From the Unitat de Bioquímica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907 Hospitalet de Llobregat, Spain
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ABSTRACT |
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Bone morphogenetic proteins (BMPs) constitute a
family of multifunctional growth and differentiation factors
structurally related to transforming growth factor-. BMPs were first
identified by their osteoinductive effects, inducing ectopic bone
formation when implanted in skeletal muscle, and have an important role as regulators of skeletal development in vivo. In
vitro, BMP-2 is able to transdifferentiate myogenic C2C12 cells
into the osteoblastic phenotype. In this report, we show that the
osteoinductive effects of BMP-2 in C2C12 cells are mediated by bone
morphogenetic protein receptor type-IA in combination with both activin
receptor type II and bone morphogenetic protein receptor type II. We
also analyzed the expression levels of nuclear protooncogenes to
understand early transcriptional events induced by BMP-2. We show that
junB is an immediate early gene induced by BMP-2 and
transforming growth factor-
. BMP-2 induces transcriptional
activation of JunB expression as early as 30 min after ligand addition,
reaching maximal levels after 90 min. Increase of JunB mRNA
correlates with a higher AP-1 binding activity. Furthermore, ectopic
overexpression of JunB is sufficient to inhibit expression of myoblast
differentiation markers in C2C12 cells. These data, taken together,
show the involvement of JunB in the early steps of inhibition of
myogenic differentiation induced by transforming growth factor-
family members.
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INTRODUCTION |
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During mammalian development, pluripotent mesenchymal cells give
rise to several specialized cell types, including cells with myogenic,
osteogenic, or adipogenic potential. This differentiation process
consists of at least two stages. In the first step or commitment, upon
certain stimuli, undifferentiated cells become committed to a
particular lineage. Later, during terminal differentiation, cells
acquire specific phenotypes by expressing genes encoding a set of
proteins unique to a certain type of cells. The best known example of
master genes governing cell fate are the family of myogenic basic
helix-loop-helix (bHLH)1
transcription factors (1). In muscle, the bHLH factors MyoD and Myf-5
play a role in lineage determination, whereas myogenin and MRF4 appear
soon thereafter to execute the differentiation program (1-2). Other
examples of such factors are the peroxisome proliferator-activated
receptor and CCAAT/enhancer-binding protein family members, which
control adipocyte differentiation or the transcription factor Cbfa1,
recently found as an activator of osteoblast differentiation (3-5).
The fact that ectopic expression of each these factors is sufficient to
induce a unique differentiation program or even to transdifferentiate
cells from other lineages emphasizes their key function (6-8).
Each specific cell commitment and differentiation program is ultimately controlled by signals and regulatory pathways that converge to activate a small number of transcription factors. These mechanisms involve cell-cell and cell-matrix interactions, as well as extracellular diffusible factors. Among the latter, bone morphogenetic proteins (BMPs) have been shown to be regulators of skeletal development (9). They were originally identified by their osteoinductive effects, inducing ectopic bone formation when exogenously implanted in rat or mouse muscle (10). Furthermore, knock-out mice showed marked skeletal defects (11-14), and they are expressed in the embryo at sites of cartilage and bone formation (15). BMP-2/4 also play a role in inductive interactions during early development, regulating dorso-ventral patterning and mesoderm induction (16). In addition to their function during morphogenesis and determination of the body axis, BMPs are also involved in later stages of development. BMPs not only stimulate maturation of osteoblast precursor cells, they also convert the differentiation pathway of myoblastic cells into the osteoblast lineage (17).
BMPs belong to the transforming growth factor- (TGF-
) superfamily
encompassing TGF-
s, activins, and Mullerian inhibiting substance.
BMPs and other members of the TGF-
superfamily exert their
biological function by interacting with two types of related transmembrane serine/threonine kinase receptors, known as type I and
type II (18). For both TGF-
and activin, receptor activation requires binding of ligand to the type II receptor, which then recruits
and phosphorylates the type I receptor (19). Phosphorylation on the GS
region activates the kinase activity of the type I receptor, which
propagates the signal to downstream substrates (19-20). The BMP
receptor system is somewhat different. They also require heteromeric complex formation for signaling, but, unlike TGF-
or activin, BMP
type I receptors are able to bind the factor without coexpression of
type II receptors; however, both together achieve high affinity binding
(21-23). Downstream signaling by these receptors is mediated by the
recently identified family of Smad proteins (18, 24). In response to
receptor activation, Smad proteins become phosphorylated at their
carboxyl termini and translocate into the nucleus (25-27). Recent
results support the notion that distinct Smad family members transduce
signaling from specific receptor combinations. Smad1 is a BMP signal
transducer, and the closely related Smad2 and Smad3 are TGF-
/activin
signal transducers. Smad4/DPC4, originally found as a gene mutated in
pancreatic carcinomas (28), functions as a shared partner, which
hetero-oligomerizes with all other Smads upon phosphorylation (29).
Once into the nucleus, Smad complexes probably exert a direct role in
transcriptional control. Consistent with this function, transcriptional
activity of various Smad proteins and association of Smad2 with the
DNA-binding protein FAST1 have recently been shown (30).
Although the molecular mechanisms involved in BMP signaling are beginning to emerge, the precise BMP receptor combinations as well as early transcriptional events that mediate the osteogenic differentiation effects of BMP in muscular tissues have remained elusive. Here, we report that the osteoinductive response to BMP-2 in C2C12 cells is mediated by the type I receptor BMPR-IA in combination with both type II receptors BMPR-II and ActR-II. Additionally, to understand early events induced by BMP-2, we analyzed the expression level of nuclear protooncogenes. Results presented here indicate that junB is an immediate early gene transcriptionally induced by BMP-2. Moreover, ectopic overexpression of JunB is sufficient to inhibit expression of myogenic markers on C2C12 cells. Therefore, these data suggest the involvement of JunB in the inhibition of myogenic differentiation induced by BMP-2.
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EXPERIMENTAL PROCEDURES |
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Plasmids, Cell Lines, and Transfections--
-Actin and
-acetylcholine receptor promoter regions fused to a CAT reporter
were kindly provided by Dr. Kocniezcny (Purdue University; West
Lafayette, IN) (31). T
R-IHA and ActR-IHA
vectors have been described previously (32). BMPR-IAHA
construct containing the hemagglutinin (HA) epitope at the carboxyl terminus was generated by PCR. A full-length JunB cDNA was
subcloned into pCDNA3 for mammalian expression. A 3-kilobase mouse
JunB promoter fused in a CAT reporter (JB3000CAT) was kindly provided by Dr. Nakajima (Osaka University; Osaka, Japan). C2C12 were cultured in DMEM supplemented with 20% fetal bovine serum and transiently transfected with the indicated vectors by the calcium phosphate method
(33). Cell lines COS-1 and R1B/L17 were cultured and transiently
transfected with the indicated vectors as described previously
(32).
Immunofluorescence--
C2C12 cells were grown on coverslips and
treated with BMP-2 (Genetics Institute, Cambridge, MA) or TGF-1
(Sigma) for 3 days in DMEM supplemented with 2% inactivated horse
serum. Cells were fixed in 4% paraformaldehyde and processed as
described in Ref. 34, using rabbit anti- myosin antiserum as primary
antibody (1/100 dilution) (Sigma) and Texas red-labeled anti-rabbit
antibody as secondary antibody (Amersham Life Science Inc.).
Alkaline Phosphatase and CAT Assays-- Alkaline phosphatase was assayed as follows. Cells were washed with phosphate-buffered saline and lysed in 50 mM Tris-HCl, 0.5% Nonidet P-40, pH 7.5. Cell lysates were incubated in a buffer containing 0.1 M glycylglycine, 5 mM MgCl2, and 10 mM p-nitrophenyl phosphate, pH 9.5. The reaction was stopped with 0.5 M NaOH, and absorbance was measured at 405 nm. CAT activity from transiently transfected cells was measured as described in Ref. 33.
RNA Preparation and Northern Blots-- Total RNA was extracted from C2C12 cells according to the Chomczynski method (33). Northern analysis was done as described in Ref. 33.
Affinity Labeling in Vivo and in Vitro-- Receptor affinity labeling assays in vivo were performed as described in Ref. 32. For in vitro affinity labeling, cell extracts from COS-1 cells expressing epitope-tagged receptors were immunoprecipitated using anti-HA antibody (12CA5 Babco) and protein A-Sepharose (Pharmacia Biotech Inc.). Immunoprecipitates were washed five times in 50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, pH 7.5. After washing twice in Krebs-Ringer Hepes buffer, bound receptors were incubated with 125I-BMP-2 for 3 h at 4 °C, followed by four washes in the same buffer. Receptors were cross-linked to bound ligand with disuccinimidyl suberate (Pierce) for 15 min, washed twice, and subjected to SDS-PAGE and autoradiography.
Immunoprecipitations-- Antibodies against ActR-I, BMPR-IA, and BMPR-IB were kindly provided by Dr. P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden) (35). Antibodies against ActR-II were kindly provided by Dr. W. Vale (The Salk Institute, La Jolla, CA) (36). Affinity-labeled receptors from C2C12 cells were solubilized in 50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, pH 7.5. Cell extracts, clarified by centrifugation, were incubated with each antibody for 1 h at 4 °C, followed by incubation with protein A-Sepharose for another 1 h. Bound receptors were then washed five times in solubilization buffer, and subjected to SDS-PAGE and autoradiography.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared by the method of Andrews and Faller (33). Pelleted cells were
resuspended in 400 µl of cold Buffer A (10 mM Hepes-KOH,
pH 7.9 at 4 °C, 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) by flicking the tube.
After a 10-min incubation on ice, cells were vortexed for 10 s and
then centrifuged. The pellet was resuspended in 20-100 µl of cold
buffer C (20 mM Hepes-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min for high salt extraction. Cellular debris was removed by centrifugation for 2 min, and the supernatant fraction were stored at
70 °C. The protein content was determined using the Bradford protein concentration assay (Bio-Rad) with bovine serum albumin as
standard. The AP-1 oligonucleotide 5
-CGCTTGATGAGTCAGCCGGAA-3
(Promega) was labeled with [
-32P]ATP and T4
polynucleotide kinase.
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RESULTS |
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C2C12 myoblasts constitute an in vitro model system to
study the ability of BMPs to alter cell lineage from the myogenic to the osteogenic phenotype. C2C12 cells differentiate into multinucleated myotubes when concentration of serum is reduced from 20% to 2%. BMP-2
inhibits myotube formation, inducing osteoblastic markers such as
alkaline phosphatase activity or osteocalcin production (17). Treatment
with 1 nM BMP-2 or 1 nM TGF- completely
inhibited myotube formation and myosin expression on C2C12 cells
exposed to differentiation media (Fig.
1A). This
BMP2-dependent inhibition of myogenic differentiation could
be also seen using reporter constructs containing muscle-specific
promoter regions of myogenic markers such as
-actin or
-acetylcholine receptor. Addition of 1 nM BMP-2 to cells
treated with differentiation media inhibited 50-70% of their CAT
reporter activity (Fig. 1B). Inhibition of reporter
expression is not a general transcriptional effect of BMP-2 since
expression from constitutive promoters did not show any change (data
not shown). C2C12 cells express extremely low levels of alkaline
phosphatase and other osteoblastic markers when cultured both in
proliferative or differentiation media. Time-course studies show that,
of all the TGF-
superfamily members tested, only BMP-2 is able to
stimulate alkaline phosphatase expression, beginning to appear within 2 days, and increasing until day 4 (Fig. 1C). These data
indicate that different members of of the TGF-
superfamily have
distinct biological effects on selection of C2C12 terminal
differentiation.
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We analyzed the expression and binding affinity of distinct activin and BMP receptors in C2C12 cells to determine which receptor combinations are involved in osteogenic BMP responses. Northern analysis indicates that C2C12 cells express BMPR-II and ActR-II as activin/BMP type II receptors and ActR-I and BMPR-IA as type I receptors (Fig. 2A). In contrast, both ActR-IIB and BMPR-IB were expressed below detection limits of either Northern or reverse transcription-PCR analysis (data not shown).
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We also tested binding of BMP-2 to the endogenous receptors expressed in C2C12 cells. Immunoprecipitation of the cross-linked receptor complexes using antiserum against BMPR-IA gave a strong signal of about 75 kDa (Fig. 2B). A labeled band could be seen in immunoprecipitates using an antiserum against ActR-II. In addition, a weak signal was detected when antiserum against BMPR-IB was used. This signal could be generated by a small number of BMPR-IB receptor mRNA that could not be detected by reverse transcription-PCR. Alternatively, since BMPR-IB antibody was raised against peptides derived from its juxtamembrane region (35), and BMPR-IA and -IB have closely related juxtamembrane regions, it is likely that the antibody against BMPR-IB cross-reacts with small amounts of BMPR-IA. Although it has been reported that ActR-I is able to bind BMP-2/4 when overexpressed in COS-1 cells (23), using the same immunoprecipitation approach, we could not detect any BMP-2 binding to ActR-I at physiological receptor levels.
Some cross-linking studies showed that when transiently transfected,
type I receptors for BMP are capable of binding to BMP2/4 without
coexpression of the type II receptor (21, 23). To confirm these data
and exclude the possibility that endogenous type II receptor expressed
in these cells could allow binding to type I receptors, we analyzed the
ability of distinct purified type I receptors to bind BMP-2 in
vitro. Epitope-tagged BMP-2 receptors expressed in COS cells were
immunopurified and subjected sequentially to binding and cross-linking
to 125I-BMP-2. This binding assay showed specific binding
of the ligand to BMPR-IA compared with control lanes derived from
mock-transfected cells or cells transfected with transforming growth
factor- receptor type I (T
R-I), which is unable to bind BMP-2. In
agreement with the in vivo assays, purified ActR-I was also
unable to bind BMP-2 by itself (Fig. 2C). Altogether, these
results suggest that the major receptor complexes involved in
osteogenic responses of BMP-2 are those including BMPR-IA in
combination with ActR-II and probably BMPR-II.
TGF- has been shown to modulate expression of several nuclear
protooncogenes in epithelial and fibroblastic cells (37-38). In
addition, knock-out mice for transcription factors involved in AP-1
function, such as c-Fos or ATF2, exhibited profound defects in bone
formation (39-40). These two lines of evidence led us to investigate
the expression levels of nuclear protooncogenes to understand early
transcriptional events in myogenic/osteogenic transdifferentiation
processes induced by BMP-2. RNAs from C2C12 cells cultured with or
without 1 nM BMP-2 in differentiation media were analyzed
by Northern blotting. As shown in Fig. 3,
expression of c-jun, junD, or c-myc
was not significantly modified at any time point, either with or
without ligand addition. However, 1 nM BMP-2 transiently
increased JunB mRNA. Induction of JunB is detected as early as 30 min after BMP-2 addition, reaching maximal (9-fold over control
levels) levels after 90 min and decreasing thereafter. Interestingly,
we also detected a sharp induction of c-fos transcription at
30 min, followed by a rapid decline to control levels. Nevertheless,
this induction was observed both in BMP-2-treated and untreated cells,
suggesting this effect was likely due to media replacement and is not a
consequence of BMP-2 addition.
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We next assessed whether induction of JunB is dependent on new protein
synthesis. C2C12 cells were treated with BMP-2 for 90 min, together
with the protein synthesis inhibitor, cycloheximide (10 µg/ml), or
the RNA synthesis inhibitor, actinomycin D (1 µg/ml). Northern
analysis revealed an even higher induction of JunB mRNA in the
presence of cycloheximide and a complete block of induction when
actinomycin D was added (Fig. 4). We also
tested the effects of other members of the TGF- superfamily on JunB
mRNA levels. TGF-
at 1 nM strongly induced JunB
mRNA, whereas no significant induction was detected in RNA from
C2C12 cells treated with 1 nM activin A (Fig. 4).
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Transient cell transfections with a JunB promoter/CAT reporter gene
construct were performed to confirm whether BMP-2 and TGF- regulate
JunB mRNA steady state levels through modulation of its
transcription rate. R1B/L17 clone, which is unresponsive to TGF-
due
to mutations at its receptor type I (32), and C2C12 cells were
transfected with a CAT construct containing a 3-kilobase proximal
region of mouse JunB promoter and treated with BMP-2 or TGF-
both at
1 nM. Assay of CAT activity after 16 h of incubation indicated that both factors induced promoter activity 2-3-fold as
compared with control cultures in C2C12 cells, whereas only BMP-2 was
able to stimulate CAT activity in L17 cells (Fig.
5). Thus, these results indicate that the
enhancement of junB gene expression in myogenic cells does
not require protein synthesis and is mediated, at least in part, by
modulation of its promoter activity.
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AP-1 is a collective term referring to homo- and heterodimeric transcription factors composed of members of the Jun, Fos, or ATF families that bind to a common DNA site known as the AP-1 binding site. Distinct combinations of AP-1 factors regulate different target genes and thus execute distinct biological functions (41). We tested if higher expression levels of JunB mRNA induced by BMP-2 correlate with higher AP-1 function. Gel mobility shifts were performed with a radiolabeled AP-1 consensus oligonucleotide incubated in the presence of nuclear extracts from cells treated with or without 1 nM BMP-2 at different times. As shown in Fig. 6, we obtained a single shifted band. This band was completely competed with a 50-fold molar excess of unlabeled probe (data not shown). There was a binding increase in extracts from cells treated or not with factor for 1 h. This BMP-independent increase is likely to be due to the strong activation of c-Fos transcription, seen at 30 min in Fig. 3, since it has been described that c-Fos can be rate-limiting in the formation of heterodimers with the other AP-1 members (41). At later times, the intensity of the shifted band progressively increased in extracts from cells treated with BMP-2 in respect to their control counterparts. Preincubation of the extracts with antibody against JunB partly abrogates the binding, further suggesting the involvement of JunB in these AP-1 complexes (Fig. 6). These data suggest that induction of JunB results in formation of transcriptionally active AP-1 complexes specific for JunB-regulated genes.
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Whereas both BMP-2 and TGF- induce JunB transcription and inhibit
myogenic differentiation of C2C12 cells, only BMP-2 is able to
transdifferentiate these cells to the osteoblastic lineage. Thus, we
analyzed the relationship between JunB expression and myogenic
differentiation. We used regulatory sequences of
-actin and
-acetylcholine receptor genes fused to a CAT reporter gene. These
enhancers contain binding sites for myogenin and MyoD that are strictly
muscle-specific (31). Fig. 7 shows that
CAT activities were induced 2-4-fold in C2C12 cells transfected with
those reporters when shifted to differentiation media for 3 days. These
transcriptional activations were efficiently blocked by addition of
both BMP-2 or TGF-
. More interestingly, when cotransfection assays
were performed with a JunB expression vector, transactivation of both reporters by differentiation media was completely suppressed. No
effects were seen using SV40-
-galactosidase as reporter or when
other unrelated genes were cotransfected with the CAT reporters (data
not shown). Thus, we conclude that induction of JunB is sufficient and
could be, at least partly, responsible of the inhibition of myogenic
differentiation of C2C12 cells by BMP-2 and TGF-
.
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DISCUSSION |
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Our results show that the ability of BMP-2 to transdifferentiate
myogenic cells to the osteoblastic lineage is mediated by pathways
activated by BMPR-IA. We also demonstrate that activation of these
pathways lead to early transcriptional activation of junB
gene expression with a concomitant increase in
JunB-dependent AP-1 binding activity. TGF-, another
member of the TGF-
superfamily, shares with BMP-2 both the ability
to inhibit myogenesis and to activate transcription of JunB. On the
basis of this evidence and the observation that ectopic expression of
JunB in myogenic precursor cells is sufficient to inhibit myogenic
differentiation, we propose that the induction of JunB has an important
role in the control of myogenic differentiation by BMP-2 and
TGF-
.
During the process of ectopic bone formation, after bone-inducing
factors are implanted into muscular tissues, these factors appear to
alter the differentiation of muscle cells around the implantation sites
(9). BMPs have been shown to be potent bone inducers in vivo
(9-10). Although related factors failed to induce ectopic bone
formation, some of them are able to act as negative regulators of
muscle cell development. In this regard, it has recently been shown
that knock-out mice for GDF-8, a member of the TGF- superfamily,
have 2-3-fold increased body mass (42). In the present study, we show
that the TGF-
-related factors studied differ in their
transdifferentiation effects in C2C12 cells. Whereas, in agreement with
Ref. 17, BMP-2 both inhibits myogenesis and activates osteogenesis,
TGF-
is only capable of inhibit myogenesis (Fig. 1). The above data
suggest that osteogenic effects are elicited by a unique signaling
pathway initiated by BMP-2.
BMPR-IA Is the Main Receptor for BMP-2 in C2C12 Cells--
BMP
signaling at the plasma membrane is mediated by a subset of
transmembrane serine/threonine kinase receptors (18). However, a
certain redundancy in the ligand binding of the type I receptors has
been reported (18, 21-23). For this reason, the assignment of precise
receptor combinations that mediate each activin or BMP response remains
tentative. For example, BMP-4 binds to BMPR-IA and BMPR-IB, whereas
BMP-7 binds to BMPR-IB and less efficiently to BMPR-IA (21). Both BMP-2
and BMP-7 can also bind to one of the activin type I receptors, ActR-I,
in the presence of a suitable receptor type II (23). In respect to type
II receptors, BMPR-II has been shown to bind BMP-2, -4, and -7 and is
required for signaling in combination with certain type I receptors
(22-23). Additionally, BMP-7 has been shown to bind to ActR-II/ActR-I
complexes, signaling certain activin-like effects (43). The present
identification of ActR-II as a BMP-2 binding receptor at physiological
expression levels further extends the notion of versatility in this
receptor system. Our conclusion that the osteogenic responses induced
by BMP-2 in C2C12 cells arise from activation of BMPR-IA rests on several lines of evidence. First, immunoprecipitation assays show that
BMPR-IA is the expressed receptor on C2C12 cells (BMPR-IB is not
expressed or expressed at very low levels) with higher binding affinity
for BMP-2 (Fig. 2). Second, although required for signaling, its
ability to bind BMP-2 is independent of the presence of a type II
receptor, suggesting that in the cellular context it could combine with
both BMPR-II and ActR-II acting as activating receptors. Furthermore,
constitutively active BMPR-IA is sufficient to mediate osteoinductive
responses in chicken wing development (44), suggesting that, as with
other TGF- superfamily receptors, signaling depends on the
specificity of receptor type I kinase.
junB Is an Immediate Early Gene Induced by
BMP-2--
Developmental studies performed in vitro and
in vivo implicate the Fos and Jun family of transcription
factors in the regulation of bone tissue formation (39-41). Here we
show that, from all AP-1 transcription factors studied, only JunB is
highly induced at early times after BMP-2 or TGF- addition (Fig. 3).
Regulation of the steady-state levels of JunB is likely to be due to
regulation of its transcription rate since addition of actinomycin D
completely blocks induction of its mRNA by BMP-2. Furthermore,
BMP-2 and TGF-
are able to induce expression from an heterologous
reporter construct. R1B/L17 cells lack functional type I receptors for TGF-
, but they have receptors for BMPs (21-22). Thus, the fact that, in these cells, only BMP-2 activated reporter expression indicates that the signal transduction elicited by BMP-2 and TGF-
are initiated by distinct subsets of receptors at the cell membrane, that at some later point, would converge in this specific JunB transcriptional response.
Ectopic Expression of JunB Is Sufficient to Inhibit Myogenic
Differentiation--
Several studies reported differences in the
pattern of expression and response to stimuli between Jun family
members (41). In addition, considerable differences in their
transactivation and transforming activities have been reported (38, 41,
45-46); c-Jun is an effective activator of human collagenase and
synthetic promoters containing a single
12-O-tetradecanoylphorbol-13-acetate response element,
whereas JunB transactivates promoters with multiple 12-O-tetradecanoylphorbol-13-acetate response elements (46). In addition, c-Jun/
mouse showed embryonic lethality,
further suggesting Jun family members have non-equivalent
transcriptional functions (39-40). In addition, overexpression of JunB
has been shown to counteract activation of c-Jun-responsive genes,
suggesting a role for JunB as a negative regulator of c-Jun (45, 46).
Protein interaction assays showed, in the absence of c-Fos,
preferential formation of c-Jun/JunB heterodimers that have decreased
DNA binding activity in respect to their homodimeric counterparts,
whereas, in the presence of c-Fos, preferential formation of JunB/c-Fos
heterodimers should contribute to AP-1 activity (45). Therefore,
modification in the relative levels of Fos and Jun family members by
extracellular factors, such as BMP-2 and TGF-
, would result in the
presence of a specific subset of AP-1 dimers with altered
transcriptional activity. This hypothesis has been demonstrated in the
differential regulation of collagenase transcription by cell-specific
induction of JunB or c-Jun and could also be the case in the C2C12
model presented here since ectopic overexpression of JunB, altering the
c-Jun/JunB ratio within the cell, is sufficient to mimic inhibition of
myogenic differentiation by BMP-2 and TGF-
.
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ACKNOWLEDGEMENTS |
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We thank Esther Adanero, Cristina Sanchez, and Mònica Vallés for technical assistance and Drs. S. Ambrosio, J. Gil, C. Harvey, S. Konieczny, J. Massagué, K. Nakajima, G. Pons, A. Tauler, P. ten Dijke, and W. W. Vale for discussion and gifts of plasmids, antibodies, and reagents. We also thank the Genetics Institute for recombinant BMP-2.
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FOOTNOTES |
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* This work was supported by Grant PM-0171 from the Ministerio de Educación y Ciencia, Grant 1995-SGR00427 from the Generalitat de Catalunya, and fellowships from Fundació Pi i Sunyer (to E. C.) and Ministerio de Educación y Ciencia (to T. L.).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 should be addressed. Tel.: 34-3-4024281;
Fax: 34-3-4024213; E-mail: fventura{at}bellvitge.bvg.ub.es.
1
The abbreviations used are: bHLH, basic
helix-loop-helix; BMP, bone morphogenetic protein; TGF-,
transforming growth factor-
; BMPR-I, bone morphogenetic protein
receptor type I; T
R-I, transforming growth factor-
receptor type
I; ActR-II, activin receptor type II; BMPR-II, bone morphogenetic
protein receptor type II; PCR, polymerase chain reaction; CAT,
chloramphenicol acetyltransferase; PAGE, polyacrylamide gel
electrophoresis; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's
medium.
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REFERENCES |
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