF-
actions.
--
In the absence of TGF-
, Smad proteins are
distributed in the nucleus and cytoplasm (2). Upon stimulation by
TGF-
, however, R-Smad proteins (Smad2/3) are phosphorylated by the
activated TGF-
type I receptor and heterodimerize with Smad4 (1-5).
Therefore, we reasoned that the inhibitory effect of lefty may involve
blockage of TGF-
-mediated heterodimerization of Smad proteins and
subsequent nuclear translocation of these heteromeric complexes (1-6).
P19 cells were treated with TGF-
in the presence and absence of
lefty. After 1 h of treatment, the cytosolic and nuclear lysates
of these cells were subjected to Western blotting using antibodies to
Smad2/3, Smad4, and Smad 5. As compared with the control cells, TGF-
led to the accumulation of Smad2 and Smad4 in the nuclei of treated cells (Fig. 2A,
arrows). Endogenous Smad3 was not detected. This could be
due to lower levels of Smad3 in P19 cells as well as our
electrophoretic conditions. Similar differential endogenous expression
of Smad2 and Smad3 proteins has been previously reported (37). TGF-
did not have any effect on nuclear translocation of Smad5, which is an
intracellular mediator of the BMP signaling pathway (54). Although
lefty did not change the amount of Smad proteins in the cytosol or the
nuclei of the treated cells, it inhibited the nuclear translocation of
both Smad2 and Smad4 induced by TGF-
(Fig. 2A).
Immunolocalization of Smad4 in P19 cells treated with TGF-
and lefty
showed nuclear accumulation of Smad4 upon TGF-
treatment. As
assessed by the intensity of staining, lefty did not increase the
amount of nuclear Smad4 on its own, but inhibited the TGF-
-induced
nuclear accumulation of Smad4 (Fig. 2B). These findings show
that lefty prevents the TGF-
-mediated nuclear accumulation of the
Smad2·Smad4 complex required for gene transcriptional activity.
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Fig. 2.
Lefty inhibits
TGF- -mediated nuclear translocation of R-Smad
proteins and Smad4. P19 cells were treated for 30 min with
medium alone (control), TGF-
(5 ng/ml), recombinant lefty (5 ng/ml),
and TGF-
(5 ng/ml) plus lefty (5 ng/ml). A, the cytosolic
and nuclear fractions were prepared from the untreated (
) and treated
(+) cells, and equal amounts of proteins (10 µg/lane) were subjected
to Western blot analysis for Smad2/3, Smad4, Smad5, and histone 3. The
localization of histone 3 in the nuclear fraction and its absence in
the cytosolic preparation showed that these preparations were not
cross-contaminated. Arrows point to Smad2 and Smad4
accumulated in the nuclear lysates. B, Smad4 was localized
by immunoperoxidase staining in the treated cells. Panel a,
control cells treated with medium alone; panel b, cells
treated with TGF-
; panel c, cells treated with lefty;
panel d, cells treated with TGF-
and lefty. Percentages
of cells showing nuclear staining were as follows: control, 2%;
TGF-
, 25%; lefty, 3%; and TGF-
+ lefty, 1%. Arrows
point to nuclear Smad4.
--
Biological signaling of TGF-
involves
heterodimerization of R-Smad proteins with Smad4 (5-7). In light of
the findings, we assessed the extent that lefty can inhibit the
TGF-
-mediated heterodimerization of R-Smad proteins with Smad4.
R-Smad proteins were immunoprecipitated using anti-Smad2/3 antibody
from the cytosol of P19 cells that were treated with TGF-
and/or
lefty. The immunoprecipitates were subjected to Western blotting for
Smad4 (Fig. 3). As expected, in the
TGF-
-treated cells, Smad4 was present in the R-Smad
immunoprecipitates, showing that it had heterodimerized with these Smad
proteins (Fig. 3). Lefty alone had no effect on this event, but
prevented the TGF-
-mediated heterodimerization of Smad4 with R-Smad
proteins (Fig. 3). Treatment of cells with TGF-
or lefty did not
lead to any detectable change in the total amount of R-Smad or Smad4 (Fig. 3). These findings show that lefty inhibits the
heterodimerization of R-Smad with Smad4 induced by TGF-
.
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Fig. 3.
Lefty inhibits
TGF- -mediated heterodimerization of R-Smad
proteins with Smad4. P19 cells were treated for 30 min with medium
alone (control), TGF-
(5 ng/ml), lefty (5 ng/ml), and TGF-
(5 ng/ml) plus lefty (5 ng/ml). The proteins in the nuclear preparations
of untreated (
) and treated (+) cells were immunoprecipitated with an
antibody to R-Smad (Smad2/3), and the immunocomplexes were subjected to
Western blot analysis for Smad4 (upper panel). The
arrow points to Smad4. Other bands are the heavy chain of
the immunoglobulin in the immunocomplexes. The cell lysates were
analyzed by Western blotting for R-Smad and Smad4 to assess the overall
amount of these proteins (middle and lower
panels).
--
The findings suggested that the effect of lefty may be
directly exerted on R-Smad activity. To test this, the phosphorylation of R-Smad was assessed in P19 cells in the presence of both lefty and
TGF-
. P19 cells were treated with TGF-
, lefty, or both in the
presence of [32P]orthophosphate to label phosphorylated
proteins. R-Smad proteins were immunoprecipitated, and the
immunocomplexes were subjected to SDS-PAGE, blotting, and
autoradiography. The analysis showed that although TGF-
led to the
phosphorylation of R-Smad proteins, lefty had no discernible effect.
However, lefty reduced the TGF-
-mediated R-Smad phosphorylation
(Fig. 4A). Densitometric
analysis of the bands showed that lefty inhibited Smad phosphorylation
by 80%.
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Fig. 4.
Lefty inhibits R-Smad phosphorylation.
A, P19 cells were incubated for 30 min in culture medium
alone (control) and in culture media supplemented with TGF- (5 ng/ml), lefty (5 ng/ml), and TGF-
(5 ng/ml) and lefty (5 ng/ml) in
the presence of [32P]orthophosphate (10 µCi/ml). R-Smad
proteins were immunoprecipitated with anti-Smad2/3 antibody, and the
immunoprecipitates were subjected to SDS-PAGE, followed by
autoradiography (upper panel). The overall amount of R-Smad
was assessed by Western blotting of cell lysates (lower
panel). B, P19 cells were transfected without (
) and
with (+) the constitutively active TGF-
type I receptor
(TGF-beta RI), and then transfected cells were treated for
30 min in the presence of [32P]orthophosphate (10 µCi/ml) in cell culture medium alone (control;
) or supplemented
with lefty (5 ng/ml; +). Smad2/3 proteins were immunoprecipitated from
the cell lysates, and the immunoprecipitates were subjected to
SDS-PAGE, followed by blotting and autoradiography (upper
panel). The overall amount of TGF-
type I receptor and R-Smad
was assessed by Western blotting (middle and lower
panels). C, P19 cells were transfected with the
reporter construct pSBE-Lux. Twenty-four hours after
transfection, cells were treated with lefty for 30 min. Cells were
removed 24 h after treatment, and luciferase activity was
quantitated and is expressed in arbitrary units. Values presented are
the means ± S.D. of triplicate determinations.
to its receptors leads to several events, including
oligomerization of the type I and type II receptors and transphosphorylation of the type I receptor by the constitutively active serine/threonine kinase type II receptors (20). To assess whether the effect of lefty is dependent on these initial events during
TGF-
signaling, we transfected P19 cells with a constitutively active TGF-
type I receptor and examined the effect of lefty on
phosphorylation of R-Smad proteins. Lefty inhibited the phosphorylation of Smad2 by this receptor (Fig. 4B). Densitometric analysis
of the bands showed that lefty inhibited Smad2 phosphorylation by 75%.
These findings show that inhibition of R-Smad phosphorylation by lefty
is independent of binding of TGF-
to its receptor, does not appear
to require complex formation between type I and II receptors, and is
not dependent on the phosphorylation of the type I receptor.
type I
receptor. P19 cells were cotransfected with the receptor and the
pSBE-Lux construct, and the activity of the reporter was assayed in the
presence of an increasing amount of lefty. Lefty inhibited the activity
of the reporter induced by the receptor in a dose-dependent
fashion (Fig. 4C). Taken together, these data show that the
effect of lefty is initiated downstream of TGF-
receptor activation.
Is Not Mediated by Inhibitory Smad Proteins and Does Not
Require Protein Synthesis--
One possibility for the inhibitory
effect of lefty on TGF-
-mediated activities is induction of
expression of inhibitory Smad (anti-Smad) proteins. To determine
whether the inhibitory activity exhibited by lefty involves synthesis
of Smad7, P19 cells were treated with lefty for various periods of time
(0-60 min), and the cell lysates were subjected to Western blot
analysis for Smad7 (Fig. 5A).
Lefty did not induce any change in the total amount of Smad7. Likewise,
treatment of cells with lefty failed to induce any change in the amount
of Smad6 (data not shown). These data show that inhibition of the
activity of TGF-
by lefty does not require synthesis of Smad6 or
Smad7.
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Fig. 5.
Lefty does not induce Smad7, and inhibition
of phosphorylation of R-Smad proteins by lefty does not require protein
synthesis. A, P19 cells were treated with 5 ng/ml lefty
for 0, 15, 30, and 60 min. The proteins in the cell lysates were
subjected to Western blot analysis for Smad7. Equal loading was
assessed by Western blotting of the cell lysates for actin.
B, P19 cells were transfected without ( ) and with (+) a
constitutively active form of the TGF-
type I receptor
(TGF-beta RI). Twenty-four hours after transfection, cells
were treated with cycloheximide (20 µg/ml) for 1 h. Cells were
then treated for 30 min in the presence of
[32P]orthophosphate (10 µCi/ml) in culture medium alone
(control;
) or supplemented with recombinant lefty (5 ng/ml; +).
R-Smad proteins (Smad2/3) were immunoprecipitated from the cell
lysates, and the immunocomplexes were subjected to SDS-PAGE and
autoradiography (upper panel). The overall amount of Smad2
was assessed by Western blotting (lower panel).
type I receptor in the presence and
absence of lefty. Lefty inhibited R-Smad phosphorylation by the active
TGF-
type I receptor in the presence of cycloheximide (Fig.
5B). These findings show that inhibition of the activity of
TGF-
by lefty does not require synthesis of a protein that blocks
phosphorylation of R-Smad proteins.
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Fig. 6.
Lefty inhibits BMP signaling.
A, P19 cells were incubated for 30 min in culture medium
alone (negative control; ) or supplemented (+) with BMP-4 (5 ng/ml),
lefty (5 ng/ml), and with BMP-4 (5 ng/ml) and lefty (5 ng/ml) in the
presence of [32P]orthophosphate (10 µCi/ml). Smad5 was
immunoprecipitated with anti-Smad5 antibody, and the immunoprecipitates
were subjected to SDS-PAGE, followed by autoradiography (upper
panel). The overall amount of Smad5 was assessed by Western
blotting of cell lysates (lower panel). B, P19
cells were transfected with the Vent2-luciferase reporter construct.
Twenty-four hours after transfection, cells were treated for 30 min
with BMP-4 (5 ng/ml) and/or various concentrations of lefty as shown.
Cells were removed 24 h after treatment and analyzed for
luciferase activity. The values presented are the means ± S.D. of
triplicate determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a potent cytokine capable of modifying and regulating
many different cell functions. In view of these diverse activities, there is a great interest in understanding how the signaling by TGF-
is regulated. In view of the potent and diverse activities of TGF-
,
the action of this cytokine is tightly controlled by a number of
negative feedback mechanisms (29-33). Here, we show that lefty is an
integral part of these feedback loops. Lefty exerts a significant
inhibition of R-Smad phosphorylation by TGF-
and BMP in a
dose-dependent fashion. This action of lefty appears to be
dependent on events that are downstream from receptor activation since
lefty inhibited R-Smad phosphorylation by a constitutively active
TGF-
receptor. Inhibition of R-Smad phosphorylation by lefty is
associated with inhibition of events that follow R-Smad activation,
including the heterodimerization of R-Smad with Smad4, nuclear
accumulation of the R-Smad·Smad4 complexes, and repression of the
transcriptional activity of genes responsive to TGF-
and BMP. Thus,
the effects of TGF-
, BMP, and lefty are channeled through a common
Smad-mediated pathway, and the magnitude of the response to TGF-
or
BMP depends on the amount of available lefty.
signaling by interaction and association with
receptor-activated Smad (29-31). On the other hand, Smad7 binds the
activated TGF
type I receptor, blocking the association, phosphorylation, and activation of Smad2 (28, 32). For these reasons,
we considered that inhibition of phosphorylation of R-Smad by lefty
might be mediated by induction of Smad6 or Smad7. However, lefty did
not alter the synthesis of these inhibitory proteins. SnoN and Ski are
unlikely candidates for mediating the actions of lefty since they both
are nuclear oncoproteins. SnoN maintains the repressed state of
TGF-
-responsive genes in the absence of ligand and participates in
negative feedback regulation of TGF-
signaling in the presence of
ligand. Under basal conditions, the SnoN oncoprotein interacts with
Smad2 and Smad4 and represses their abilities to activate transcription
through recruitment of the transcriptional corepressor N-CoR. Ski
directly interacts with Smad2, Smad3, and Smad4 on a TGF-
-responsive
promoter element and represses their abilities to activate
transcription through recruitment of the nuclear transcriptional
corepressor N-CoR and possibly its associated histone deacetylase
complex (55). Thus, the actions of lefty complements the actions of
these intracellular inhibitors of TGF-
responsiveness. The
inhibitory effect of lefty is not due to synthesis of a protein,
including induction of Smad6 or Smad7, suggesting that lefty acts
through an as yet unidentified protein that interacts with either the
intracytoplasmic domain of the TGF-
receptor or more likely the
R-Smad proteins.
and/or BMP and the Smad
signaling pathway is likely to play a major role during implantation
and embryogenesis as well as oncogenesis. Lefty is a novel subfamily of
the TGF-
protein superfamily and is comprised of lefty-1
and lefty-2 in mouse (39, 41) and their homologs, lefty A (ebaf) and lefty B, in human
(48, 56). Both lefty-1 and lefty-2 are key
embryonic signals that drive development of an asymmetric body plan.
Both of these genes are asymmetrically expressed on the left side of
gastrulating mouse embryos. However, the major expression domains of
the two genes are different. lefty-1 expression is
predominantly confined to the left side of the ventral neural tube,
whereas lefty-2 is strongly expressed in the left lateral
mesodermal plate (39, 41). In zebrafish embryos, lefty-1 is
expressed in the central nervous system in the left diencephalon, whereas lefty-2 is expressed in the left heart field (57).
Antivin/lefty-1 is also expressed asymmetrically on the left
side of the prospective floor plate, notochord, and lateral plate
mesoderm of the chick embryo (58). Asymmetric expression of
lefty and nodal is perturbed in mouse
mutants with laterality defects, and mutation of lefty A is seen in
humans who exhibit left/right axis malformations (58-60). Furthermore,
the knockout mutation of lefty-1 induces a variety of
L/R positional defects in visceral organs. The most notable feature of
lefty-1-deficient mice is bilateral expression of
nodal and lefty-2, which leads to a left lung
isomerism (40). These and other observations support the concept that
during embryogenesis, lefty proteins encode a signal for leftness and
appear to be involved in neurogenesis. Like lefty, the signaling
molecules of TGF-
, including its receptors and Smad proteins, have
been implicated in embryo development. TGF-
is intrinsically
involved in patterning during developmental processes and neurogenesis.
In the chick embryo, TGF-
is involved in the death of ciliary,
dorsal root, and spinal motor neurons as well as in neuronal losses
that follow limb bud ablation (62). Both overexpression and deletion of TGF-
genes cause significant developmental abnormalities and death
in embryos (63-68). Mice genetically deficient in the TGF-beta receptor type II gene die around embryonic day 10.5 from
abnormalities that are reminiscent of those described in
TGF-
1
/
embryos, including defects in
hematopoiesis and visceral yolk sac vasculogenesis (69). In mouse
embryos, Smad2 has been found as a key mediator that directs epiblast
derivatives toward an endodermal as opposed to a mesodermal fate (70).
Recently, some evidence has emerged that directly ties the function of
lefty to the signaling molecules of the TGF-
and BMP proteins. Some, if not all, of the developmental processes driven by TGF-
family members may be subject to modulation by lefty. We showed here that
lefty inhibits BMP signaling, suggesting that the tissue patterning
during embryogenesis might be regulated by the joint effect of these
cytokines. Consistent with this thesis, it has recently been shown that
regulation of left/right asymmetry in the gut and heart is not solely
induced by lefty; rather, it is driven by context-dependent
interactions between lefty and BMP-4 (61). Injection of lefty into
two-cell stage Xenopus blastocysts promotes neuralization,
an effect that is also inducible by factors such as noggin,
follistatin, and chordin that inhibit BMP signaling (41, 44-47). These
findings show that the development of asymmetry requires the joint
cooperation of various members of the TGF-
superfamily. Similar to
lefty, levels of phosphorylated Smad2 have been found to be
asymmetrically distributed across both the animal-vegetal and
dorsoventral axes in Xenopus embryos, suggesting that Smad2
is a candidate morphogen that regulates primary germ layer formation
and dorsoventral patterning (7). Mice
trans-heterozygous for both Smad2 and nodal mutations have
defects in left/right patterning, indicating that Smad2 is involved in
the development of left/right asymmetry and that Smad2 may mediate
nodal signaling in these developmental processes (72). Moreover, the
gene dosage of Smad2 has been found to be important in tissue
patterning, suggesting that lefty might act by regulating the function
of Smad2. The left/right asymmetric expression of
lefty-2 is controlled by a left side-specific
enhancer. The transcription factor FAST2, which mediates
signaling by TGF-
and activin, binds to a conserved sequence in this
asymmetric enhancer, which is both essential and sufficient for
left/right asymmetric gene expression (73, 74). The forkhead
transcription factor FoxH1, also known as FAST1, which associates with
Smad proteins in response to an activin/TGF-
signal, plays a key
role in the development of dorsal axial structures in zebrafish and in
specification of mesoderm by TGF-
superfamily signals during early
Xenopus embryogenesis (75-77). These findings directly tie
the lefty functions to the action of TGF-
and its signaling
molecules during embryogenesis.
signaling, and for this reason, it
is thought that TGF-
and Smad signaling molecules act as potent
tumor suppressors (78). Since the normal function of the TGF-
signaling pathway is suppression of cellular proliferation and
transformation, our results provide a model for the action of
lefty as a potential oncoprotein by showing that it
counteracts the TGF-
-mediated signaling. According to this model,
dominant repression of TGF-
-inducible genes that are negative
regulators of cell cycle progression by lefty might confer upon lefty a
transforming activity. Lefty is overexpressed in certain forms of human
cancer, including those derived from colon, pancreas, ovary, and testis (79). High levels of expression of lefty in human cancers may produce a
disruption of TGF-
-mediated signaling in a manner similar to that
induced by overexpression of inhibitory Smad proteins and SnoN proteins
or mutation of genes that encode TGF-
receptors or Smad proteins
(57, 80, 81). This could be relevant to certain human cancers that,
despite the presence of active TGF-
receptors and Smad proteins, are
unresponsive to TGF-
(82, 83). Dominant disruption of lefty function
in tumors that overexpress lefty might restore gene activation and
growth inhibition by TGF-
(79). However, a separate model for the
action of lefty in human cancers can be envisaged. According to this
second model, lefty acts as a tumor suppressor in a fashion similar to
the action of inhibitors of TGF-
signaling such as Sno. Loss of Sno
has been shown to increase susceptibility to tumorigenesis in mice (87). This model is quite conceivable since overexpression of TGF-
,
despite the loss of TGF-
sensitivity in cancer cells, is
paradoxically associated with an aggressive and invasive tumor growth
(85-87). In this scenario, TGF-
promotes tumor growth, and factors
that inhibit TGF-
signaling abate the TGF-
-mediated responses and
suppress tumor growth. TGF-
promotes late-stage tumor progression,
tumor invasion, and metastasis in a number of model systems, including
human colon (88) and breast (89) carcinomas. These actions of TGF-
in TGF-
-resistant tumors are presumably mediated by autocrine and/or
paracrine mechanisms (90, 91). Blocking these actions of TGF-
by
lefty overexpression in tumors might slow down the aggressive tumor
growth or may lead to tumor regression. Consistent with this line of
reasoning, introduction of lefty to tumor cells reduces tumor growth in
nude mice.2
-induced nuclear
accumulation of Smad2 and Smad3 and Smad-dependent
transcription (38). These findings imply that induction of MAPK
activation by lefty might modulate gene transcription by TGF-
.
Treatment of P19 cells with both BMP-4 and lefty induces a more
vigorous MAPK activity in 5 min, but this activity is much less than
that induced by lefty alone at later time points (71). Therefore,
although lefty inhibits BMP signaling along the Smad pathway, its
effect on the MAPK activity of BMP appears to be complex, being both
additive as well as antagonistic (71).
family that
does not have any discernible effect on its own on Smad signaling, but
blocks the TGF-
or BMP actions by preventing the phosphorylation of
R-Smad proteins. This inhibition is not due to synthesis of any
protein, including Smad6 and Smad7. Lefty inhibits the phosphorylation
of R-Smad by a constitutively active TGF-
type I receptor, which
suggests that its action is directed at an event downstream from
receptor activation. This action is sufficient to inhibit TGF-
- or
BMP-mediated R-Smad phosphorylation and subsequent heterodimerization
of R-Smad with Smad4, nuclear translocation of R-Smad·Smad4
heterocomplexes, and TGF-
- or BMP-mediated gene transcription. These
findings show that growth-promoting and transcriptional responses to
TGF-
or BMP actions are abated by lefty by inhibition of
phosphorylation of R-Smad proteins. The data suggest that lefty is an
integral part of the mechanisms that provide negative feedback during
TGF-
and BMP signaling.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant CA46866 from the National Institutes of Health and a grant from Lexon Inc. (to S. T.).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.
To whom correspondence and reprint requests should be addressed:
Dept. of Pathology, Biomedical Research Center, 350 Community Dr.,
Manhasset, NY 11030. Tel.: 516-484-0813; Fax: 516-484-2831; E-mail:
tabibzadeh@bioscience.org.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M010783200
2 J. Mason and S. Tabibzadeh, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
R-Smad, receptor-regulated Smad;
Erk, extracellular signal-regulated kinase;
Jak, Janus kinase;
Stat, signal
transducers and activators of transcription;
PAGE, polyacrylamide gel
electrophoresis;
MAPK, mitogen-activated protein kinase;
BMP, bone
morphogenetic protein.
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