From Deutsches Krebsforschungszentrum Heidelberg, Division of Signal Transduction and Growth Control (B0800), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
Received for publication, November 26, 2000, and in revised form, February 16, 2001
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ABSTRACT |
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The expression of MMP13 (collagenase-3), a member
of the matrix metalloproteinase family, is increased in
vivo as well as in cultured osteosarcoma cell lines by
parathyroid hormone (PTH), a major regulator of calcium
homeostasis. Binding sites for AP-1 and Cbfa/Runt transcription factors
in close proximity have been identified as cis-acting
elements in the murine and rat mmp13 promoter required for
PTH-induced expression. The cooperative function of these factors in
response to PTH in osteoblastic cells suggests a direct interaction
between AP-1 and Cbfa/Runt transcription factors. Here, we demonstrate
interaction between c-Jun and c-Fos with Cbfa/Runt proteins. This
interaction depends on the leucine zipper of c-Jun or c-Fos and the
Runt domain of Cbfa/Runt proteins, respectively. Moreover, c-Fos
interacts with the C-terminal part of Cbfa1 and Cbfa2, sharing a
conserved transcriptional repression domain. In addition to the distal
osteoblast-specific element 2 (OSE2) element in the murine and rat
mmp13 promoter, we identified a new proximal OSE2 site
overlapping with the TRE motif. Both interaction of Cbfa/Runt proteins
with AP-1 and the presence of a functional proximal OSE2 site
are required for enhanced transcriptional activity of the
mmp13 promoter in transient transfected fibroblasts and in
PTH-treated osteosarcoma cells.
Interstitial collagenase (collagenase-3, MMP13) is a member of the
large family of matrix metalloproteinases that play a decisive role in
the degradation of components of the extracellular matrix, particularly the collagens. It degrades collagen type II most efficiently but also collagens type I, III, and X, which are the major
components of cartilage and bone (1). MMP13 is expressed in
hypertrophic chondrocytes and osteoblasts during embryogenesis and in
the adult bone and is thought to be involved in endochondral ossification and bone remodeling (2-4). The level of MMP13 expression depends on the exposure to a variety of systemic and local factors including hormones and cytokines present in the bone microenvironment (5-9). High constitutive levels of MMP13 were observed in
osteosarcomas and chondrosarcomas, suggesting a critical role in the
formation of bone tumors (2, 10).
Parathyroid hormone (PTH)1
has been shown to stimulate MMP13 expression in vivo as well
as in vitro cultures of primary bone cells and
osteoblast-derived osteosarcoma cell lines (8, 9, 11). PTH is secreted
by the parathyroid gland and is a major regulator of calcium
homeostasis. Intracellular signaling initiated by ligand-activated
PTH/PTHrP receptor present on the plasma membrane of osteoblasts (12)
is mediated predominantly by activation of adenylate cyclase, resulting
in enhanced concentration of cAMP and activation of the protein kinase
A (PKA) (13, 14).
We and others have identified the minimal PTH-responsive region within
the mmp13 promoter (8, 9). This region contains a conserved
AP-1 binding site and an osteoblast-specific element 2 (OSE2), which is
recognized by members of the Cbfa/Runt family of transcription factors.
Both elements act cooperatively and are absolutely required for
PTH-dependent promoter activation. In agreement with the
crucial role of AP-1 and Cbfa/Runt transcription factors in
osteosarcoma cell lines, expression of MMP13 is reduced in mice lacking
c-fos and is completely absent in
cbfa1 While DNA binding activity of AP-1 is increased upon PTH treatment
through enhanced de novo synthesis of c-Fos and c-Jun
proteins (16, 17), a high basal level of Cbfa/Runt binding activity was
detected independently of PTH treatment, suggesting different mechanisms for transcriptional activation (9). Since no changes in
Cbfa/Runt protein or RNA levels were detectable after PTH treatment, posttranslational modifications of Cbfa1 in the signaling pathway for
PTH-induced mmp13 promoter activation was suggested. Indeed, Cbfa1 was shown to be phosphorylated in vitro by PKA, a
major target of the PTH/PTHrP receptor pathway. Additionally, PTH
stimulated the transactivation capacity of Cbfa1 through a PKA site
within the C-terminal transactivation domain
(18).3
Cbfa1-dependent transcription was also induced by
mitogen-activated protein kinase pathways that have been shown to be
stimulated by PTH via a cAMP-mediated pathway independent of Ras (19,
20).
The close proximity of the AP-1 and OSE2 sites and their cooperative
function in response to PTH in osteoblastic cells suggest a physical
interaction between AP-1 and Cbfa/Runt transcription factors. Previous
reports have already demonstrated that Cbfa/Runt proteins are promoter
organizers that cooperate and interact with neighboring factors and
recruit transcriptional co-activators or co-repressors to regulate
expression of tissue-specific genes. Specifically, functional
interaction between Cbfa/Runt proteins and transcriptional regulators
(e.g. ALY (21), Ear-2 (22), p300 (23), Groucho/TLE/R-esp and
HES-1 (24-26), Smads (27, 28), YAP (29), Ets-1 (30), C/EBPs (32-34),
and PU.1 (35)) have been described.
Here, we demonstrate for the first time a direct interaction between
AP-1 and Cbfa/Runt transcription factors in vitro as well as
in cultured cells. The leucine zipper of c-Jun or c-Fos and the Runt
domain of Cbfa/Runt proteins were sufficient for interaction.
Additionally, c-Fos was able to interact with a C-terminal part of
Cbfa/Runt proteins sharing a conserved repression domain. Furthermore,
we identified a new proximal OSE2 site overlapping with the TRE element
in the murine and rat mmp13 promoter. Transient transfection
assays in fibroblasts and PTH-treated osteosarcoma cells demonstrated
that interaction between Cbfa/Runt proteins and AP-1 and the presence
of a functional proximal OSE2 site are required for synergistic
activity on the mmp13 promoter. These data provide evidence
for a direct interaction between AP-1 and Cbfa/Runt transcription
factors in the process of PTH-induced expression of MMP13 and for a new
functional composite OSE2/TRE element.
Expression and Reporter Plasmids--
pGST-Cbfa1 fusion protein
resulted from insertion of the BamHI/EcoRI
fragment of Cbfa1 (58) into the pGex1 vector (Amersham Pharmacia
Biotech) opened with BamHI/EcoRI.
pGST-Cbfa1
pBAT-Cbfa1 resulted from the ligation between the
SalI/EcoRV fragment of Cbfa1 excised from
pBSK-Cbfa1 into the pBAT-c-Jun vector (36), where the c-Jun coding
sequence was removed by SalI/EcoRV restriction.
Plasmids for in vitro translation of full-length c-Jun and
the mutants
Expression vectors for c-Fos-HA and c-Jun-HA in which the tag is
in frame in the C-terminal end of the c-Fos and c-Jun genes were
kindly provided by D. Bohmann. pCEV is the expression vector for the
PKA catalytic subunit and is described in Uhler and McKnight (39). The
Myc-tagged Cbfa1 construct (pcDNA3.1-Cbfa1) resulted from
the insertion of the XbaI/EcoRI fragment of Cbfa1
excised from pBSK-Cbfa1 into pcDNA3.1(B) (Invitrogen) linearized
with BamHI/EcoRI. pcDNA3.1-Cbfa1
The luciferase reporter plasmids mColl-luc( Purification of Recombinant Proteins and Analysis of
Protein-Protein Interactions--
Recombinant proteins were expressed
in BL21pLysS bacteria (Stratagene) cultured in 300 ml of TY medium (10 g of tryptone/peptone, 10 g of yeast extract, 5 g of NaCl,
1 g of casaminoacids, 1 liter of H2O, and 100 µg/ml
ampicillin) in the presence of 0.1 mM
isopropyl-1-thio-
For GST pull-down assays, equal amounts of purified recombinant GST
fusion proteins were mixed with glutathione-agarose beads (Sigma) and
in vitro translated c-Jun or c-Fos proteins (TNT Coupled Reticulocyte Lysate Systems; Promega). Alternatively, cell lysates prepared with PP100 buffer from PTH-treated or untreated UMR106 cells
or transiently transfected HEK293 cells were used. After incubation (1 h at 4 °C), the beads were washed three or four times with 1 ml of
PP100 buffer, and the coprecipitated proteins were separated by
SDS-PAGE using a 15% polyacrylamide gel. GST pull-down assays with
in vitro translated proteins were analyzed by
autoradiography. GST pull-down assays with cell lysates were analyzed
by Western blot.
For co-immunoprecipitation, the cell lysates prepared with PP100 buffer
were first precleared by incubation (30 min, 4 °C) with 10 mg of
protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) and then
were mixed with 10 mg of Protein A-Sepharose CL-4B beads and 2 µg of
monoclonal anti-Myc antibody (9E10; Roche Molecular Biochemicals).
After 2 h of incubation at 4 °C with gentle rotation, the
protein A-coupled Sepharose beads were washed three times with 1 ml of
PP100 buffer, and co-precipitated proteins were separated by SDS-PAGE
using a 15% polyacrylamide gel and analyzed by Western blot.
Western blots were performed as described (40) using the following
antibodies: anti-HA (sc-805; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), anti-GST (27-4577-01; Amersham Pharmacia Biotech), anti-Myc
(9E10; Roche Molecular Biochemicals), anti-Cbfa1 (sc-8566; Santa Cruz
Biotechnology), anti-c-Jun (J31920; Transduction Biolaboratories), and
anti-c-Fos (sc-52; Santa Cruz Biotechnology).
EMSA--
Nuclear extracts from UMR106 cells were prepared
according to the protocol described (41), and protein
concentration was determined using the protein bioassay
solution (Bio-Rad). EMSA were performed with 2-4 µg of protein of
nuclear extracts and with 30,000-50,000 cpm of a
32P-radiolabeled probe as described (41, 42). The
oligonucleotides for the TRE-Coll probe were described by
Porte et al. (9), and the sequences of the other
probes were as follows: mutOSE2/mutTRE-Coll-A, 5'-AGCTAAAGTGACGACTCATCACTAT-3'; mutOSE2/mutTRE-Coll-B,
5'-AGCTATAGTGATGAGTCGTCACTTT-3'; mutOSE2/TRE-Coll-A,
5'-AGCTAAAGTACTGACTCATCACTAT-3'; mutOSE2/TRE-Coll-B, 5'-AGCTATAGTGATGAGTCAGTACTTT-3'; OSE2/mutTRE-Coll-A,
5'-AGCTAAAGTGGTGGCGCATCACTAT-3'; OSE2/mutTRE-Coll-B,
5'-AGCTATAGTGATGCGCCACCACTTT-3'.
The oligonucleotides for the Oct probe were kindly provided by T. Wirth, and sequences were previously described (68).
Cell Culture and Transfections--
Medium, growth conditions,
and treatment of cells with PTH or TPA have been described previously
(9). Human HEK293 embryonic kidney cells and mouse F9 embryonic
carcinoma cells were transiently transfected according to the calcium
phosphate method as described by Angel et al. (43). Rat
UMR106 osteosarcoma cells were transfected by electroporation using the
0.4-cm cuvettes and setting the Gene-pulser (Bio-Rad) to 0.25 kV and
960 microfarads. Transfected cells were split on two 94-mm dishes and
cultured overnight. For PTH stimulation, after a 24-h culture,
transfected cells were treated for 8 h with PTH.
Measurement of luciferase and Cbfa/Runt Family Members Directly Interact in Vitro with c-Jun and
c-Fos--
To address the question of whether AP-1 and Cbfa/Runt
transcription factors could directly interact with each other, we
performed in vitro pull-down assays using purified GST-Cbfa1
fusion proteins and in vitro translated
35S-labeled c-Jun or c-Fos proteins (Fig.
1b, lane
1). Association with c-Jun and c-Fos was observed using
GST-Cbfa1 but not using GST alone (Fig. 1b, lanes
2 and 3). Comparison of the input amount of
in vitro translated c-Jun or c-Fos with the amount of the
proteins pulled down by GST-Cbfa1 suggests a higher affinity of Cbfa1
for c-Jun as for c-Fos. Posttranslational modifications
(e.g. phosphorylation) of c-Jun or c-Fos occurred in the
rabbit reticulocyte lysate (38) and were visible as additional shifted
bands in the SDS-PAGE but did not influence the interaction with
GST-Cbfa1. Vice versa, in vitro translated
35S-labeled Cbfa1 was pulled down by recombinant GST-c-Jun
or GST-c-Fos. In agreement with the initial data, the binding activity
between Cbfa1 and c-Jun was stronger than that between Cbfa1 and c-Fos (data not shown).
Next we determined the domains of Cbfa1 responsible for the interaction
with c-Jun or c-Fos. Therefore, Cbfa1 deletion mutants fused to GST
were generated (Fig. 1a), and purified proteins were incubated with in vitro translated, 35S-labeled
c-Jun or c-Fos proteins in GST pull-down assays. c-Jun could associate
with GST-Cbfa1 deletion mutants lacking either the C- or N-terminal
region or even both, but not with mutants missing the Runt domain of
Cbfa1 (Fig. 1b). Similarly, c-Fos could interact with
GST-Cbfa1 deletion mutants lacking either the C or N terminus or both
(Fig. 1b). These data demonstrate that the Runt domain is
sufficient for interaction with c-Jun or c-Fos. However, additional
regions within the Cbfa1 protein were found to be involved in the
association; c-Fos could be pulled down by GST-Cbfa1
To investigate if c-Jun and c-Fos are able to interact simultaneously
with Cbfa1, we used either full-length GST-Cbfa1 or GST-Cbfa1
Cbfa2 is another Cbfa/Runt family member that has been postulated to
bind to the upstream OSE2 site on the mmp13 promoter (9).
The members of the Cbfa/Runt family are highly conserved within the
Runt domain and share homology in their C-terminal part. To confirm a
possible interaction of c-Jun and c-Fos with Cbfa2, we performed
pull-down assays with recombinant full-length GST-Cbfa2 and deletion
mutants missing either the C-terminal part (GST-Cbfa2 The Leucine Zipper of c-Jun and c-Fos Is Essential for the
Interaction with the Cbfa/Runt Proteins--
In order to define the
region within c-Jun required for the interaction with the Cbfa/Runt
proteins, pull-down assays were performed using wild type and deletion
mutants of c-Jun (Fig. 2, a
and b, lanes 1-6) together with
either GST alone or a GST-Cbfa1
Accordingly, full-length c-Fos and its mutants containing the bZIP
domain (Fig. 2, c and d, lanes
1-4) were pulled down by GST-Cbfa1 The Interaction between Cbfa/Runt Proteins and c-Jun Is Not
Modified by PTH or TPA Treatments in Cultured Cells--
To confirm
that exogenously expressed c-Jun and c-Fos are also able to interact
with recombinant Cbfa/Runt proteins, human embryonic kidney HEK293
cells were transiently transfected with expression vectors encoding
c-Jun or c-Fos tagged with the hemagglutinin epitope (c-Jun-HA or
c-Fos-HA). Cell lysates were first tested for the expression of the
HA-tagged proteins by Western blot (Fig. 3a). Subsequently, crude cell
extracts containing either c-Jun-HA or c-Fos-HA were used in pull-down
experiments together with GST alone, GST-Cbfa1, or GST-Cbfa2. Whereas
c-Jun-HA associated with GST-Cbfa1 and GST-Cbfa2, the pulled down
c-Fos-HA was hardly detectable and only visible after longer exposure
(Fig. 3a, lanes 3 and 5, and data not shown). This might be due to weaker binding affinity between c-Fos and the Cbfa/Runt proteins. The observation is consistent with the results obtained with in vitro translated proteins
(see Fig. 1).
To demonstrate that interaction occurs in cultured cells as well, we
transiently transfected F9 embryonal carcinoma cells with expression
vectors for Myc-tagged Cbfa1 (Cbfa1-Myc) and HA-tagged c-Jun
(c-Jun-HA). Expression of tagged proteins was confirmed by Western blot
using a monoclonal anti-Myc antibody or a polyclonal anti-HA antibody
(Fig. 3b). Interaction was investigated by
co-immunoprecipitation experiments with the anti-Myc antibody followed
by a Western blot with the anti-HA antibody. Co-precipitation of
c-Jun-HA was only detectable in cells co-transfected with Cbfa1-Myc,
confirming interaction between these two proteins in cultured cells.
Transcription from the mmp13 promoter can be induced by TPA
or PTH treatment. TPA activates PKC and the mitogen-activated protein
kinase pathway and enhances phosphorylation of c-Jun, whereas PTH
predominantly activates the PKA signaling pathway. Although c-Jun is
not phosphorylated by PKA, both the OSE2 and the TRE elements in the
promoter region are necessary for PTH-dependent activity of
MMP13 transcription (8, 9). To investigate if posttranslational
modifications of c-Jun downstream of TPA or PTH signaling pathways
would modify the interaction between c-Jun and Cbfa/Runt proteins, GST
pull-down assays were performed. To yield eventually modified c-Jun,
HEK293 cells expressing c-Jun-HA were either treated with TPA or
co-transfected with an expression vector for PKA, leading to
overexpression of the catalytic subunit, which can not be titrated by
the lower amount of endogenous regulatory subunits (39). Neither TPA
treatment triggering phosphorylation of c-Jun nor overexpression of the
catalytic PKA subunit revealed an alteration in the amount of c-Jun-HA
pulled down by GST-Cbfa1 or GST-Cbfa2 (Fig. 3a,
lanes 4 and 9). To use a more in
vivo relevant system to study the role of TPA or PTH on the
Cbfa1/c-Jun interaction, we used the UMR106 rat osteosarcoma cell line.
Treatment of these cells by TPA or PTH revealed an induced expression
of endogenous c-Jun and c-Fos (Fig. 3c, lanes
1-3, and data not shown). In crude extracts from untreated
UMR106 cells or from cells treated by TPA or PTH, c-Jun could be pulled
down by GST-Cbfa1 and GST-Cbfa2 but not by the GST control (Fig.
3c, lanes 4, 7, and
10). TPA treatment of the cells specifically increased the
amount of associated c-Jun that most likely could be due to enhanced
synthesis of the protein (Fig. 3c). Applying similar
conditions, we could see only very weak binding of c-Fos to GST-Cbfa2
appearing after a long exposure of the protein gels (data not shown).
Endogenous Cbfa1 from UMR106 cell lysates could be pulled down by
GST-c-Jun A New OSE2 Element Overlaps the TRE Motif and Affects AP-1
Binding--
Recently, we identified an OSE2 element in close
proximity to the TRE site in the mouse and rat mmp13
promoter (9). Upon close analysis, we could identify an additional
GTGGT sequence partially overlapping with the TRE site (Fig.
4a). We will refer to this
sequence as the OSE2/TRE motif. Both recombinant GST-Cbfa1 and
GST-Cbfa2 were able to bind to the OSE2/TRE-Coll probe (Fig. 4,
b and c, lanes 1-3,
7, and 8). The difference in complex formation observed between GST-Cbfa1 and GST-Cbfa2 is most likely due to slight
differences in protein amounts, which were calculated by Coomassie
staining of the purified proteins after SDS-PAGE. Alternatively, we
cannot exclude the possibility that under these conditions (e.g. in the absence of Cbf
Since Cbfa/Runt proteins can bind c-Jun and c-Fos in vitro
as well as in cultured cells, we wanted to analyze whether this association in combination with the presence of the overlapping OSE2
motif could modify AP-1 binding on the OSE2/TRE site. To investigate
whether Cbfa/Runt proteins are associated with the AP-1 complex on the
OSE2/TRE motif, we preincubated PTH-treated UMR106 cell nuclear
extracts with an anti-Cbfa1 polyclonal antibody (anti-Cbfa1#70)3 or added a nonlabeled OSE2-Coll probe
containing the conserved OSE2 motif of the distal mmp13
promoter. The addition of the competitor OSE2-Coll probe did not result
in a significant change in AP-1-DNA complex formation (data not shown),
suggesting that Cbfa/Runt proteins are able to simultaneously interact
with the OSE2 motif as well as AP-1 members bound to DNA. In contrast,
pretreatment with anti-Cbfa1#70 resulted in decreased complex formation
at the OSE2/TRE-Coll probe, similar to pre-treatment with anti-c-Jun or
anti-c-Fos antibodies (Fig.
5a, upper
panel). Anti-Cbfa1#70 specifically supershifted Cbfa1
associated with DNA (Fig. 5a, middle
panel) but did not interfere with binding of the ubiquitous transcription factor Oct1 to the consensus octamer binding site (Fig.
5a, lower panel). These data
demonstrate complex formation of Cbfa1 and AP-1 members at the proximal
OSE2/TRE motif, which additionally might be stabilized in
vivo by Cbfa/Runt-binding at the distal OSE2 element within the
mmp13 promoter.
To investigate whether the newly identified OSE2 element contributes to
the complex formation at the OSE2/TRE motif, we performed a bandshift
assay with nuclear extracts from PTH-treated UMR106 cells and
OSE2/TRE-Coll or mutOSE2/TRE-Coll probes. We observed a reduced complex
formation if the OSE2 site was mutated (Fig. 5b,
lanes 3 and 4). The reduced bandshift
activity was most likely due to interference with binding of Cbfa1 at
the mutated OSE2 element. However, we could not exclude the possibility
that the introduced mutation affects AP-1 binding at the TRE element
independent of Cbfa1 function. Therefore, we performed a bandshift
analysis with in vitro translated c-Jun and c-Fos in the
absence of Cbfa/Runt proteins. No major differences in AP-1 binding
activity to the OSE2/TRE-Coll or the mutOSE2/TRE-Coll probes was
detected (Fig. 5c, lanes 2 and
4), suggesting that in the absence of Cbfa/Runt proteins,
the heterodimer of c-Jun/c-Fos bound to both oligonucleotides with
similar affinity. This observation excluded the possibility that the
sequence flanking the TRE site at the mmp13 promoter is
required for AP-1 binding in the absence of Cbfa/Runt proteins. Therefore, we propose that Cbfa/Runt proteins bound to the overlapping OSE2 site enhance the stabilization of AP-1-DNA complex formation by
direct protein-protein interaction.
Interaction between Cbfa1 and AP-1 Increases Transcriptional
Activation by AP-1--
To assess the functional relevance of Cbfa1
binding to AP-1 and the OSE2/TRE motif, we analyzed the transcriptional
activity of Cbfa1 on a luciferase reporter gene driven by a
mmp13 promoter fragment (mColl-luc(
Transfection of HEK293 cells with an expression vector for a Cbfa1
mutant lacking the C-terminal region (Cbfa1 The New OSE2 Site Is Involved in PTH-induced Transcriptional
Activation of the mmp13 Promoter--
The TRE motif and a distal OSE2
site located 80 base pairs upstream have been previously shown to be
essential for PTH-dependent induction of MMP13 expression
(8, 9). To study the involvement of the newly identified OSE2 site of
the OSE2/TRE motif in the PTH response, we transiently transfected
UMR106 cells with control mColl-luc(0), wild-type mColl-luc( In the present work, we show for the first time that Cbfa/Runt
proteins interact directly with c-Jun and c-Fos. This interaction is
mediated by the Runt domain and the leucine zipper, respectively. Moreover, the leucine zipper of c-Fos can also interact with a region
in the C terminus of Cbfa1 and Cbfa2. Furthermore, we identified a new
OSE2 site overlapping the TRE site in the proximal promoter region of
the murine and rat mmp13 gene (OSE2/TRE motif). The newly
identified OSE2 site is involved in (i) stabilization of AP-1 DNA
binding on the TRE site, (ii) enhancement of AP-1 transcriptional activity in the presence of Cbfa1, and (iii) PTH-dependent
induction of the mmp13 promoter activity in UMR106
osteoblast-like cells.
Interaction between Cbfa/Runt and c-Jun or c-Fos proteins has been
shown in vitro by GST pull-down experiments. Furthermore, we
could demonstrate in vivo interaction between Cbfa1 and
c-Jun expressed in F9 cells by co-immunoprecipitation. GST pull-down experiments revealed no difference between monomers and dimers of
Jun/Fos proteins in their efficiency to interact with Cbfa1. Therefore,
we assume that only one component of the AP-1 dimer might interact with
the Cbfa/Runt protein. In light of the higher binding affinity between
Cbfa/Runt proteins and c-Jun, one could imagine that c-Jun might be a
better candidate for the interaction with Cbfa/Runt proteins. We
speculate that the residues in the leucine zipper involved in the
dimerization between c-Jun and c-Fos on one hand and in the interaction
with Cbfa/Runt proteins on the other hand would not be identical,
because one would expect a competition in interaction between c-Jun and
c-Fos with Cbfa/Runt.
Analysis of deletion mutants of Cbfa1 or Cbfa2 and c-Jun or c-Fos
revealed direct interaction between the Runt domain and the leucine
zipper, respectively. It has been demonstrated that the Runt domain is
specifically involved in the interaction with the co-factor Cbf Interestingly, superposition of the AML1/Cbf In addition to the requirement of the leucine zipper and the Runt
domain for interaction, we could demonstrate an interaction between the
leucine zipper of c-Fos and the C-terminal region of Cbfa/Runt proteins
(amino acids 467-514 of Cbfa1). Interestingly, the region of Cbfa1
containing residues 443-516 is highly homologous to a domain in the
C-terminus of Cbfa2 responsible for transcriptional repression function
(47, 48). It has been suggested that the repression activity is due to
the recruitment of co-repressors of the transducin-like enhancer of
split (TLE) family, which also interact with the same C-terminal region
of Cbfa1 and Cbfa2 (24-26). Therefore, it is tempting to speculate
that c-Fos could interfere with the interaction between Cbfa/Runt and
TLE proteins, thereby reducing the inhibitory effect exerted by the
TLEs on the transcriptional activity of Cbfa/Runt proteins. A similar
model has been postulated for the competitive binding to Cbfa1
C-terminal end between TLE and HES-1 (hairy/enhancer of split-1), which
can potentiate Cbfa1-mediated transactivation (26). However, a possible
competitive binding between c-Fos and TLE proteins to the C terminus of
Cbfa/Runt transcription factors remains to be demonstrated.
The interaction between Cbfa/Runt and AP-1 transcription factors
suggests that AP-1 transcriptional activity is potentiated by Cbfa/Runt
proteins. In addition, a number of observations suggest that
DNA-binding of Cbfa1 to the newly identified OSE2 site in the proximal
region of the murine mmp13 promoter would contribute to
enhanced transcriptional activity of AP-1. (i) nuclear extract from
PTH-treated UMR106 cells showed reduced DNA binding activity of AP-1 on
the OSE2/TRE motif when the OSE2 site was mutated. However, c-Jun and
c-Fos translated in vitro and used in excess for EMSA
experiments bound to the TRE site in this OSE2/TRE element with the
same efficiency regardless of the OSE2 sequence. (ii) Basal activity of
the proximal mmp13 promoter ( Cbfa1 has an essential role in bone development, since it is
required for osteoblast differentiation and is mutated in patients with cleidocranial dysplasia (57-60), whereas Cbfa2 is required for
hematopoiesis and is a frequent target of chromosomal translocations in
acute leukemias (61). Cbfa/Runt binding sites have been characterized in the promoter of different genes that are specifically expressed in
bone development, such as MMP13, osteocalcin, osteopontin, osteoprotegerin, type I collagen, bone sialoprotein, and Cbfa1 (9, 57, 62-65), or in the promoter of genes involved in hematopoiesis, such as the granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor receptor, myeloperoxidase, neutrophil elastase, and genes of the T-cell receptor subunits (reviewed in Refs. 66 and 67).
Our work demonstrates that Cbfa/Runt proteins promote transcriptional
activity of AP-1 on the promoter of the murine mmp13 gene.
In the future, it will be interesting to address the question of
whether synergistic activation mediated by AP-1 and Cbfa/Runt transcription factors also contributes to tissue-specific expression of
other genes involved in bone development, hematopoiesis, and pathological processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
embryos (9,
15).2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RN1 resulted from deletion of the
BamHI/SmaI fragment from GST-Cbfa1 and religation of blunt ends after fill-in. pGST-Cbfa1
RN2 was obtained by deletion of the BamHI/ApaI fragment from pGST-Cbfa1
followed by fill-in and religation. pGST-Cbfa1
C was cloned upon the
deletion of the HindIII/EcoRI fragment from
pGST-Cbfa1 followed by fill-in and religation. pGST-Cbfa1
RC was
derived upon deletion of the NcoI/EcoRI fragment
from pGST-Cbfa1 followed by fill-in and religation. For the
construction of pGST-Cbfa1
NC or pGST-Cbfa1
N, the
NcoI/HindIII fragment or the
NcoI/EcoRI fragment was isolated from pGST-Cbfa1 and introduced in pBluescript SK(+) (Stratagene) linearized with SmaI/HindIII or SmaI/EcoRI
(giving rise to pBl-Cbfa1
NC or pBl-Cbfa1
N). The
BamHI/HindIII fragment of pBl-Cbfa1
NC or the
BamHI/EcoRI fragment of pBl-Cbfa1
N was
introduced into pGex1 (Amersham Pharmacia Biotech) linearized with
BamHI/HindIII or
BamHI/EcoRI. pGST-Cbfa1
RN3 resulted from a
deletion of the SmaI/EcoRI sequence from the
pGST-Cbfa1
RN2 vector. The XhoI/EcoRI fragment
of Cbfa2 excised from the pBluescript clone was introduced into pGex1
(Amersham Pharmacia Biotech) linearized with SmaI and
EcoRI to generate pGST-Cbfa2. The pGST-Cbfa2
C mutant was
cloned by removal of the SmaI/EcoRI fragment,
which was used to built the pGST-Cbfa2
RN vector by insertion of this
fragment into pGex3 (Amersham Pharmacia Biotech) linearized with
SmaI/EcoRI. All constructs have been sequenced to
confirm that the Cbfa/Runt fragments are in frame with the GST
sequence, and the constructs are schematically shown in Fig.
1a.
6-194,
6-223,
146-221, and
194-223 were described previously (37, 38). For construction of pBAT-c-Jun basic D,
a HindIII/PstI fragment was excised from
pBAT-c-Jun
6-194 and a PstI/XbaI fragment was
excised from pBAT-c-Jun m1 (36). Both fragments were inserted into the
pBAT vector linearized with the restriction enzymes HindIII
and XbaI. The plasmid for in vitro translation of
full-length c-Fos was described previously (36), and the plasmids for
c-Fos-298, c-Fos-218, and c-Fos-171 were kindly provided by R. Müller (69).
C was
generated by excision of the HindIII fragment from the
pcDNA3.1-Cbfa1 and introduced into pcDNA3.1(B) (Invitrogen)
linearized by HindIII.
66/+29) and
mColl-luc(
663/+29) were generated by replacement of the
CAT-SV40poly(A) sequence of the mColl-CAT vector (9) by the
luciferase-SV40poly(A) sequence derived from the pGL3 basic vector
(Promega) with XhoI/BamHI restriction.
mColl-luc(0) was obtained after HindIII restriction of the
mColl-luc(
66/+29) construct followed by a religation that resulted in
the deletion of the promoter region containing the OSE2/TRE site.
mColl-luc(
66/+29)sdm and mColl-luc(
663/+29)sdm plasmids were
generated using the QuickChangeTM site-directed mutagenesis
kit (Stratagene). The following primers were used: TRE/sdm-A
(5'-CACACCCCAAAGTACTGACTCATCACTATC-3') and TRE/sdm-B
(5'-GATAGTGATGAGTCAGTACTTTGGGGTGTG-3'). pRSV-lacZ plasmid was kindly
provided by T. Wirth.
-D-galactopyranoside (Sigma), and lysis
was performed using the PP100 buffer (25 mM Tris/HCl, pH
7.8; 5 mM MgCl2, 100 mM KCl, 10%
glycerol, 0.1% Nonidet P-40, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/µl pepstatin). 250 µl of
glutathione-agarose beads (Sigma) were mixed with the cell lysate and
incubated for 60 min at 4 °C with gentle rotation, and subsequently
the beads were washed three or four times with 1 ml of PP100 buffer.
For electrophoretic mobility shift assay (EMSA) experiments, the
recombinant proteins were eluted from the beads using an elution buffer
(50 mM Tris/HCl, pH 7.8, 0.1% Nonidet P-40, 10% glycerol,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM glutathione). The yield of the purification
and the relative amount of recombinant proteins were estimated by SDS-PAGE and Coomassie staining.
-galactosidase activity was performed
as described (40) using a TD-20/20 Luminometer (Turner Designs).
pRSV-lacZ was co-transfected in all experiments, and
-galactosidase
activity was used to normalize for different transfection efficiencies
in the individual experiments. A minimum of three independent
transfections were performed and S.D. values were calculated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction between purified recombinant
Cbfa/Runt proteins and in vitro translated c-Jun or
c-Fos. a, schematic representation of GST-Cbfa/Runt
constructs used for pull-down analysis. QA, glutamine- and
alanine-rich region; Runt, Runt domain; AD,
activation domain; ID, inhibitory domain. b,
pull-down experiments were performed using full-length GST-Cbfa1 or
deletion mutants and in vitro translated c-Jun and c-Fos
labeled with [35S]methionine. The input amount of
in vitro translated c-Fos or c-Jun proteins is shown in
lane 1. c, pull-down experiments were
performed with GST-Cbfa1 or GST-Cbfa1 C and in vitro
translated c-Jun and c-Fos labeled with [35S]methionine.
Lanes 1 and 2 show in vitro
translated c-Jun and c-Fos proteins. d, pull-down experiment
with full-length GST-Cbfa2 or deletion mutants and in vitro
translated c-Jun and c-Fos labeled with [35S]methionine.
The quality and the quantity of recombinant GST fusion proteins used
for these GST pull-down assays was estimated by Coomassie staining, and
the amount of GST proteins used in the experiment was calculated to be
equal (data not shown).
RN1 and
GST-Cbfa1
RN2, the mutants lacking the Runt domain but containing the
C terminus of Cbfa1 (Fig. 1b, lanes 6 and 9). The interaction between Cbfa1 and c-Fos was
abolished when, in addition to the Runt domain, the extreme C-terminal
end of Cbfa1 was deleted (Fig. 1b, lane
10). Furthermore, deletion of the N terminus of Cbfa1
resulted in decreased binding of c-Jun but enhanced binding of c-Fos,
suggesting that binding affinity for either c-Jun or c-Fos could be
specifically regulated by motifs in this region.
C
fusion proteins together with in vitro translated c-Jun
and/or c-Fos labeled with [35S]methionine for pull-down
assays (Fig. 1c). Consistent with the previous results, both
c-Jun and c-Fos associate with GST-Cbfa1 as well as GST-Cbfa1
C (Fig.
1c). Although c-Jun and c-Fos interact with the Runt domain,
no difference between the pull-down assays with c-Jun and c-Fos alone
or with the combination of the two proteins was detectable, even if the
GST-Cbfa1
C mutant was used, which lacks the additional binding site
for c-Fos. Consequently, we suggest that c-Jun and c-Fos could
associate with Cbfa1 as a monomer or as a Jun-Fos heterodimer and
possibly as a Jun homodimer.
C) or the
N-terminal part together with the Runt domain (GST-Cbfa2
RN). c-Jun
could associate with GST-Cbfa2 and GST-Cbfa2
C (Fig. 1d,
lanes 1-3). Similar to Cbfa1, the deletion of
the Runt domain resulted in a strong reduction of the association
between c-Jun and Cbfa2 (Fig. 1d, lane
4). c-Fos also interacted with GST-Cbfa2 (Fig.
1d, lanes 5 and 6), but
binding activity of Cbfa2 to c-Fos was again weaker in comparison with
c-Jun. In contrast to c-Jun, interaction of c-Fos and Cbfa2 was not
affected by the deletion of the Runt domain but was strongly impaired
upon the removal of the C-terminal region (Fig. 1d,
lanes 7 and 8). In conclusion, these
results demonstrate that c-Jun mainly associates with the Runt domain
of Cbfa/Runt proteins, whereas c-Fos can also interact with a region in
the C terminus of Cbfa1 or Cbfa2.
C fusion protein. Full-length c-Jun
and all mutants containing the complete bZip domain associate with
GST-Cbfa1
C but not with GST alone (Fig. 2b,
lanes 7-16). In contrast, the c-Jun basic D
mutant lacking the leucine zipper domain was not able to interact with GST-Cbfa1
C, suggesting that this structure is essential for the association with the Runt domain of Cbfa1 (Fig. 2b,
lane 18). Consistent with these results, the N
terminus of c-Jun containing only the transactivation domains did not
interact with GST-Cbfa1
C (data not shown).
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Fig. 2.
The leucine zipper of c-Jun and c-Fos is
required for interaction with Cbfa/Runt proteins. a,
schematic representation of the constructs for full-length c-Jun and
deletion mutants used for pull-down experiments with GST-Cbfa1 C.
I, II, and III, N-terminal
transactivation domains;
,
domain; bZIP, basic region
and leucine zipper. b, pull-down experiments were performed
with GST or GST-Cbfa1
C and in vitro translated
full-length c-Jun or deletion mutants labeled with
[35S]methionine. Lanes 1-6 show
the expression level of in vitro translated Jun proteins.
c, schematic representation of the constructs for
full-length c-Fos and deletion mutants used for pull-down experiments
with GST-Cbfa1
C. N-TAD, N-terminal transactivation
domain; C-TAD, C-terminal transactivation domain;
TRD, C-terminal transrepressor domain; bZIP,
basic region and leucine zipper. d, pull-down experiments
were performed with GST or GST-Cbfa1
C and in vitro
translated full-length c-Fos or deletion mutants labeled with
[35S]methionine. Lanes 1-4 show
expression of in vitro translated Fos proteins.
C but not by the GST
control (Fig. 2d, lanes 5-10). As for
c-Jun, the removal of the leucine zipper of c-Fos resulted in the loss
of the interaction (Fig. 2d, lane 12).
Binding to c-Fos-171 was also lost when the full-length GST-Cbfa1 or
GST-Cbfa1
RN2 fusion proteins were used, showing that interaction
with the C-terminal region of Cbfa1 might involve the leucine zipper of
c-Fos (data not shown). In summary, these results demonstrate that
Cbfa-AP-1 protein complex formation is mediated by the leucine
zipper of c-Jun or c-Fos and the Runt domain of Cbfa/Runt proteins.
Additionally, the leucine zipper of c-Fos is required for the
association with the C terminus of Cbfa1.
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Fig. 3.
Interaction of Cbfa/Runt proteins and c-Jun
expressed in mammalian cells. a, pull-down experiments
were performed with GST-Cbfa/Runt proteins and crude extracts prepared
from HEK293 cells transiently transfected with 5 µg of
expression vectors encoding c-Jun-HA or c-Fos-HA. The transfected cells
were treated or not treated with 100 ng/ml TPA for 2 h or
co-transfected with 5 µg of an expression vector encoding the
catalytic subunit of PKA. Expression of exogenous c-Jun-HA or c-Fos-HA
and co-precipitation of these proteins was analyzed by Western blot
using an anti-HA antibody. Equal amounts of GST-Cbfa1 and GST-Cbfa2
used in pull-down assays was also tested by Western blot with an
anti-GST antibody. b, co-immunoprecipitation of Cbfa1-Myc
and c-Jun-HA expressed in F9 cells. F9 cells were transiently
transfected with 10 µg of a Cbfa1-Myc expression vector and 5 µg of
a c-Jun-HA expression vector. Expression of the proteins was analyzed
by Western blot with anti-Myc or anti-HA antibodies (*, nonspecific
signal). Immunoprecipitation (IP) was performed with
100-200 µg of crude extract and 2 µg of the anti-Myc antibody, and
co-precipitation of c-Jun-HA was analyzed by Western blot with the
anti-HA antibody. c, pull-down experiments were performed
with purified GST-Cbfa/Runt proteins and 200-300 µg of crude
extracts from UMR106 cells. UMR106 cells were left untreated or were
treated with 10 8 M rat PTH
fragment (residues 1-34) or 100 ng/ml TPA for 2 h.
Expression of endogenous c-Jun in crude cell extracts or c-Jun
interacting with GST-Cbfa1 or GST-Cbfa2 was analyzed by Western blot
using an anti-c-Jun antibody. Equal amounts of GST-Cbfa/Runt proteins
used were confirmed by Western blot using an anti-GST antibody.
d, pull-down experiment with GST-c-Jun
6-223 and 200-300
µg of cell extracts from untreated or PTH-treated UMR106 cells. The
expression of endogenous Cbfa1 and of the co-precipitated protein was
analyzed by Western blot using an anti-Cbfa1 antibody.
6-223, a c-Jun GST fusion protein lacking its
transcriptional activation domain, but not by GST alone, showing that
this protein-protein interaction was affected neither by the c-Jun
transcriptional activation domain nor by posttranslational
modifications on Cbfa1 induced upon PTH treatment (Fig.
3d).
) GST-Cbfa2 exhibits a lower
binding affinity to the OSE2/TRE element compared with GST-Cbfa1.
Mutations within the putative OSE2 site (mutOSE2/TRE-Coll and
mutOSE2/mutTRE-Coll) interfered with binding of GST-Cbfa/Runt to the
OSE2/TRE motif (Fig. 4c, lanes 4-6,
9, and 10). Upon mutation of the TRE site (OSE2/mutTRE), the GST-Cbfa/Runt proteins were still able to bind the
OSE2 site and to shift the probe (Fig. 4c, lanes
11 and 12). As a control, the recombinant
GST-Cbfa/Runt proteins were preincubated with an anti-GST
antibody, or unlabeled OSE2-Coll probe containing the distal
OSE2 element of the mmp13 promoter was added in excess. Both
resulted in impaired binding of GST-Cbfa/Runt proteins to this OSE2/TRE
site (data not shown).
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Fig. 4.
Identification of a new OSE2 site overlapping
with the conserved TRE motif in the proximal mmp13
promoter. Shown are schematic representations of the
OSE2/TRE motif in the proximal mmp13 promoter region of the
mouse and rat genes (a) and of the mutations within the
OSE2/TRE motif used for EMSA experiments (b). c,
EMSA experiments were performed with equal amounts of purified GST or
GST-Cbfa/Runt fusion proteins and 32P-labeled wild type or
mutant OSE2/TRE-Coll probes.
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Fig. 5.
Binding of the Cbfa/Runt proteins to the AP-1
complex and the newly identified OSE2 site in the OSE2/TRE motif is
required for efficient AP-1-DNA complex formation at the TRE site.
EMSA experiments were performed with 2 µg of nuclear extracts from
PTH-treated UMR106 cells, which were preincubated with anti-c-Jun,
anti-c-Fos, or anti-Cbfa1 antibodies (a), 2 µg of nuclear
extracts from untreated ( PTH) or PTH-treated
(+PTH) UMR106 cells (b), or in vitro
translated c-Jun and c-Fos proteins together with
32P-labeled wild type (OSE2/TRE-Coll) or mutant
(mutOSE2/TRE-Coll) probes (c). To confirm the specificity of
the antibodies used in a, supershift experiments with a
32P-labeled OSE2-Coll probe spanning the distal OSE2
element of the mmp13 promoter or a
32P-labeled Oct probe containing a consensus octamer
binding site were done.
66/+29)) containing the
OSE2/TRE motif. HEK293 cells were transiently transfected with an
expression vector encoding Cbfa1 together with mColl-luc(
66/+29).
Expression levels of endogenous c-Jun and c-Fos were not influenced by
the presence of the Cbfa1-Myc protein (Fig.
6b), showing that the effects
on luciferase activity were due to Cbfa1 expression and not due to
changes in c-Jun or c-Fos levels. Increasing amounts of exogenous Cbfa1
resulted in a concomitant increase of mmp13 promoter
transcriptional activity (data not shown). A 3-fold stimulation of
transcriptional activity was measured by transfection of 10 µg of
Cbfa1 expression vector together with mColl-luc(
66/+29). In contrast,
the control luciferase vector (mColl-luc(0)) was not activated by Cbfa1
(Fig. 6, c and d). Upon mutation of the OSE2 site
in the
66/+29 promoter, the basal activity was not changed compared
with the wild-type
66/+29 promoter (Fig. 6c). However, in
the presence of Cbfa1, the inducibility is reduced to 30-50% (Fig.
6c). Similar results were obtained upon transient
transfection of GM637 human fibroblasts (data not shown). Therefore, we
conclude that Cbfa1 can induce transcription of the
66/+29
mmp13 promoter fragment through the newly identified OSE2
site. Nevertheless, when the OSE2 site is mutated, Cbfa1 can still
interact with AP-1 and trigger partial stimulation of the promoter.
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Fig. 6.
Cbfa1 enhances AP-1-mediated transactivation
in HEK293 cells. a, schematic representation of the
luciferase reporter plasmids regulated by the 66/+29 mmp13
promoter fragment. mColl-luc(0) represents a control vector lacking the
mmp13 promoter fragment. mColl-luc(
66/+29) contains the
66/+29 promoter fragment of the murine mmp13 gene with the
proximal OSE2/TRE element in front of the luciferase reporter gene.
mColl-luc(
66/+29)sdm was generated by mutating the OSE2 site in the
mColl-luc(
66/+29) vector. b, HEK293 cells were transiently
transfected with 10 µg of an expression vector for Cbfa1-Myc.
Expression of exogenous Cbfa1-Myc or endogenous c-Jun and c-Fos
proteins were detected by Western blot analysis. Similar amounts of
endogenous c-Myc protein detected with the anti-Myc antibody indicate
similar quality and quantity of loaded protein extracts. c,
Cbfa1 was co-transfected with 5 µg of the indicated luciferase
vectors. Relative luciferase activity obtained with mColl-luc(0)
reporter construct alone was brought to the value of 1. d,
10 µg of Cbfa1 or Cbfa1
C expression vectors were co-transfected
with 5 µg of mColl-luc(
66/+29). Relative luciferase activity
obtained with mColl-luc(
66/+29) reporter construct alone was brought
to a value of 1. For c and d, a minimum of three
independent transfections were performed, and S.D. values were
calculated.
C) stimulated luciferase
expression to a similar extent as the full-length Cbfa1 (Fig.
6d). This observation suggests that the C-terminal region of
Cbfa1 containing activation and repression domains is not involved in
transcriptional activation of the
66/+29 promoter. Although we cannot
exclude the involvement of the remaining N-terminal region of Cbfa1, it
is more feasible that the interaction between Cbfa1 and AP-1 is
responsible for enhanced transcription due to stabilization of the
complex formation on DNA.
663/+29),
or mutant mColl-luc(
663/+29)sdm reporter constructs (Fig.
7a) and measured reporter gene
expression in untreated and PTH-treated cells. In agreement with the
transient transfections in fibroblasts, mutation of the OSE2 site in
the OSE2/TRE motif did not influence basal transcriptional activity (Fig. 7b). PTH treatment of UMR106 cells transfected with
mColl-luc(
663/+29) resulted in a 2-fold induced luciferase activity
compared with the basal level (Fig. 7b). In contrast,
PTH-induced luciferase expression was significantly reduced if the OSE2
site in the OSE2/TRE motif was mutated, confirming that this site is
crucial for full activation of the mmp13 promoter after PTH
treatment.
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Fig. 7.
The new OSE2 site is involved in
PTH-dependent activation of the mmp13
promoter in UMR106 cells. a, schematic
representation of luciferase reporter constructs. mColl-luc( 663/+29)
contains the
663/+29 promoter fragment of the murine mmp13
gene with the distal OSE2 sites and the proximal OSE2/TRE element in
front of the luciferase reporter gene. mColl-luc(
663/+29)sdm was
generated by mutating the proximal OSE2 site in the OSE2/TRE element of
the mColl-luc(
663/+29) vector. b, 5 µg of the indicated
luciferase reporter constructs were transiently transfected in UMR106
cells. 24 h after transfection, cells were treated with
10
8 M PTH for 2 h. Relative
luciferase activity obtained with mColl-luc(
663/+29) reporter
construct or mColl-luc(
663/+29)sdm reporter construct in the absence
of PTH treatment was brought to the value of 1. The inset
graph shows similar basal activity of mColl-luc(
663/+29)
and mColl-luc(
663/+29)sdm in transient transfected UMR106 cells in
the absence of PTH.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(44)
and other transcription factors, like Ets-1 (30), PU.1 (35), and C/EBP
proteins (31, 34, 35). For example, the Runt domain of Cbfa2 interacts
with PU.1 and C/EBP
in the synergistic activation of the human
macrophage colony-stimulating factor receptor promoter in myeloid cell
lines (31, 35). Moreover, synergistic transcriptional activity due to
direct interaction between the Runt domain of Cbfa1 and C/EBP
has
been shown in osteoblasts (34).
coordinates (Protein
Data Bank code 1e50) onto the NFAT-AP-1-DNA complex (Protein Data Bank
code 1a02) suggests that the Jun/Fos interaction site on the Runt
domain overlaps with that of Cbf
(44-46).4 Thus, it will be
interesting to investigate whether Cbf
and c-Jun or c-Fos can bind
simultaneously or in a competitive fashion to the Runt domain. Since
there exists evidence that Cbf
is constitutively present in the
Cbfa1 complex bound on the distal OSE2 sites independently of PTH
treatment (8), we propose that Cbf
, Cbfa/Runt, and AP-1 proteins can
interact simultaneously. This idea was further supported by the
observation that we were able to co-precipitate in vitro
translated c-Jun, c-Fos, and Cbf
using GST-Cbfa1. Interestingly, the
affinity between Cbfa1 and the AP-1 members appeared to be enhanced in
the presence of the co-factor (data not shown).
66/+29), harboring the
OSE2/TRE element, was identical in fibroblasts regardless of the
presence or absence of a functional OSE2 site. Nevertheless, transactivation of this promoter by co-transfection of a Cbfa1 expression vector was 30-50% higher when the OSE2 site was intact. (iii) Mutation of the newly identified OSE2 site inhibits
PTH-dependent induction of the mmp13 promoter by
more than 50% in transiently transfected UMR106 cells. Therefore,
PTH-dependent induction of the mmp13 promoter
does not only require the previously characterized distal OSE2 and
proximal TRE sites (8, 9) but seems to require also the binding of
Cbfa1 to the proximal OSE2/TRE site. It has been shown that the level
of Cbfa1 protein in osteoblasts is not affected by PTH treatment and
that the distal OSE2 sites are constitutively bound by Cbfa/Runt
proteins (8, 9, 18). These observations do not exclude the possibility
of a synergistic binding of Cbfa/Runt and AP-1 transcription factors at
the proximal OSE2/TRE element. Numerous examples have been reported
describing direct interaction and cooperation of AP-1 with other
transcription factors, like Ets (49-51), MyoD, (52), Smads (53, 54),
Rb (55), and NF-
B (56). Thus, a model could be proposed, in which
Cbfa/Runt and AP-1 transcription factors synergistically bind to the
proximal OSE2/TRE motif. Protein-protein interaction as well as DNA
binding of Cbfa/Runt proteins to the OSE2/TRE motif could be
responsible for the observed synergism. In addition to
PTH-dependent synergy between the OSE2 and TRE sites at the
composite OSE2/TRE element in the proximal promoter region, the distal
OSE2 sites and the TRE site in the OSE2/TRE element may also contribute
to functional synergism. At present, both possibilities seem to be
important for PTH-dependent expression of MMP13 in
osteoblasts, since mutation of the proximal OSE2 site did not result in
the complete loss of PTH-induced transcriptional activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to D. Sandmaier for excellent technical assistance. We thank R. Müller for plasmids encoding c-Fos deletion mutants, D. Bohmann for c-Fos-HA and c-Jun-HA expression vectors, and W. Schmid for the PKA expression vector. We thank S. Andrecht for help with the figures and M. Schorpp-Kistner, B. Hartenstein, and A. Warren for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by European Community Marie Curie Research Training Grant ERB4001GT962949 and grants from the Training and Mobility of Researchers (ERB-FMRX-CT96-0044) and Biomed-2 (BMH4-CT98-3505) programs of the European Community.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Laboratoire Arago (UMR 7628 du CNRS), BP44, 66651 Banyuls sur Mer, Cedex, France.
§ To whom correspondence should be addressed. Tel.: 49-6221-42-4570; Fax: 49-6221-42-4554; E-mail: p.angel@dkfz.de.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M010601200
2 J. Tuckermann, S. Mundlos, and P. Angel, unpublished data.
3 D. Porte and P. Angel, unpublished data.
4 A. Warren, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTH, parathyroid hormone; PKA, protein kinase A; OSE2, osteoblast-specific element 2; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; TPA, 12-O-tetradecanoylphorbol-13-acetate; HA, hemagglutinin; TLE, transducin-like enhancer of split; HA, hemagglutinin.
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REFERENCES |
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