From the Cell & Molecular Biology Graduate Group, the
§ Department of Medicine, and the ¶ Howard Hughes
Medical Institute, the University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania 19104
Received for publication, October 14, 2002
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ABSTRACT |
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Bone morphogenetic proteins (BMPs) are members of
the transforming growth factor- Bone morphogenetic proteins
(BMPs)1 are members of the
transforming growth factor- The importance of BMP-related factors in embryonic development has been
elucidated by extensive analysis in model organisms. For example, in
Drosophila, the BMP orthologue decapentaplegic (dpp) plays a critical role in dorsal-ventral patterning of
the embryo as well as patterning of the imaginal discs (8). In C. elegans, BMPs regulate the size of the organism as well as patterning of the male tail (9). A role for BMPs in vertebrate development was first suggested by observations that ectopic expression of BMPs causes expansion of ventral tissues and reduction of dorsal and
anterior structures in Xenopus embryos (10, 11) and
ectodermal explants (12). In support of this, BMP-4 is
expressed in a ventral-lateral crescent in the marginal zone of
gastrula stage embryos (12). BMPs have also been implicated in neural
cell fate determination in Xenopus (13, 14).
The importance of BMP signaling in bone formation and in vertebrate
development has been confirmed by genetic analyses in mouse and
zebrafish (2, 3, 5, 15). Thus, mutation of the BMP-5 (short
ear mutant) and BMP-7 genes in mice disrupt multiple aspects
of skeletal development (16), whereas mice lacking BMP-2, BMP-4, or the BMP type I receptor, ALK-3, die
in utero prior to the onset of bone formation, supporting an
additional role for BMPs in earlier developmental processes, including
mesoderm induction and neural-epidermal cell fate decisions (2). In
zebrafish, a forward genetic screen for defects in early embryonic
development yielded a series of dorsalized mutants, including mutations
in genes encoding BMP 2b and 7 (swirl and
snailhouse), the BMP receptor ALK8 (lost-a-fin),
and other extracellular and intracellular components of this tightly
regulated signaling pathway (e.g. Smad5/somitabun and tolloid/mini-fin) (15, 17).
Similar to other TGF- Smad proteins can be divided into three broad groups, the
pathway-restricted Smads, the common-mediator Smads, and the
antagonistic Smads (4, 18). The pathway restricted Smads are regulated by specific TGF- BMP signaling is highly regulated in Drosophila and in
vertebrates (20), as evidenced by the marked defects in embryonic development observed when these regulatory components are mutated in
flies, zebrafish, or mice (2-5,15). Thus extracellular signaling by
BMPs is regulated by a number of secreted proteins, including BMP
binding factors such as chordin, noggin, follistatin, gremlin, and
cerberus (4), as well as upstream molecules that regulate chordin
function, such as twisted gastrulation and tolloid (15, 27, 28). In
addition, intracellular regulators of BMP signaling have been
identified, including inhibitory Smads, BAMBI, Smurf1, and a number of
nuclear proteins, including Tob and OAZ (4, 29, 30).
To identify novel molecules that regulate the intracellular BMP/Smad1
signaling pathway, we have undertaken a yeast two-hybrid protein
interaction screen using Smad1 as bait. In this work, we describe the
Smad1 interacting protein SANE (Smad1
Antagonistic Effector), which is expressed in
early vertebrate embryos in a pattern that overlaps Smad1, binds to
type I BMP receptors as well as Smad1, and specifically antagonizes
BMP/Smad1 signaling in embryos as well as in a mammalian model of bone
formation. Our data show that SANE functions by inhibiting
BMP-dependent Smad1 phosphorylation and thus blocking the
nuclear translocation of Smad1.
Yeast Two-hybrid Screen and Cloning of
SANE--
Xenopus Smad1 was used as bait to screen a
Xenopus oocyte cDNA library in the Saccharomyces
cerevisiae strain Y190 as previously described
(Clontech). Approximately 3 × 106
clones were screened, and of twelve positive clones, three independent, but overlapping clones coded for the carboxyl-terminal portion of SANE.
A SANE partial cDNA clone was used to screen a UniZAP Xenopus oocyte cDNA library. The SANE coding
region was cloned into the expression vector, pCS2MT. The SANE sequence
has been submitted to the GenBankTM databases under
accession number AF115498. SANE- Co-immunoprecipitation Analysis--
Synthetic mRNA was
generated by in vitro transcription (Ambion) and injected
into Xenopus oocytes (5 ng) or embryos (1 ng). Xenopus oocyte or embryo (blastula stage) extracts were
prepared as previously described (31) and immunoprecipitated with
either anti-myc 9E10 monoclonal antibody or anti-Smad1 polyclonal
antibody (T-20 or H-465) (Santa Cruz Biotechnology). Immunoprecipitates were analyzed by immunoblotting using antibodies to the myc epitope (9E10), Smad1 (H-465 or T-20), Smad2 (N-19), Smad5 (D-20)
(Santa Cruz Biotechnology), and Smad4 and pSmad1 (Upstate
Biotechnology). The SANE monoclonal antibody was generated by
immunizing mice with the SANE-C fragment fused in-frame to the carboxyl
terminus of glutathione S-transferase (GST) according to
standard procedures. Four hybridoma clones were selected from initial
screening and tested for specificity to SANE. The monoclonal antibodies
react with the endogenous SANE protein of 98.5 kDa (as a single band) in Xenopus in Western blots, with myc-tagged full-length
SANE (lower electrophoretic mobility), and with the SANE-C fragment but
not with SANE- Xenopus Embryo Manipulations, RT-PCR Analysis, and in Situ
Hybridization--
mRNA for SANE constructs (1 ng) was
injected into a single ventral blastomere of Xenopus
four-cell embryos. Embryos were allowed to develop until tadpole stage
for analysis of dorsal-ventral patterning phenotypes. For ectodermal
explant assays, mRNA for SANE constructs (0.5 and 1 ng)
was injected into the animal pole of Xenopus one-cell
embryos, and ectoderm was explanted at stage 8. Explants were harvested
at stage 23 for analysis of neural markers (NCAM,
otx2, and XAG) or for the dorsal mesodermal
marker (muscle actin) as previously described (14). For rescue of SANE, Smad1 mRNA (4 ng) was co-expressed with SANE
mRNA (1 ng) in explants, and RT-PCR was performed as described above.
For mesoderm induction assays, BMP-4 (0.5 ng) or
activin Stable C2C12 Cell Lines--
C2C12 myoblasts were transfected
with pCS2MT-SANE constructs or pCS2LacZ as control with pCR3.1
(Invitrogen), which contains the neomycin-selectable marker, in a ratio
of 10:1 µg of DNA using LipofectAMINE (Invitrogen). Myoblasts were
mock treated or treated with BMP-4 protein (50 µg/ml) or TGF- Luciferase Reporter Assays--
XVent.2 luciferase
reporter or XMix.2 luciferase reporter plasmids (50 pg) were
co-injected with either BMP-4 or activin
Smad1 Nuclear Translocation Assay--
NIH 3T3 cells were
transiently transfected with expression vectors containing myc-tagged
SANE. After 24 h to allow for gene expression, cells were starved
and treated with BMP-4 protein (20 ng/ml, R&D Systems). Cells were
fixed and stained by immunofluorescent methods as described previously
(25) using an anti-Smad1-specific antibody (Upstate Biotechnology).
Nuclear versus cytoplasmic localization of endogenous Smad1
was assessed by counting 200-400 cells per group. This experiment was
repeated three times with similar results.
Cloning of Xenopus SANE--
Full-length Xenopus Smad1
was used as bait in a two-hybrid screen of a Xenopus oocyte
cDNA library (36). Partial cDNAs from twelve positive clones
were isolated and sequenced. One of the partial clones, which was
isolated three times, was sufficient to interact with Smad1 by
co-immunoprecipitation analysis in Xenopus embryos. This
partial clone was used to isolate a cDNA that includes the complete
SANE open reading frame (Fig. 1), which
encodes an 870-amino acid protein with a predicted molecular
mass of 98.5 kDa. SANE shows 55% overall identity to a human
protein named MAN1, a predicted nuclear envelope protein, with greater
similarity (up to 85%) in the carboxyl-terminal half of the sequence
(37). SANE also contains an LEM motif in the N-terminal region, similar to lamina associated protein 2 (LAP2), emerin,
and MAN1 (37), but there is significant divergence from
MAN1 in the N-terminal sequence outside of the LEM domain. SANE shows
up to 85% sequence similarity with several expressed sequence tags
(EST) from mouse and other vertebrates, including Xenopus
laevis and Xenopus tropicalis. SANE is also similar to
a MAN1 homologue from C. elegans (46%) (38) and an EST from
Drosophila melanogaster (38%), suggesting that SANE may be
conserved evolutionarily. Hydropathy analysis reveals that SANE
contains a strongly hydrophobic region between amino acids 438 and 458, suggesting that SANE is a membrane protein; a second less hydrophobic
region is found between residues 596 and 608. Although this second
hydrophobic domain is also similar to MAN1, it appears to be shorter
than a typical membrane-spanning SANE Interacts with Smad1--
Endogenous SANE protein, which is
present throughout early embryogenesis, associates with Smad1 in
Xenopus embryos (Fig.
2A), as determined by
immunoprecipitation of Smad1 and immunoblotting for SANE; thus embryo
lysates were immunoprecipitated with a Smad1-specific antibody, with an
antibody that recognizes multiple Smad family members (including Smad1,
2, 3, 5, and 8) or with a control antibody. Western blotting with
monoclonal antibodies to SANE (Fig. 2A, upper
panel, lanes 2 and 3) shows that SANE
co-immunoprecipitates with Smad1, confirming that endogenous SANE and
Smad1 interact in vivo. (Note that Smad1 protein migrates
close to and is partially obscured by the mouse IgG heavy chain; Fig.
2A, lower panel, lane 1.) Furthermore,
the region of SANE that interacts with Smad1 lies within the
carboxyl-terminal 130 amino acids of SANE. This was shown by
co-expressing a myc-tagged SANE carboxyl-terminal fragment (SANE-C) or
SANE lacking the carboxyl-terminal region (SANE-
Smad1, like Smads 2, 3, and 5, is regulated by receptor-mediated
phosphorylation of carboxyl-terminal serine residues (4, 18, 19). To
examine whether this region of Smad1 is involved in SANE binding, Smad1
deletion constructs (Fig. 3A)
were expressed with SANE in Xenopus oocytes and Smad1·SANE
complexes were immunoprecipitated with anti-Smad1 antibodies
followed by immunoblotting for SANE. This analysis demonstrates that
SANE interacts with the carboxyl-terminal third of Smad1, which
includes the MH2 domain and the conserved serines that are
phosphorylated by the BMP type I receptor (Fig. 3B).
Furthermore, SANE fails to interact with a Smad mutant in which the
carboxyl-terminal serines were mutated to alanines
(Smad1-ala) but does interact if these serines are changed
to aspartic acid (Smad1-asp) (Fig. 3C), suggesting that the
carboxyl-terminal sequence of Smad1 is important for interaction with
SANE. However, neither BMP-4 nor chordin overexpression affected the
level of SANE·Smad1 interaction (data not shown).
To address the specificity of SANE interaction with Smad1, we also
performed co-immunoprecipitation analysis with the activin/Vg1 and
TGF- SANE Specifically Inhibits BMP Signaling--
To test the effect
of SANE on BMP- and Smad1-regulated processes, we used several well
established assays of BMP/Smad1 signaling in Xenopus.
Activators of the BMP pathway such as BMPs and Smad1 cause expansion of
ventral mesoderm, whereas inhibitors, such as chordin, noggin,
follistatin, gremlin, dominant-negative BMP receptor, and Smad6, cause
expansion of dorsal mesoderm (4, 5, 39). Expression of SANE
mRNA in ventral blastomeres (Fig. 4A), over a narrow dose range,
results in partial duplication of the dorsal axis, like other BMP
pathway inhibitors. Similarly, ventral expression of SANE-C (Fig.
4C), which alone is sufficient to bind Smad1, results in
partial axis duplication. Injection of SANE-
To test whether SANE can block ectopic BMP signaling and to assess
further the functional specificity of inhibition by SANE, we examined
whether SANE could inhibit mesoderm induction by either BMP-4 or
activin in ectodermal explants. Fertilized eggs were injected with
mRNA encoding BMP or activin with or without
SANE mRNA. Animal pole explants were dissected at the
blastula stage, cultured until stage 10, and expression of mesodermal
markers was measured by RT-PCR. SANE blocks BMP-dependent
induction of ventral mesoderm (Fig. 4E), as assessed by
expression of the ventral mesodermal marker Xwnt8 and the
pan-mesodermal marker brachyury (40, 41) but has no effect
on mesoderm induction by activin (Fig. 4F), assessed by expression of
brachyury and the dorsal mesodermal marker
goosecoid (42). Thus SANE specifically interferes with the
BMP/Smad1 pathway and does not disrupt activin signaling.
To test whether SANE inhibits activation of direct targets of BMP, we
utilized luciferase reporters containing response elements from the
Xenopus Vent.2 gene, a direct transcriptional
target of BMP signaling (43), and the Mix.2 gene, a direct
target of activin signaling (44, 45). SANE inhibits
BMP-4-dependent activation of the
XVent.2-luciferase reporter in a dose-dependent manner (Fig. 4G) but does not inhibit
activin-dependent activation of Mix.2 (Fig. 4H).
SANE also does not inhibit TGF-
Additionally, BMP signaling has been shown to induce epidermal cell
fate at the expense of neural cell fate in Xenopus
ectodermal explants (13, 14). Thus, expression of inhibitors of the BMP signaling pathway causes neural induction in ectodermal explants, and
this has been widely used to characterize putative BMP inhibitors. Expression of SANE in ectodermal explants mimics other BMP antagonists, with formation of a cement gland (Fig.
5A) and induction of multiple neural markers (Fig. 5B), including NCAM,
a pan-neural marker, otx2, an anterior neural marker, and
XAG, a marker of anterior neural and cement gland cell types.
Expression of SANE-C also causes neural induction, whereas the
SANE- SANE Inhibits BMP Signaling in Mammalian Cells--
SANE also
inhibits BMP signaling in a mammalian cell culture model of osteoblast
differentiation. The mouse C2C12 myoblast cell line responds to BMP by
differentiating into an osteoblast lineage (35). Thus, stable C2C12
cell lines expressing either full-length myc-tagged SANE, SANE-C, or
SANE Inhibits Phosphorylation and Nuclear Translocation of
Smad1--
To begin to understand the mechanism by which SANE inhibits
BMP signaling, we examined the effect of SANE on Smad1 phosphorylation. BMP-4 mRNA was injected alone or with SANE
mRNA into fertilized eggs, embryos were harvested at stage 10.5, and Smad1 phosphorylation was assessed by Western blotting using
antibodies specific to Smad1 phosphorylated at the carboxyl terminus
(46). BMP-4 increased phosphorylation of endogenous Smad1 as described
previously (Fig. 7, lane 2),
and SANE reduced BMP-dependent Smad1 phosphorylation in a
dose-dependent manner (Fig. 7, lanes 3 and
4, upper panel) without changing the level of
Smad1 protein (Fig. 7, middle panel).
Phosphorylation of Smad1 leads to its nuclear translocation and
activation of BMP/Smad1-dependent gene expression; thus, to address the effect of SANE on Smad1 nuclear translocation, we utilized
the Smad nuclear translocation assay that has been established in COS-1
and NIH3T3 cells (25, 47). NIH3T3 cells were transiently transfected
with the full-length SANE expression plasmid. After allowing time for
expression, cells were starved and then treated with BMP-4 protein to
promote Smad1 nuclear translocation. Endogenous Smad1 is readily
detected in the cytoplasm of ~95% of untreated cells by
immunofluorescence (Fig. 8A,
upper left, Fig. 8B) and over 95% of Smad1
translocates to the nucleus in control cells after treatment with BMP
(Fig. 8, A and B). SANE inhibits
BMP-dependent nuclear translocation of endogenous Smad1 in
80% of the SANE-transfected cells. SANE also blocks nuclear
translocation of overexpressed Smad1 in COS-1 cells but does not block
TGF- SANE Interacts with BMP Receptors--
The predicted amino acid
sequence of SANE reveals a hydrophobic region between residues 438 and
458, indicating a potential transmembrane domain, and biochemical
fractionation in Xenopus embryos (Fig.
9A) and COS-1 cells (not
shown) shows that SANE protein localizes primarily to a
detergent-soluble membrane fraction and not cytosolic or nuclear
fractions. We therefore tested whether SANE can also interact with BMP
receptors. SANE was expressed in Xenopus embryos with
HA-epitope-tagged type I receptors specific for BMPs (ALK-3 and ALK-6),
an activin/BMP type I receptor (ALK-2), or with an
activin/TGF- Our data show that SANE specifically interacts with and regulates
the BMP pathway. In support of this, SANE binds directly to BMP
pathway-specific Smads1 and 5, but weakly, if at all, with activin/TGF- BMP signaling is highly regulated by a number of extracellular
inhibitors, including chordin, noggin, follistatin, and gremlin, which
were first identified in Xenopus as dorsalizing factors that
specifically inhibit BMP function (2-5). Similar to these proteins,
SANE blocks BMP-mediated ventral mesoderm induction and leads to
formation of a partial secondary dorsal axis when expressed in ventral
cells. In addition, SANE, like these other inhibitors (51-53), induces
neural tissue in ectodermal explants and blocks BMP function in
mammalian cells as well.
SANE bears superficial similarity to other proteins that interact with
the TGF- Intracellular Smad antagonists, such as Smads 6 and 7, are induced in
response to BMP or TGF- SANE shows no apparent sequence similarity to other proteins that
interact with BMP-pathway specific Smads or BMP receptors, including
BAMBI, Tob, Smurf1, OAZ, and BRAM1 (29, 54-56, 58, 60, 64).
However, SANE does show sequence similarity with the nuclear envelope
protein MAN1 (37). Like MAN1, SANE has an LEM domain near the amino
terminus. In other LEM domain proteins, this region has been shown to
interact with the DNA binding protein BAF (barrier to
autointegration factor) and with DNA (65).
Except for the LEM domain, however, much of the N-terminal domain of MAN1, which is required for localization of the protein to the inner
nuclear membrane, diverges significantly from the corresponding region
of SANE (66). In addition, unlike MAN1, the hydropathy analysis of SANE
is more consistent with a single transmembrane domain, placing the SANE
amino terminus topologically outside the cell. The greatest sequence
similarity between MAN1 and SANE is found in the carboxyl-terminal
region, which includes the Smad1 binding domain of SANE, suggesting
that this may represent a functionally conserved domain, and it is
interesting to consider that the MAN1 carboxyl-terminal region could
also interact with Smad1 to regulate BMP signaling, perhaps in the
nucleus. Indeed, regulation of transcription has been suggested as a
possible function for other LEM domain proteins (67). The presence in
mouse and Xenopus of several expressed sequence tags
encoding related, but distinct genes raises the intriguing
possibilities that SANE is one of a family of TGF- SANE is expressed in cells that are likely to respond to BMP signaling,
because the expression of SANE parallels Smad1 expression in most
tissues. However, at the gastrula stage SANE is enriched in
the dorsal marginal zone, similar to other BMP antagonists, such
as chordin, noggin, and
follistatin, whereas Smad1 mRNA is ubiquitous
at this stage, consistent with a role for SANE as an inhibitor of BMP
signaling in the dorsal marginal zone.
Our model for the mechanism of SANE action suggests that SANE interacts
with the BMP type I receptor through its amino-terminal domain and with
Smad1 through its carboxyl-terminal domain to inhibit BMP signaling.
Inhibition of BMP signaling by SANE is supported by biochemical data,
analysis in cultured cells and embryonic explants, and phenotypic
analysis in intact embryos. Thus, biochemically, we have shown that
SANE blocks phosphorylation of endogenous Smad1 by the BMP type I
receptor. As a consequence, SANE prevents translocation of Smad1 to the
nucleus and blocks BMP-dependent gene expression. This
inhibition is clearly specific to the BMP/Smad1 pathway, because Smad2
translocation and Smad2-dependent transcriptional
activation are not affected by SANE. We have also demonstrated that
SANE antagonizes BMP-induced bone formation in C2C12 cells, BMP-induced
ventral mesodermal gene expression and neural to epidermal cell fate
specification in embryonic explants, and endogenous ventral mesoderm
formation in intact embryos, but that it does not inhibit biological
responses to activin or TGF- In summary, SANE interacts with two components of the BMP pathway, is
expressed in the embryo in a pattern that closely parallels Smad1
expression, and specifically inhibits Smad1-dependent
signaling in Xenopus embryos and mammalian cells. Inhibition
by SANE in Xenopus embryos is functionally similar to the
effects of other BMP inhibitors, such as chordin, noggin, follistatin,
gremlin, and tob, yet SANE defines a new class of genes to regulate
this pathway that uniquely interacts with both BMP receptors and BMP pathway specific Smads.
(TGF-
) superfamily that play
important roles in bone formation, embryonic patterning, and
epidermal-neural cell fate decisions. BMPs signal through pathway
specific mediators such as Smads1 and 5, but the upstream regulation of
BMP-specific Smads has not been fully characterized. Here we report the
identification of SANE (Smad1 Antagonistic
Effector), a novel protein with significant sequence
similarity to nuclear envelop proteins such as MAN1. SANE binds to
Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE
specifically blocks BMP-dependent signaling in
Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-
/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because
a SANE mutant that does not bind Smad1 does not inhibit BMP signaling.
Furthermore, inhibition appears to be mediated by inhibition of
BMP-induced Smad1 phosphorylation, blocking
ligand-dependent nuclear translocation of Smad1. These
studies define a new mode of regulation for intracellular BMP/Smad1 signaling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) superfamily that were first
identified by their ability to induce ectopic bone formation in the
soft tissue of rats (1). Because then BMP family members have been identified in diverse organisms from Drosophila and
Caenorhabditis elegans to mammals and, in addition to their
roles in bone formation, have been shown to play critical roles in
embryonic patterning and neural induction (2-7).
superfamily members (4, 5), BMPs
bind to a heteromeric receptor complex composed of type II and type I
receptors; the type II receptor phosphorylates the type I receptor,
thus activating the type I receptor kinase, which in turn
phosphorylates cytoplasmic Smad proteins (4, 18, 19). Phosphorylated
Smad proteins form a multimeric complex that translocates to the
nucleus and activates transcription through interaction with DNA
binding proteins or through direct DNA binding.
superfamily members (20). For example, Smad1 and
Smad5 are specifically activated by BMPs, which lead to phosphorylation of these Smads on carboxyl-terminal serines (21). Similarly, activin/nodal/TGF-
ligands cause phosphorylation of analogous serines in Smad2 and Smad3 (22, 23). Overexpression of Smad1 in
Xenopus mimics ectopic BMP signaling, inducing ventral
mesoderm from ectodermal explants (24-26), whereas Smad2 can induce
dorsal mesoderm, similar to the effect of activin- or nodal-related
proteins (reviewed in Ref. 5).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C was generated by digesting
pCS2MT-SANE with SnaB1 (position 2242) and religating the vector
resulting in a truncation at amino acid 747. SANE-C was generated by
subcloning a SANE EcoRI insert from the pGAD10 yeast clone
into pCS2MT.
C, indicating that the antibodies specifically react
with an epitope in the SANE carboxyl terminus. Antibody binding to
endogenous SANE in Western analysis is blocked by co-incubation with
GST-SANE-C protein (data not shown).
B (10 pg) mRNA was co-injected
with SANE mRNA (1 ng) into Xenopus one-cell
embryos, ectoderm was explanted at stage 8, and explants were harvested
at stage 10.5 explants for analysis of mesodermal markers
(brachyury, wnt8, and goosecoid) as
previously described (32). Gene expression was assessed by RT-PCR, as
described, using primers for neural markers, NCAM,
otx2, and XAG, or for the mesodermal markers,
muscle actin, goosecoid (gsc),
brachyury (Xbra), or Xwnt8. In
situ hybridization was performed as described previously (33,
34).
1 (1 ng/ml) and stained for alkaline phosphatase as previously described
(35). Alkaline phosphatase-positive cells were scored as a percentage
of total cell number as determined by counting cell nuclei. At least
300 cells in multiple fields were counted.
B mRNA alone or with SANE or
SANE-
C mRNA (500-1500 pg) in the animal pole of one-cell Xenopus embryos. Extracts were made at late
blastula stage (stage 9), and luciferase activity was measured
according to standard protocols using the Monolight 3010 luminometer
(BD Pharmingen).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix. Xenopus SANE
mRNA is expressed in oocytes, fertilized eggs, and throughout early
development. SANE is enriched in a dorsal-lateral crescent in
presumptive dorsal mesodermal cells at the gastrula stage and is
restricted primarily to the anterior central nervous system in the
tadpole in a pattern that is similar to the distribution of
Xsmad1 mRNA at this stage (data not shown) (12).
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Fig. 1.
SANE sequence. The predicted amino acid
sequence of Xenopus SANE is shown and compared with human
MAN1, which is 55% identical overall to SANE. The predicted
transmembrane domain is underlined, and the LEM domain is
indicated by a cross-hatched box.
C) with Smad1
(Fig. 2, B and C). While all three constructs are
expressed, only full-length SANE and the carboxyl-terminal region
(SANE-C) co-immunoprecipitate with Smad1 (Fig. 2C). This region of SANE is necessary and sufficient for Smad1 interaction. This
interaction is likely direct, because GST-SANE associates with Smad1
protein in vitro (data not shown).
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Fig. 2.
Interaction of SANE with Smad1.
A, endogenous SANE and Smad1 interact in vivo in
Xenopus embryos. Lysates from stage 9 embryos were
immunoprecipitated with antibodies to SANE (lane 1), a
Smad1-specific antibody (lane 2), an antibody that
recognizes Smads1, 2, 3, 5, and 8 (lane 3), or a control
antibody (anti-myc, lane 4); immunoprecipitates were Western
blotted with the SANE (upper panel) or Smad1 (lower
panel) antibodies. (The presence of mouse IgG heavy chain in SANE
immunoprecipitates, which runs in a position close to Smad1, partially
obscured the Smad1 band in lane 1, lower panel).
B, SANE constructs used in co-immunoprecipitation analysis.
All SANE constructs are tagged at the amino terminus with the myc
epitope. The carboxyl-terminal 123 amino acids are deleted in
SANE- C. SANE-C encode the carboxyl-terminal 130 amino acids.
C, Smad1 interacts with the carboxyl-terminal region of
SANE. myc-tagged SANE and Smad1 were co-expressed in Xenopus
oocytes. Extracts were immunoprecipitated with an anti-Smad1 antibody
and Western blotted with anti-myc antibody (upper panel).
Extracts were blotted with anti-myc antibody to confirm expression of
all three SANE constructs (lower panel).
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Fig. 3.
a, Smad1 constructs used in
co-immunoprecipitation analysis. Smad1 N contains only the
MH1 domain (amino acids 1-200), Smad1 C encodes the MH2
domain (amino acids 310-464), and Smad1 LC
encodes the linker and the MH2 domain of Smad1 (amino acids 201-464).
In Smad1-asp and Smad1-ala, the three
carboxyl-terminal serines (amino acids 461, 462, and 464) are changed
to aspartic acid or alanine residues, respectively. b, SANE
interacts with the carboxyl-terminal domain of Smad1.
Smad1-N (lane 1), Smad1-C (lane
2), or Smad1-LC (lane 3) was expressed with
SANE in Xenopus oocytes. Extracts were Western blotted with
an anti-Smad1 antibody (middle panel) or with anti-myc
antibody for SANE expression (lower panel). Extracts were
immunoprecipitated with an anti-Smad1 antibody, and analyzed by Western
blot using an anti-myc antibody (upper panel). c,
SANE interaction requires Smad1 carboxyl-terminal residues. mycSANE was
expressed alone (lane 1) or with either Smad1-ala
(lane 2) or Smad1-asp (lane 3) in
Xenopus oocytes. Extracts were immunoprecipitated with an
anti-Smad1 antibody and Western blotted either with an anti-Smad1
antibody (middle panel) or with anti-myc antibody
(upper panel). Extracts were Western blotted with an
anti-myc antibody to confirm equal SANE expression (lower
panel). d, specificity of SANE interaction with Smad
proteins. Myc-SANE was expressed in Xenopus oocytes or
embryos either alone or with hemagglutinin (HA)
epitope-tagged Smads 1, 2, 4, and 5 (lanes 3-8, and
10) or FLAG-tagged Smad3 (lanes 11 and
12). Extracts were immunoprecipitated with anti-HA or
anti-FLAG antibodies and Western blotted with an anti-myc antibody to
detect co-immunoprecipitation of myc-SANE (upper panel) or
with anti-HA or FLAG antibodies to confirm immunoprecipitation of the
respective Smad proteins (middle panel; note that Smad 3 migrated close to the IgG band). Cell lysates removed prior to
immunoprecipitation were Western blotted with an anti-myc antibody to
confirm equal SANE expression (lower panel).
-specific Smads 2 and 3, the TGF
superfamily-shared Smad4,
and the BMP-specific Smad5 (Fig. 3D). Smads 1, 2, 3, 4, or 5 were co-expressed with myc-epitope tagged SANE (mycSANE) in
Xenopus embryos. Smads were immunoprecipitated followed by immunoblotting for mycSANE. This analysis shows that SANE strongly interacts with the BMP-specific Smads, Smad1 and Smad5, and binds weakly, if at all, with TGF-
specific Smad2 or Smad3 (Fig.
3D). These findings are supported by functional studies in
Xenopus and mammalian cells (see below).
C mRNA into ventral
blastomeres does not alter dorsal-ventral patterning (Fig.
4B), although this was limited by toxicity at higher doses.
Secondary axis induction by SANE is reversed by co-injection of Smad1
(Fig. 4D), suggesting that SANE specifically interferes with
the BMP/Smad1 pathway and inhibits the pathway at or upstream of the
level of Smad1. Expression of SANE-FL, SANE-
C, and SANE-C in dorsal
blastomeres did not affect dorsal-ventral patterning (Table
I) consistent with the low affinity of
SANE for activin/TGF-
specific Smad2.
View larger version (41K):
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Fig. 4.
SANE specifically inhibits BMP signaling
(A-D). Induction of partial secondary
axes by ventral expression of SANE in Xenopus embryos.
A, SANE-FL (full-length SANE); B,
SANE- C; or C, SANE-C mRNA (1 ng) was injected into a
single ventral blastomere of four-cell embryos. D, SANE-FL
mRNA was co-injected with Xenopus Smad1
mRNA (4 ng). SANE had no effect when injected into dorsal
blastomeres (see Table I). E and F, SANE inhibits
BMP but not activin-mediated mesoderm induction in Xenopus
ectodermal explants. BMP-4 (E) or
activin (F) mRNA were injected with or
without SANE into the animal pole of fertilized eggs, animal
cap explants were removed at stage 9, and expression of mesodermal
markers was assessed by RT-PCR at stage 10+. Induction of
Xbra and Xwnt8 by BMP-4 (E, lane
4) was inhibited by co-expression of SANE (lane 5), but
activin-mediated induction of gsc and Xbra
(F, lane 9) was not affected by SANE (lane
11). SANE mRNA alone (lanes 3 and
10) did not induce mesodermal markers. Lanes 1 and 6 show expression of Xbra, Xwnt8,
and gsc in whole embryos (stage 10.5). EF-1
is
used as a loading control. G and H, direct
inhibition of BMP signaling by SANE. G, an
XVent.2 promoter luciferase construct was co-injected with
BMP-4 mRNA (lane 1) and together with
increasing doses of SANE mRNA (lanes 2-4).
H, similarly, an activin-responsive Xmix.2
promoter luciferase construct was co-injected with
activin-
B mRNA (lane 6) and
together with increasing doses of SANE mRNA (lanes
7-9).
-Galactosidase mRNA (lanes 5 and 10) was co-injected as control. Embryos were harvested
at stage 10.5 for analysis of luciferase activity.
Axis duplication by SANE in xenopus
-dependent activation of
the 3TP-Lux promoter reporter in Mv1Lu cells (data not shown),
an assay that has been used extensively to measure direct
transcriptional response to TGF-
(23). These data support a specific
inhibitory role for SANE in the regulation of direct transcriptional
targets of BMP signaling.
C construct, which does not bind Smad1, does not induce neural
markers, suggesting that this effect is mediated through interaction
with Smad1. SANE did not induce dorsal mesoderm, as seen by the absence
of muscle actin expression, suggesting that the induction of neural
fates by SANE is direct, rather than a secondary consequence of dorsal mesoderm induction. These results in the Xenopus ectodermal
explant assay are consistent with the inhibition of BMP signaling by
SANE.
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Fig. 5.
Neuralization of ectodermal explants by
SANE. Embryos were injected with SANE, SANE- C, or
SANE-C mRNA; control is from un-injected embryos. Animal caps were
explanted at stage 9, cultured until stage 23, photographed
(A) and then RNA was extracted for RT-PCR (B).
A, explants expressing SANE-FL or SANE-C underwent slight
elongation and formed prominent cement glands (arrows), as
seen with other neutralizing agents such as noggin and chordin.
B, induction of neural markers: lane 1 shows
expression of neural markers (XAG, NCAM, and
otx2) and dorsal mesodermal marker (muscle actin) in whole
embryos (stage 23). Lane 3 shows control explants from
un-injected embryos. Lanes 4 and 5 show explants
expressing SANE-FL, lanes 6 and 7 show explants
expressing SANE-
C, and lanes 8 and 9 show
explants expressing SANE-C. EF-1
alpha is used as a
loading control.
-galactosidase as control were established, and populations of
transfected C2C12 cells were screened for transgene expression by
Western blot analysis. These cell lines were then treated with BMP-4
protein and stained for alkaline phosphatase activity, indicative of
osteoblast differentiation (Fig. 6). As
reported previously, BMP-4 induces bone-specific alkaline phosphatase
in control cells expressing
-galactosidase (Fig. 6, A and
D). However, expression of SANE-C inhibited BMP-4 mediated
induction of alkaline phosphatase by more than 10-fold (Fig. 6,
B and D). Expression of full-length SANE also
inhibited alkaline phosphatase induction (~5- to 6-fold; Fig. 6,
C and D). SANE did not block formation of
myotubes upon serum withdrawal and did not block TGF-
1-mediated
inhibition of myogenic differentiation (data not shown). Inhibition of
BMP-4 signaling in C2C12 myoblasts by SANE and SANE-C is consistent
with their inhibitory activity in Xenopus and again supports
the specificity of SANE for Smad1 and the BMP pathway.
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Fig. 6.
SANE inhibits BMP-mediated osteoblast
differentiation. C2C12 cell lines stably expressing:
A, -galactosidase; B,
SANE; or C, SANE-C were treated with BMP-4
protein or TGF-
1 protein and tested for alkaline phosphatase
activity, a marker of osteoblasts. D, quantitation of
alkaline phosphatase positive cells per total nuclei is shown for
samples from A-C.
View larger version (32K):
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Fig. 7.
SANE inhibits BMP-dependent Smad1
phosphorylation. BMP-4 mRNA (0.9 ng) was injected
into embryos alone or with SANE-FL (lane 3, 0.75 ng;
lane 4, 1.5 ng), and embryos were harvested at stage 10.5 for Western blot with antibodies specific for phosphorylated Smad1
(upper panel), Smad1 (middle panel), or SANE
(lower panels). Endogenous Smad1 phosphorylation is
increased by BMP-4, and this effect is blocked by expression of
SANE-FL.
1-dependent Smad2 nuclear translocation under
similar conditions (data not shown). These data are also consistent
with the specificity of SANE for antagonizing the BMP pathway and offer
a mechanism to explain SANE inhibition of BMP/Smad1 signaling.
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Fig. 8.
SANE prevents BMP mediated Smad1 nuclear
translocation. A, SANE was expressed in NIH3T3 cells,
and after 24 h, cells were treated with BMP-4 protein and analyzed
by immunofluorescence for nuclear translocation of endogenous Smad1.
4',6-Diamidino-2-phenylindole staining was used to identify nuclei
(blue). Transfected SANE was visualized with
rhodamine-labeled secondary antibody (red), and Smad1 was
visualized with an fluorescein isothiocyanate-labeled secondary
antibody (green). Endogenous Smad1 in nontransfected cells
served as a positive control for BMP-dependent nuclear
translocation. Arrows indicate cells transfected with SANE.
B, quantitation of the effect of SANE on Smad1 nuclear
translocation in the presence of BMP in NIH3T3 cells as shown in Fig.
9A. The number of cells counted is indicated at the
top of each column. For the SANE-transfected
group, only SANE-positive cells were counted.
-specific type I receptor (ALK-4); SANE was
immunoprecipitated and the type I receptors were detected by
immunoblotting with HA antibodies. The BMP-specific type I receptors
(ALK-3 and ALK-6) strongly associate with SANE (Fig. 9B,
lanes 3 and 5), but interaction of SANE with
ALK-2 and ALK-4 is barely detectable (Fig. 9B, lanes
7 and 9). This interaction is independent of the Smad1
binding region of SANE, because SANE-
C also associates with ALK-3 or
ALK-6 whereas SANE-C, the Smad1 binding domain of SANE, does not (Fig.
9C, lanes 4-6). Thus, SANE interacts
specifically with two components of the BMP signaling pathway.
View larger version (25K):
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Fig. 9.
SANE interacts with the BMP type I
receptor. A, SANE is a membrane protein: Stage 10 embryos were lysed in the presence (+) or absence ( ) of nonionic
detergent (1% Triton X-100) and centrifuged, and supernatants were
analyzed by Western blotting for endogenous SANE. In the presence of
detergent, SANE is in the soluble fraction. B, SANE
interacts with BMP-specific type I receptors: mRNA for HA-tagged
type I BMP-specific receptors ALK-6 and ALK-3, the activin and BMP
receptor ALK-2, and the activin/TGF-
receptor ALK-4 was injected
with (+) or without (
) myc-tagged SANE-FL mRNA. Receptor
complexes were immunoprecipitated with anti-HA antibodies and then
Western-blotted for myc-SANE (upper panel). Expression of
myc-SANE (middle panel) and HA-tagged receptors (lower
panel) is shown by Western blotting of lysates. C,
interaction between SANE and BMP receptor does not require the Smad1
interaction domain of SANE. SANE-FL (S), SANE-
C
(
C), or SANE-C (C) was co-expressed with
ALK-6-HA or with
-galactosidase (
-gal). Receptor
complexes were immunoprecipitated with anti-HA antibodies as above and
then blotted for myc-SANE (left panel). ALK-6 associates
with SANE-FL (lane 4) and SANE-
C (lane 5) but
not with SANE-C (lane 6), which contains the Smad1
interaction domain of SANE. Expression of myc-SANE in lysates is shown
by Western blot (right upper panel). The
lower panel shows expression of ALK-6-HA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-specific Smad2 and 3; binds strongly to type I
BMP-specific receptors (ALK-3 and ALK-6); inhibits BMP signaling
(but not activin or TGF-
) in multiple established assays of BMP
signaling; and is expressed in a pattern consistent with a role in
BMP/Smad1 signaling. Furthermore, the dorsalizing effects of SANE are
reversed by co-expression of Smad1, indicating that the effect of SANE is specific to the BMP/Smad1 pathway and acts at or upstream of Smad1.
Although ALK-2 is also a BMP receptor, the sequence of ALK-2 is
sufficiently distinct from ALK-3 and -6 that it was not recognized as a
BMP receptor until recently, suggesting that its structure is
significantly different than ALK-3 and -6 (23, 48). ALK-3 and ALK-6 are
type I receptors for BMP4 and BMP7, and ALK-2 is a type I receptor for
BMP6 and 7 but not BMP4, which may suggest that ALK-3 and -6 have
biological roles that are similar to each other but distinct from ALK-2
(48-50). This is consistent with our observation that SANE interacts
with ALK-3 and ALK-6 and not with ALK-2.
and BMP pathways. The BMP and activin membrane bound
inhibitor (BAMBI) is a transmembrane pseudoreceptor that antagonizes
both activin and BMP receptors and, like SANE, mimics other BMP
inhibitors when overexpressed in Xenopus embryos (54). However, unlike SANE, BAMBI is not specific for the BMP pathway and is
not known to interact with Smads. SARA, like SANE, interacts with both
receptors and Smads but is specific for the activin/TGF-
pathway and
does not interact with BMP pathway-specific Smads or BMP receptors
(55). Furthermore, SARA shows no sequence similarity to SANE, and
ectopic SARA expression does not inhibit TGF-
signaling, whereas
SANE inhibits BMP signaling. Smurf1 and Tob, like SANE, specifically
bind BMP pathway Smads and antagonize BMP signaling (29, 56). However,
unlike SANE, Smurf1 and Tob do not interact with BMP receptors and do
not inhibit Smad1 nuclear translocation; Smurf1 acts by accelerating
Smad1 protein turnover and Tob sequesters Smads in nuclear bodies.
signaling and are proposed to act as
feedback inhibitors of the respective pathways (57-60). These proteins
appear to mediate their effects by binding to type I receptors and
preventing pathway-specific Smad activation, although Smad6 has also
been reported to sequester Smad1 in a nonfunctional complex (60-63).
Thus, while SANE and Smad6 do not share obvious sequence similarity,
they appear to have similar activities in the regulation of BMP signaling.
/Smad interacting
genes and that other SANE-like molecules may regulate the TGF-
/Smad2 pathway.
. This evidence for SANE as an inhibitor
of BMP signaling comes from gain-of-function studies in
Xenopus and mammalian cells, similar to analysis of other
BMP antagonists. However, these experiments do not rule out the
possibility that SANE could act as an adaptor that mediates interaction
between BMP type I receptor and Smad1 and that could either positively
or negatively regulate signaling. In this scenario, SANE could recruit
Smad1 to the type I receptor for its subsequent activation, thus acting
in a positive role in BMP signaling. This adaptor model would predict
that expression of SANE at lower doses may potentiate BMP signaling if
endogenous SANE is limiting. However, we have not detected any
enhancement of BMP-4 activity over a wide dose range of SANE
RNA in the induction of mesodermal markers such as Xbra and
XWnt8 or in the induction of
Xvent.2-luciferase.2
Similarly, expression of SANE in dorsal blastomeres does not ventralize Xenopus embryos at any dose tested (Table I and
data not shown). These data do not support a role for SANE as a
positive regulator of BMP signaling; however, further studies,
including loss-of-function experiments, will be needed to validate this hypothesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Deardorff, C. Phiel, and K. Tremblay for reviewing the manuscript and A. Liu for technical assistance. Special thanks to Dr. P. Rao for his assistance. We also thank the late Dr. A. Wolffe for generously providing the oocyte cDNA library, Dr. P. ten Dijke for the phospho-Smad1 antibody, and Drs. J. Graff, D. Melton, J. Massague, A. Suzuki, K. Cho, J. Epstein, M. Hazama, R. Rupp, D. Turner, E. Shore, F. Kaplan, and E. Olmstead for plasmids and reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from The Center for Research in FOP and Related Disorders and the American Cancer Society and by Grant AR45587 from the National Institutes of Health (to H. C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF115498.
An Assistant Investigator of the Howard Hughes Medical Institute.
** To whom correspondence should be addressed. Tel.: 215-898-2319; Fax: 215-573-4320; E-mail: hchuang@mail.med.upenn.edu.
Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M210505200
2 G. P. Raju, P. S. Klein, and H.-C. Huang, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BMP, bone
morphogenetic protein;
TGF-, transforming growth factor-
;
SANE, Smad1 antagonistic effector;
EST, expressed sequence tags;
SANE-C, SANE
carboxyl-terminal fragment;
SANE-
C, SANE lacking the
carboxyl-terminal region;
GST, glutathione S-transferase;
SANE-FL, SANE full-length;
RT, reverse transcriptase.
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