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INTRODUCTION |
Osteoblasts are cells of mesenchymal origin required to form the
skeleton during development and to maintain bone mass thereafter. Their
unique ability to deposit a matrix that can eventually mineralize explains that abnormal osteoblast differentiation and/or osteoblast function have dramatic consequences. Indeed, arrest of osteoblast differentiation often leads to lethal skeletal dysplasia whereas impaired osteoblast activity causes bone fragility with risk of fracture, a hallmark of osteoporosis, the most common degenerative disease in the western hemisphere (1-3). This pivotal role of the
osteoblasts in bone biology justifies the importance of identifying molecular regulators of osteoblast differentiation and function. However as of today only a few factors playing such a role have been
identified, and no genetic cascade can yet be drawn showing how the
differentiation of a mesenchymal cell to a fully functional osteoblast
is controlled. With the long-term goal to delineate such cascade we
have embarked on studying the regulation of expression of Runx2/Cbfa1
(thereafter Cbfa1), which is to date the best characterized regulator
of osteogenesis (4).
Cbfa1 is one of the three mammalian members of the runt family of
transcription factors (5). Gene deletion experiments as well as
overexpression studies have shown that Cbfa1 is necessary and
sufficient for osteoblast differentiation (4). The finding that humans
or mice heterozygous for Cbfa1 inactivation develop an
identical phenotype termed Cleidocranial dysplasia (3, 4) established
the remarkable conservation of Cbfa1 function during mammalian
evolution. At the molecular level, Cbfa1 has been shown to directly
regulate the expression of major osteoblastic marker genes such as the
type I collagen genes, osteopontin, bone sialoprotein, and osteocalcin
(6, 7). These findings, as well as the fact that impairing Cbfa1
function postnatally leads to osteopenia (8), indicate that it acts
beyond development as a major regulator of bone mass via its control of
the rate of bone matrix deposition by differentiated osteoblasts. This
notion was recently extended to the resorption side of bone remodeling
(9, 10). Indeed, overexpression of Cbfa1 in osteoblasts was
shown to increase their ability to sustain osteoclastogenesis and
therefore to regulate, although indirectly, bone resorption as well as
bone formation.
The Cbfa1 gene potentially gives rise to two major
transcripts, TypeII/Cbfa1p57/Osf2/MASNSL and
TypeI/Cbfa1p56/MRIPVD (11-13). These transcripts differ by the
promoter they originate from (P1 or P2, respectively), by their
5'-untranslated region and N-terminal coding sequence and by their
pattern of expression in cell lines (11, 12). However, by in
situ hybridization on mouse embryos, the same pattern of
expression is observed when using a probe specific to the Type II form
and a probe common to the two Cbfa1 transcripts, indicating the
existence of a predominant pattern of expression in vivo
(6). Throughout development and in adult tissues, Cbfa1
expression appears to be strictly restricted to cells of the
osteochondrogenic lineage, although its level of expression varies
according their stage of differentiation (14). Cbfa1 is
initially expressed in all cells of the mesenchymal condensations prefiguring each of the future skeletal elements, beginning at 10.5 days post-coitum in mouse (4). When chondrocyte differentiation proceeds in these structures, Cbfa1 expression becomes
restricted to pre-hypertrophic chondrocytes and to the osteoblast
progenitor cells present in the bone collar (14). Postnatally,
osteoblasts will become the only cell type expressing Cbfa1
(6, 14).
Considering that Cbfa1 expression is restricted to the
skeletal cell lineage and that Cbfa1 is an inducer of osteoblast
differentiation, it is likely that the factors controlling its
expression would trigger the early steps of skeletal cell commitment
and/or those of osteoblast differentiation. We therefore embarked in
the analysis of the mouse Cbfa1 promoter with the long-term
goal to identify cis-acting elements and trans-acting factors that
induce and regulate Cbfa1 expression specifically in the
osteoblastic lineage. In this study we report that such a cis-acting
element is present at 
415/375 in the mouse Cbfa1 P1
promoter. Despite the fact that this region binds AP1 and NF1 nuclear
activities, two classes of transcription factors whose members are
broadly expressed, this element acts as an enhancer only in
osteoblastic cells. Our analysis therefore shows that an
osteoblast-specific activity can be achieved through a specific
interaction between broadly expressed factors.
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EXPERIMENTAL PROCEDURES |
Cell Culture and DNA Transfection--
UMR106 and ROS17/2.8
cells were cultured in Dulbecco's modified Eagle's medium/F12
(DMEM/F12; Invitrogen)/10% fetal bovine serum (Invitrogen). NIH3T3,
C2C12, 3T3L1, and COS cells were cultured in DMEM/10% fetal bovine
serum. F9 cells were cultured in Eagle's minimal essential medium
(EMEM; Invitrogen)/10% fetal bovine serum. Transfections were
performed using the calcium phosphate co-precipitation method as
previously described (15) or using FuGENE 6 (Roche Molecular
Biochemicals) according to the manufacturer's instructions. An equal
amount of pSV/
-galactosidase plasmid was added to each DNA mix to
monitor the efficiency of transfection (15). Luciferase and
-galactosidase activities were assayed as previously described (15).
All transfections were repeated at least 6 times with two different
preparations of plasmid DNA.
DNA Constructions--
All promoter fragments were cloned in the
pGL2basic promoter-less luciferase expression vector
(Clontech). The 2800/+111 mouse Cbfa1
promoter region was cloned as an EcoRI/PstI
fragment excised from a mouse Sv/ev genomic clone
(Clontech). This initial fragment was used to
generate all deletion mutants using as 5'-ends its internal
NdeI (p2164-luc), XbaI (p1389-luc),
BglII (p976-luc), SacI (p780-luc),
ScaI (p89-luc) restriction sites and as 3'-end its terminal
PstI site. Footprint analyses were performed using as
templates the BglII/HindIII (976/734),
HindIII/StuI (733/342), and
StuI/ScaI (341/89) fragments cloned in
pBluescript. Multimers (five copies) of the wild-type or mutant CE1
oligonucleotides (Table I) were cloned in front of the 46/+10
adenovirus type 2 major late promoter region itself fused to the
luciferase reporter gene (pAMLP-luc). The FosB, Fra1, Fra2, JunB, and
JunD expression vectors were a generous gift from Dr. G. Karsenty
(Baylor College of Medicine, Houston, Texas). pCMV-
FosB was
generated by cloning a PCR fragment amplified with the following
primers: 5'-CAGAGAGCGGAACAAGCTGGCTGC-3' and
5'-GCTCTAGATCACCTCGGCCAGCGGGC-3' into pCMV5. NF1 expression vectors were a kind gift from Dr. J. Rosen (Baylor College of Medicine,
Houston, TX).
Northern Blot, RT-PCR, and Western Blot
Analyses--
Extractions of total RNA and Northern blot experiments
were performed using standard protocols (16). Membranes were
successively hybridized with a Cbfa1 (6) and a
glyceraldehyde-3-phosphate dehydrogenase probe used as a control for
equivalence of RNA loading. For RT-PCR analysis, DNase I-treated total
RNAs were first reverse transcribed using oligo-dT primers. PCR
amplifications were then carried out according to a previously
described protocol (17) using the following NF1-specific
degenerated primers: Deg1
(5'-TTCCGGATGA(A/G)TT(C/T)CA(C/T)CITT(C/T)AT(C/T)GA(A/G)GC-3') and
Deg2 (5'-AATCGAT(A/G)TG(A/ G)TG(C/G)GGCTGIA(C/T)(A/G)CAIAG-3') where
I is inosine. Simultaneous amplifications of Hprt exon 2 were used as internal control of the quality and equal loading of
cDNAs (Hprt primers were: 5'-GTTGAGAGATCATCTCCACC-3' and
5'-AGCGATGATGAACCAGGTTA-3'). SDS-PAGE and immunoblotting were
performed according to standard procedures (16) using commercial
antibodies (Santa Cruz Biotechnology) and the ECL detection kit
(Amersham Biosciences).
Nuclear Extracts and DNA Binding Assays--
Buffers for the
preparation of nuclear extracts contained the following protease
inhibitors: 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and pepstatin. Nuclear extracts were prepared as previously described (15). DNase I
footprint assays were performed as previously described (15) except
that 50 µg of nuclear extracts and 0.4 µg of
poly(dI·dC)·poly(dI·dC) were used per reaction. For
electromobility shift assays
(EMSAs),1 double-stranded
oligonucleotides containing the wild type or mutant CE1 sites (Table I)
were labeled with [
-32P]ATP and the T4 polynucleotide
kinase, as described (15). Each reaction contained 5 fmoles of probe
and 3 µg of nuclear extracts in 10 µl of a buffer containing 35%
glycerol, 20 mM HEPES pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 8 mM MgCl2, 2 mM dithiothreitol, 4 mM spermidine, 20 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
of leupeptin and pepstatin. Poly(dI·dC)·poly(dI·dC) (1 µg) and
single-stranded DNA (500 fmoles) were used as nonspecific competitors.
For supershift experiments antibodies (Santa Cruz Biotechnology) were
incubated with 8 µg of nuclear extracts for 30 min at 4 °C prior
to addition of the probe. Reactions were left for 20 min at room
temperature then electrophoresed on a 5% polyacrylamide gel in 0.25×
Tris borate/EDTA at 160 V for 2 h. Gels were dried and autoradiographed.
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RESULTS |
A 976/+111 Cbfa1 Promoter Fragment Is Active Only in
Osteoblastic Cells--
Cbfa1 is encoded by a single gene whose
expression is restricted to cells of the osteochondrogenic lineage
in vivo (4). To analyze the mechanisms controlling
Cbfa1 promoter cell-specificity we therefore relied on
comparing its activity in osteoblastic and in non-osteoblastic cells.
ROS17/2.8 (18) and UMR106 cells (19) were initially chosen as
osteoblastic references because they express high levels of
Cbfa1 (Fig. 1A).
Both cell lines derive from rat osteosarcomas and represent a mature
osteoblastic phenotype, expressing the PTH/PTHrP receptor and
Osteocalcin (15, 18, 19). UMR106 is however expressing Cbfa1
at a higher level than ROS17/2.8 (Fig. 1A). As
non-osteoblastic references we used cell lines that are, like
osteoblasts, of mesenchymal origin such as fibroblasts (NIH3T3),
preadipocytes (3T3L1), and myoblasts (C2C12), and cells of different
embryonic origin such as F9 teratocarcinoma and COS monkey kidney
cells. Cbfa1 expression was not detectable in any of these
cell lines (Fig. 1A).
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Fig. 1.
Osteoblast-specific activity of the
976/+111 mouse Cbfa1 promoter region in DNA
transfection experiments. A, Northern blot analysis (15 µg of total RNA per lane) of Cbfa1 expression in cell
lines (upper panel). The blot was re-probed with a
glyceraldehyde-3-phosphate dehydrogenase probe to account for RNA
loading and transfer efficiency (lower panel). B,
osteoblast-enriched activity of the 2.8-kb proximal region of the mouse
Cbfa1 promoter (p2800-luc). C, activity of
5'-deletion mutants of the p2800-luc construct transfected in UMR106
osteoblastic cells. A schematic representation of each reporter
construct is shown on the left. D,
osteoblast-specific activity of the 976-bp proximal region of the mouse
Cbfa1 promoter (p976-luc). Data represent ratios of
luciferase to -galactosidase activities (Rlu) and values are means
of 8 to 12 independent transfection experiments, with error bars
representing the S.E. of the mean.
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Using the first Cbfa1 exon as a probe we screened a mouse
genomic library and isolated a clone containing 2.8 kb lying upstream of the Cbfa1 start site of transcription (20). We first
performed DNA transfection experiments using a reporter construct
harboring this region (2800/+111) fused to the luciferase gene
(p2800-luc). This construct was significantly more active in
osteoblastic cells than in any non-osteoblastic mesenchyme-derived
cells, and it was virtually inactive in F9 and COS cells (Fig.
1B). This result suggested that the 2800/+111 region
contained regulatory element(s) favoring its expression in osteoblastic
cells. To localize them more precisely, activities of 5'-deletion
mutants of this 2.8-kb promoter fragment were compared in UMR106
osteoblastic cells. Deletions from 2800 to 976 did not alter
significantly the level of activity (Fig. 1C). In contrast,
deletion from 976 to 89 decreased by 6-fold the level of luciferase
expression, indicating that critical cis-acting elements are present in
this region (Fig. 1C). To determine whether such elements
could be cell-specific we compared the activity of p976-luc in
osteoblastic and non-osteoblastic cell lines. As shown in Fig.
1D, p976-luc displayed a tight osteoblast-specific activity.
Consistent with the difference of Cbfa1 expression in these
cells (Fig. 1A), p976-luc was more active in UMR106 than in
ROS17/2.8 cells (Fig. 1D). We therefore chose the UMR106
cell line as reference in all subsequent experiments. These results thus established the osteoblast-specificity of the 976/+111
Cbfa1 promoter fragment in DNA transfection and indicated
that at least one osteoblast-specific regulatory element lied between
976 and 89.
An Osteoblast-specific Binding Site, CE1, Is Present in the Cbfa1
Promoter between 415 and 375--
DNase I footprint assays were
performed to define the sites of binding of osteoblast-specific nuclear
proteins within the 976/89 area, searching for areas differentially
protected by nuclear extracts from UMR106 osteoblastic cells compared
with non-osteoblastic mesenchyme-derived NIH3T3 and 3T3L1 cells or F9
cells. Analysis of the 976/734 region did not show any protected area (data not shown). In agreement with such absence of cis-acting elements, a reporter construct missing this 976/734 region
displayed a similar activity that p976-luc in UMR106 cells (p976-luc,
98260 ± 12782 Rlu; p780-luc, 106752 ± 12116 Rlu).
Analysis of the 733/342 region delineated three protected regions.
First, on its lower DNA strand nuclear extracts from NIH3T3 fibroblasts
protected an area between 470 and 415 (Fig. 2A, white box).
This area was not protected by any other non-osteoblastic nuclear
extracts and more importantly it was not protected by any extracts on
the opposite strand (Fig. 2, A-C). For these two reasons
this region was not further analyzed. Two other regions, we named Cbfa1
element 1 (CE1) and Cbfa1 element 2 (CE2), were also found footprinted
(Fig. 2A, gray boxes). Independently of the DNA
strand analyzed CE1 laid between 375 and 415 and was protected by
all three mesenchymal cell-derived but not by F9 cells nuclear extracts
(Fig. 2, A-C). However, on both strands CE1 pattern of
protection was different between UMR106 extracts and NIH3T3 or 3T3L1
extracts, suggesting that this region bound different
nuclear proteins in osteoblastic and non-osteoblastic cells. CE2, which lies between 558 and 470 on the lower DNA strand,
was protected in a similar fashion by nuclear extracts from the three
mesenchymal cell-derived but was not protected by F9 cells nuclear
extracts (Fig. 2A). This suggested that this region was
binding a factor widely expressed in mesenchymal cells rather than an
osteoblast-specific factor. For this reason and because no protected
area could be observed when we analyzed the opposite strand (Fig.
2C) this region was thus not further analyzed.
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Fig. 2.
Identification of a cell-specific protected
region (CE1) by DNase I footprint assay of the 733/342
Cbfa1 promoter region. 32P-labeled
lower strand (A) or upper strand (B and
C) of the 733/342 Cbfa1 promoter region were
incubated in the absence (Naked) or presence of nuclear
extracts (UMR106, NIH3T3, 3T3L1, F9), digested with DNase I and
separated on a 6% sequencing gel. Boxes indicate protected
regions. Arrows indicate DNase I hypersensitive sites. G+A lanes
show Maxam and Gilbert corresponding sequencing reactions.
D, summary of the DNaseI footprinting results. A
single 40-bp protected region, CE1, was identified
(brackets). It was protected equally on both strands of
DNA.
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Lastly, when the 341/89 region was analyzed no cell-specific
protection could be observed (data not shown). Taken together, the
results of the footprinting analysis of the 976/89 region of the
mouse Cbfa1 promoter indicate that CE1, a 40-bp area lying between 415 and 375 (Fig. 2D), is the only region
binding distinct factors in osteoblastic and non-osteoblastic cells.
CE1 Binds NF1-like and AP1-like Nuclear Activities--
A
double-stranded oligonucleotide covering CE1 (Fig.
3A and Table
I) was used as a probe in EMSA using
UMR106 nuclear extracts as a source of proteins. Four DNA-protein
complexes were observed (Fig. 3B, lane 1).
Computer analysis of CE1 showed that it contains two core binding
sites: a 80% conserved NF1 binding site (site a,
410/396) and a 100% AP1 consensus sequence (site b,
388/382) (Fig. 3A). Based on this observation we
designed a series of mutant CE1 oligonucleotides harboring respectively
an insertional mutation known to impair NF1 binding (Insa) (21), a
mutation abolishing AP1 binding (mb) (22) or both mutations (Insa/mb)
(Fig. 3A and Table I), and tested them in EMSA. The Insa
mutation dramatically reduced (although did not abolish) formation of
complexes *1 and *4 while it did not affect formation of complexes *2
and *3 (Fig. 3B, lane 2). This result suggested
that the factors forming *2 and *3 do not bind to the NF1 site. In
contrast, the mb mutation of the AP1 site led to the sole formation of
complex 4 (Fig. 3B, lane 3), suggesting that this
complex does not bind to the AP1 element. Interestingly, both single
mutations abolished the presence of complex *1 (Fig. 3B,
lanes 2 and 3), suggesting that it is formed by
simultaneous binding of NF1 and AP1 factors. No factors bound to CE1
independently of the NF1 and AP1 sites since simultaneous mutation of
these two sites abolished the binding of all complexes (Fig.
3B, lane 4).
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Fig. 3.
CE1 binds NF1- and AP1-related nuclear
activities. A, presence of a NF1 binding site
(bracket, site a) and an AP1 binding site
(bracket, site b) in the CE1 sequence. Core
binding sequences for each site are boxed. Stars
indicate nucleotides conserved compared with the respective consensus
core sequences. Schemes represent mutant oligonucleotides;
gray boxes represent wild type sites, crossed
boxes represent mutant sites. B, electromobility shift
assays (EMSA). Labeled double-stranded wild type (lane 1)
and mutant (lanes 2-4) CE1 oligonucleotides were used as
probes in presence of UMR106 nuclear extracts. The four complexes
formed are marked as *1, *2, *3, and *4. C, competition
experiments in EMSA. Labeled double-stranded wild type CE1
oligonucleotide was used as probe in presence of UMR106 nuclear
extracts along with indicated molar excess of cold oligonucleotide
competitors containing wild type (lanes 2,
3, 6, 7) or mutant (lanes
4, 5, 8, 9) consensus binding
sites for NF1 (lanes 2-5) or AP1 factors (lanes
6-9). D, EMSA. Equivalent amounts (3 µg) of nuclear
extracts from osteoblastic cells (UMR106: lanes 1,
4, 7) or non-osteoblastic mesenchymal cells (NIH3T3:
lanes 2, 5, 8 and 3T3L1: lanes
3, 6, 9) were compared for binding to
labeled wild type CE1 (lanes 1-3), mutant Insa (lanes
4-6), and mutant mb (lanes 7-9) probes. The four
osteoblastic complexes are indicated as in B, the two
non-osteoblastic AP1-related complexes are indicated by black
dots and marked as *2/3.
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We next performed competition experiments in EMSA. Labeled wild type
CE1 oligonucleotide was used as probe in the presence of 0-, 10-, or
100-fold molar excess of cold oligonucleotides containing consensus
wild type or mutant NF1 and AP1 binding sites (Table I). NF1 wild type
consensus oligonucleotide competed away both *1 and *4 complexes (Fig.
3C, lanes 2 and 3). When mutated using
the Insa insertion (Table I) this same core sequence did not compete
binding at 10-fold molar excess, but did reduce formation of complexes
*1 and *4 at higher concentrations (Fig. 3C, lanes 4 and 5). This indicates that the Insa mutant site can
still bind NF1, although with a lower affinity than the wild type site.
This result is consistent with the direct binding experiment presented in Fig. 3B showing that formation of complexes *1 and *4 is
reduced but not abolished by the Insa mutation (Fig. 3B,
lane 2). When we used as cold competitor an AP1 consensus
site, only complex *4 formed (Fig. 3C, lanes 6 and 7). In contrast, introducing the mb mutation in this
sequence (Table I) did not impair the formation of any complex, even at
high concentrations (Fig. 3C, lanes 8 and
9), indicating that the mb mutation completely abolishes the ability of AP1 factors to bind to CE1. Together, the results of our
direct and competition EMSA experiments indicate that complex *2 and *3
are formed by an AP1 binding activity while complex *4 binds a
NF1-related nuclear protein. Complex *1 appears to contain both types
of factors, suggesting that none of them inhibits the ability of the
other to bind to its recognition site within CE1.
To determine whether any of the complexes binding to CE1 was
osteoblastic-specific, we compared EMSA patterns obtained using nuclear
extracts from UMR106, NIH3T3, and 3T3L1 cells. Using wild type CE1 the
major difference we could observe was the absence of AP1 binding
activities *2 and *3 in non-osteoblastic cell lines (Fig.
3D, lanes 1-3). Complexes of intermediate
mobility were instead present (Fig. 3D, lanes 2 and 3, black dots). Their formation was abolished
by the mb mutation indicating that, like osteoblastic complexes *2 and
*3, they contained AP1-binding activities (Fig. 3D,
lanes 8 and 9). We thus named them complexes *2/3
to reflect their intermediary mobility compared with complexes *2 and
*3. There was a difference of migration between NIH3T3 and 3T3L1 *2/3 complexes suggesting that different AP1 activities bound to CE1 in
these cells. We also observed that formation of complex *4, that binds
a NF1 nuclear activity, migrated slightly faster with osteoblastic
nuclear extracts than with non-osteoblastic extracts (Fig.
3D, lanes 7-9), suggesting that CE1 may bind
different NF1 isoforms in these two types of cells.
CE1 Functions as an Osteoblast-specific Enhancer--
To
investigate the function of CE1 and of the two putative binding sites
it contains we performed a series of DNA transfection experiments.
Multimers of either the wild type, Insa mutant, mb mutant, or Insa/mb
double mutant CE1 oligonucleotides were cloned upstream of an inactive
heterologous promoter fused to the luciferase reporter gene (Fig.
4A). These different
constructs were transfected in UMR106 cells, NIH3T3 cells, and 3T3L1
cells. Five copies of wild type CE1 (p5CE1-luc) increased the activity
of an inactive heterologous promoter 44-fold in UMR106 cells compared
with less than 4-fold in non-osteoblastic cells (Fig. 4B).
This stimulatory effect was independent of the orientation of the
oligonucleotides, demonstrating that CE1 functions as a true enhancer.
When we introduced an inactivating mutation in the AP1 site (mb) or
when we mutated simultaneously the NF1 and AP1 sites (Insa/mb), CE1
activity was totally abolished in all 3 cell types (Fig. 4,
C-E). In contrast, mutation of the NF1 site alone (Insa)
did not significantly decrease CE1 activity in osteoblasts (Fig.
4C), indicating that the NF1 nuclear protein(s) binding to
CE1 in these cells do not have a major impact on its activity. This
mutation however increased more than 5-folds CE1 activity in NIH3T3
cells and 3T3L1 cells (Fig. 4, D and E),
suggesting that the NF1 nuclear factor(s) binding to CE1 in
non-osteoblastic cells repress its activity. Considering that Insa did
not modify CE1 activity in osteoblastic cells, this result also
strongly suggests that these particular NF1 factors are not present in
osteoblastic cells.
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Fig. 4.
Functional analysis of CE1 by DNA
transfection. A, schematic representation of
multimerized wild-type (wt) CE1 oligonucleotides fused to
the AMLP-luc reporter cassette. B, comparison of CE1
multimers activity in osteoblastic cells (UMR106) and in
non-osteoblastic mesenchymal cells (NIH3T3 and 3T3L1). Values are given
above the basal activity of pAMLP-luc empty vector. C-E,
effect of mutations in the NF1 site (Insa), in the AP1 site
(mb), or in both sites (Insa/mb) on CE1 activity
in osteoblastic cells (C) and in non-osteoblastic cells
(D and E). Values are given in fold activation
relative to pAMLP-luc basal activity. All transfection data represent
ratios of luciferase versus -galactosidase activities,
and values are means of at least five independent transfection
experiments.
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Together, these experiments demonstrate that CE1 acts as an activator
of transcription specifically in osteoblastic cells. They also suggest
that this cell-specific activity results from the binding to CE1 of NF1
inhibitory factors present in non-osteoblastic cells but not in
osteoblastic cells, and from the binding of AP1 enhancing activities
specific to osteoblastic cells.
CE1 Is Evolutionary Conserved--
We next searched the
Cbfa1 promoter of the rat (12) and human (23)
Cbfa1 genes for CE1-related elements. At 399/359 in the
rat promoter and at 257/217 in the human promoter we located
identical regions that share a 93% sequence conservation with mouse
CE1 (Fig. 5A). More
specifically, these regions harbored a 100% conserved AP1 site and an
87% conserved NF1 site compared with mouse CE1. To determine if they
had similar binding abilities we performed EMSA using nuclear extracts
from UMR106 and mouse CE1 (mCE1) or rat/human putative CE1 (hCE1) as
probes. As shown in Fig. 5B, identical binding patterns were
obtained with these 2 probes, indicating that the same nuclear
activities can bind to these elements despite their 2-bp difference in
the NF1 site. Consistent with this result DNA transfection experiments
in UMR106 cells showed that 5 copies of hCE1 were as active as 5 copies of mCE1 (Fig. 5C). That the CE1 enhancer had been conserved
structurally and functionally in the Cbfa1 gene during
mammalian evolution underlines its importance.
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Fig. 5.
Functional conservation of CE1 during
mammalian evolution. A, alignment of CE1 putative
sequences present in the mouse, rat, and human Cbfa1
promoters. The NF1 and AP1 core sites are boxed. Nucleotide
differences are indicated, dashes mark a conserved
nucleotide. B, EMSA comparison of labeled double-stranded
wild type mouse CE1 (lane 1, mCE1) and human/rat
CE1 (lane 2, hCE1) oligonucleotides used as
probes in presence of UMR106 nuclear extracts. C, similar
activity of mCE1 and hCE1 multimers fused to the AMLP-luc reporter
cassette in osteoblastic cells (UMR106). Values are given in activity
above pAMLP-luc basal activity. Data represent ratios of luciferase
versus -galactosidase activities and values are means of
at least six independent transfection experiments.
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NF1-A Acts as an Inhibitor of CE1 Activity in Non-osteoblastic
Cells--
To establish that a bona fide NF1 nuclear
activity bound to CE1, we performed antibody supershift experiments in
EMSA. Addition of an antibody recognizing specifically all NF1 proteins
to nuclear extracts from UMR106, NIH3T3 and 3T3L1 cells induced the
formation of an additional band of slow mobility (Fig.
6A), indicating that NF1
proteins bind to CE1 both in osteoblastic and non-osteoblastic cells.
Four NF1 genes (NF1-A, -B,
-C, and -X) have been identified, encoding
nuclear factors binding the same DNA core sequence (24). The
transfection experiments presented Fig. 4 indicated that the NF1
isoforms binding to CE1 in osteoblastic cells had no significant impact
on its activity while those present specifically in non-osteoblastic cells were playing a major inhibitory role. We therefore compared NF1 gene expression in osteoblastic and non-osteoblastic
cells, searching for an NF1 isoform only expressed in the latter. All NF1 isoforms display a well-conserved N-terminal region and within this
region a common 486-bp product can be generated using degenerated oligonucleotides (24). When subjected to particular restriction digests, this product however leads to fragments specific of each NF1
subtype (17). Using total RNA from UMR106, NIH3T3, and 3T3L1 cells
co-amplification of the 486-bp common product with Hprt in a
semiquantitative RT-PCR assay showed that non-osteoblastic cells have a
higher level of total NF1 gene expression compared with UMR106 cells
(Fig. 6B, upper panel). More importantly, when this 486-bp common product was digested to reveal each NF1 isoform we
could never detect the presence of NF1-A transcripts in osteoblastic cells while they were abundantly expressed in non-osteoblastic cells
(Fig. 6B, lower panel, star). This
result suggested that NF1-A could be the NF1 isoform specifically
present and inhibiting CE1 activity in non-osteoblastic cells.
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Fig. 6.
Identification of NF1-A as a repressor of CE1
activity in non-osteoblastic cells. A, supershift
experiments in EMSA using a NF1-specific antibody and the wild type
CE1-labeled probe in presence of nuclear extracts from osteoblastic
cells (UMR106) and non-osteoblastic cells (NIH3T3 and 3T3L1).
Stars indicate supershifted complexes. B, RT-PCR
analysis of NF1 isoforms expression in osteoblastic cells (UMR106) and
non-osteoblastic cells (NIH3T3 and 3T3L1). Note the absence of NF1-A
expression in UMR106 cells compared with NIH3T3 and 3T3L1 cells
(star). C, repression of p976-luc activity by
co-transfection of a NF1-A expression vector in osteoblastic cells
(UMR106) and non-osteoblastic cells (3T3L1). Forced expressions of
NF1-B, -C, or -X do not induce significant effects. D,
repression of p5CE1-luc activity by co-transfection of a NF1-A
expression vector in osteoblastic cells (UMR106) and non-osteoblastic
cells (3T3L1). The Insa mutation abolishes this effect in osteoblastic
cells. In non-osteoblastic cells NF1-A forced expression blocks the
effect of the Insa mutation but does not induce a decrease below the
level of wild type p5CE1-luc activity. All transfection data represent
ratios of luciferase versus -galactosidase activities,
and values are means of at least six independent transfection
experiments.
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To test this hypothesis we analyzed which impact each NF1 isoform had
on the Cbfa1 promoter activity by co-transfecting p976-luc with expression vectors for NF1-A, -B, -C, and -X. Both in osteoblastic and non-osteoblastic cells, expression of NFA-1 decreased p976-luc activity whereas expression of the 3 other NF1 isoforms had no significant effect (Fig. 6C). This effect was specifically
mediated by CE1 because: 1) expression of NF1-A in osteoblasts
decreased the activity of p5CE1-luc but not of its Insa mutant
counterpart, and 2) increased expression of NF1-A in non-osteoblastic
cells decreased p5CE1-luc activity and completely abolished the
de-repressing effect induced by its Insa mutation (Fig. 6D).
Taken together, these experiments indicated that CE1 was negatively
regulated by NFA-1 in non-osteoblastic cells. This isoform being
expressed in non-osteoblastic cells but not in osteoblastic cells
explained the NF1-mediate repression of CE1 occurring specifically in
the former cells.
Binding of JunD/FosB AP1 Complex to CE1 Is Restricted to
Osteoblastic Cells--
We next investigated which Fos/Jun AP1 family
members were binding to CE1 by using antibodies specific for each of
the 4 Fos-related factors and the 3 Jun-related factors in EMSA. These
supershift experiments were performed using wild type CE1
oligonucleotide and nuclear extracts from UMR106, NIH3T3, and 3T3L1
cells. As shown in Fig. 7A,
cFos and cJun antibodies did not have any effect, indicating that these
AP1 family members do not bind CE1. Conversely, addition of a
Fra2-specific antibody produced a supershifted band with all 3 nuclear
extracts (Fig. 7A, lanes 5, 13, and
21), indicating that Fra-2 binds equally to CE1 in
osteoblastic and non-osteoblastic cells and is therefore unlikely to
mediate a cell-specific role. More importantly, three major differences
were observed when comparing osteoblastic and non-osteoblastic
supershift patterns. First, a FosB-specific antibody prevented the
formation of osteoblastic complex *2 (Fig. 7A, lane
3, diamond) while it did not affect formation of
non-osteoblastic complexes *2/3 (Fig. 7A, compare lane
3 to lanes 11 and 19). Second, a
JunD-specific antibody reduced formation of osteoblastic complexes *1,
*2 and *3 (Fig. 7A, lane 8) while
non-osteoblastic complexes *2/3 were not affected (Fig. 7A,
compare lane 8 to lanes 16 and 24).
Third, addition of a Fra-1-specific antibody did not have any effect on
osteoblastic nuclear extracts whereas it reduced formation of complexes
*1 and *2/3 in non-osteoblastic cells (Fig. 7A, compare
lanes 12 and 20 to lane 4). These 3 differences between osteoblastic and non-osteoblastic binding factors
were further analyzed by Western blot analysis and DNA co-transfection experiments.
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Fig. 7.
Identification of
FosB(FosB)/JunD as an activator of CE1 in
osteoblastic cells. A, supershift experiments in EMSA
using antibodies specific of each AP1 family member, the CE1-labeled
probe and nuclear extracts from osteoblastic cells (UMR106) and
non-osteoblastic cells (NIH3T3 and 3T3L1). The open diamond
and empty and black dots indicate differences
between osteoblastic and non-osteoblastic patterns of supershift.
B, Western blot analysis of nuclear extracts from
osteoblastic cells (UMR106) and non-osteoblastic cells (NIH3T3 and
3T3L1) using antibodies specific of each AP1 family member. Note the
weak Fra1 expression in UMR106 cells compared with NIH3T3 and 3T3L1
cells. C, induction of p976-luc and p5CE1-luc activities by
co-transfection with a JunD expression vector in non-osteoblastic
cells. The mb mutation abolishes this effect. D, forced
expressions of Fra family members in osteoblastic cells (UMR106) do not
have any effect on p976-luc or p5CE1-luc activities. E,
induction of p976-luc and p5CE1-luc activities by co-transfection with
FosB-related expression vectors in non-osteoblastic cells. The mb
mutation abolishes this effect. All transfection data represent ratios
of luciferase versus -galactosidase activities, and
values are means of at least six independent transfection
experiments.
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According to the above supershift analysis JunD bound CE1 only when
UMR106 extracts were used (Fig. 7A, empty
circles). To determine whether this specificity resulted from JunD
osteoblast-specific expression, osteoblastic and non-osteoblastic
extracts were analyzed by Western blot. As shown in Fig. 7B,
JunD was detected at similar levels in both types of cells, indicating
that the osteoblast-specific binding of JunD to CE1 is not associated
with its exclusive presence in these cells. We therefore hypothesized
that binding of JunD to CE1 could be inhibited in non-osteoblastic
cells and that overcoming this inhibition by increasing JunD level in
these cells would reveal its potential. To test this hypothesis we
co-transfected p5CE1-luc or p976-luc with a JunD expression vector or
its empty counterpart in NIH3T3 and 3T3L1 cells. JunD forced expression induced a 2-fold increase of p976-luc activity (Fig. 7C). A
weaker but significant increase was also observed using the p5CE1-luc construct (Fig. 7C, 5CE1). This effect was
specific since it could be abolished by the mb mutation (Fig.
7C, mb). Together, these results suggest that
JunD is an activator of CE1 whose absence of binding to this element in
non-osteoblastic cells could explain CE1 inactivity in these cells.
However, the mild activation observed upon JunD co-expression with both
the Cbfa1 promoter fragment and CE1 multimeric constructs
suggests that this is not a major mechanism regulating CE1
cell-specific activity.
The antibody supershift analysis also detected that Fra1 bound CE1 only
when non-osteoblastic nuclear extracts were used (Fig. 7A, black dots). Furthermore, Western blot
analyses indicated that Fra1 was much less abundant in UMR106 nuclear
extracts than in non-osteoblastic extracts (Fig. 7B). These
two results suggested that CE1 could be repressed by Fra1 in
non-osteoblastic cells only because they highly express this factor. To
test whether Fra1 could indeed act as an inhibitor of CE1 activity we
co-transfected Fra family members with p5CE1-luc or p-976-luc in
osteoblastic cells. Neither Fra1 nor Fra2 expression decreased CE1 (or
CE1mb) activity (Fig. 7D). This result indicates that Fra1
is not a repressor of CE1 activity whose higher expression in
non-osteoblastic cells compared with osteoblastic cells would explain
CE1 inactivity in the former.
Lastly, the EMSA supershift experiments showed that FosB bound to CE1
only when osteoblastic nuclear extracts were used (Fig. 7A, diamond). Because a Western blot analysis
demonstrated that FosB is expressed as well in non-osteoblastic cells
as in osteoblastic cells (Fig. 7B), we concluded that FosB
ability to bind CE1 could be specifically inhibited in non-osteoblastic
cells. As for JunD, this observation raised the hypothesis that FosB
could be an activator of CE1 whose binding to this element was
specifically inhibited in non-osteoblastic cells. If this hypothesis
was correct it would be possible to overcome such mechanism by raising
the level of FosB in these cells and thereby reveal its function. We
therefore analyzed the effect of overexpressing FosB (or its truncated
isoform FosB, whose overexpression in vivo stimulates
bone formation (25) in non-osteoblastic cells). Both in NIH3T3 and
3T3L1 cells activity of the Cbfa1 promoter fragment was
increased by co-transfection with the FosB and FosB expression
vectors (Fig. 7E). This increase was specifically mediated
by CE1 since it also occurred when p5CE1-luc was used as reporter but
not when its mutated counterpart was used (Fig. 7E). The
large extent of induction observed with both reporter constructs upon
FosB expression suggests that this AP1 family member is a major
regulator of CE1 activity.
In summary, our analysis of the AP1 complexes binding to CE1 suggests
that binding of JunD/FosB(FosB) is responsible for CE1 activity in
osteoblastic cells and that the absence of binding of this particular
complex in non-osteoblastic contributes to the absence of CE1 activity
in these cells.
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DISCUSSION |
To study the early transcriptional events controlling osteochondro
progenitor differentiation, we have embarked in the systematic analysis
of the promoter of mouse Cbfa1. In this study we show that
the first 976 bp of this promoter are active only in osteoblastic cells. We then narrowed down this cell specificity to a 40-bp region,
CE1, located at 415 compared with the Cbfa1 start site of
transcription and show that CE1 acts as an osteoblast-specific enhancer
in DNA transfection experiments. CE1 contains a NF1 and an AP1 binding
sites, functioning respectively as a repressor specifically in
non-osteoblastic cells and as an activator only in osteoblastic
cells. Further analyses determined that binding of particular AP1 and
NF1 factors triggers these cell specificities. Indeed, NF1-A is the
only NF1 isoform able to repress CE1 activity and it is only expressed
in non-osteoblastic cells, therefore repressing CE1 activity only in
these cells. Likewise, FosB(FosB)/JunD is an activator of CE1 that
can bind to its AP1 site only in osteoblastic cells.
Our analysis also showed that CE1 is present in mouse, rat, and human
Cbfa1 promoters where it binds NF1 and AP1 nuclear
activities in a similar fashion and activate transcription to a similar
extent. These observations suggest that CE1 plays the same role in
regulating Cbfa1 expression in these 3 species. Considering
that to fulfill the same function in mouse and human Cbfa1
has to be expressed in a similar fashion such a conservation of its
transcriptional regulation of expression during mammalian evolution is
not surprising. We predict however that in addition to CE1 other
cis-acting elements are likely to be involved in controlling
Cbfa1 expression. Indeed, Cbfa1 is highly
expressed in undifferentiated osteochondro progenitor cells but it is
down regulated in differentiating non-hypertrophic chondrocytes, only
to be re-expressed at high levels in pre-hypertrophic chondrocytes
(14). We believe that achieving such variation of expression is likely
to require more than one cis-acting element and in particular should
involve both enhancer and repressor elements.
AP1 family members have already been implicated in the regulation of
osteoblast differentiation. Notwithstanding that AP1 family members are
differentially expressed during osteoblast differentiation (26), AP1
binding sites have been identified in the promoter of several genes
expressed in osteoblasts, where they often mediate the responsiveness
of these promoters to growth factors (27-29). More recently, the role
of AP1 family members has been emphasized in vivo by two
studies showing respectively that Fra1 or FosB overexpression in
mice enhanced bone formation by osteoblasts and led to osteosclerosis
(25, 30). Sabatakos et al. (25) additionally showed that
overexpression of a short form of FosB (2FosB) in osteoblasts
induces a significant increase of the expression of Cbfa1 as
well as of bone matrix protein encoding genes. By showing that FosB
isoforms are major activators of CE1 our results provide a molecular
explanation for this observation. Interestingly, although Fra1
transgenic animals also displayed an osteosclerotic phenotype, Fra1
overexpressing osteoblasts did not show a significant increase of
Cbfa1 expression (30), nor did we observe an increase of CE1
activity upon Fra1 co-transfection experiments. These observations and
our results together suggest that the high bone mass phenotype observed
in the Fra1- and FosB-overexpressing mice may have different
molecular bases.
To our knowledge this study for the first time implicates NF1
transcription factors in the regulation of osteoblast-specific gene
expression. NF1 isoforms are encoded by 4 distinct genes whose patterns
of expression are broad although some isoforms are preferentially
expressed in specific tissues (24). Functional NF1 sites have been
identified in the promoter of multiple genes, some of them
cell-specific (31-33). In most cases, NF1 cis-acting elements acted as
activators of transcription, although some repressor activities have
been reported (24). Although CE1 binds a NF1 nuclear activity in all
cell types we analyzed, our DNA transfection experiments show that
within CE1 the NF1 binding site is neutral in osteoblastic cells or
repressive in non-osteoblastic cells. Indeed, upon mutation of the NF1
binding site in osteoblastic cells, CE1 activity is not significantly
impaired, indicating that the NF1 isoforms expressed in these cells do
not contribute significantly to its positive activity. In contrast,
this mutation de-repressed CE1 activity in non-osteoblastic cells,
indicating that these cells express a specific NF1 isoform inhibiting
CE1 activity. Because NF1-A can repress Cbfa1 promoter
activity and is expressed in non-osteoblastic cells but not in
osteoblastic cells we propose that binding of this isoform to CE1
contributes to its absence of activity in non-osteoblastic cells. That
NF1-A had already been shown to function as a repressor (34) brings further support to this hypothesis.
With CE1 being formed by the juxtaposition of two cis-acting elements
one could hypothesize that interactions between NF1 and AP1 occur and
may mediate for instance the specific binding of JunD/FosB(FosB)
complexes to CE1 in osteoblasts. Our EMSA analyses showing that NF1 and
AP1 complexes can bind to CE1 either alone or in presence of each other
along with the observation that NF1-A can specifically interfere with
JunD oncogenic ability (35) would support such hypothesis. However, the
following evidence strongly argues against it. When its NF1 binding
site is mutated CE1 activity is not modified in osteoblasts, nor is the
binding of AP1 factors (data not shown), indicating that NF1 binding is not required for the recruitment or stabilization of JunD/FosB(FosB) complexes. Conversely, when NF1 binding is inhibited in
non-osteoblastic cells CE1 activity is increased but does not however
reach the level it shows in osteoblastic cells. Together with the fact
that this mutation does not modify the binding of AP1 factors in EMSA (data not shown), this result indicates that the formation of JunD/FosB(FosB) is not actively inhibited in non-osteoblastic cells
and that this mechanism cannot account for the absence of CE1 activity
in these cells.
In summary, two pecularities characterize CE1. First, it is activated
in a cell-specific manner via non-cell-specific cis-acting elements.
Indeed, both NF1 and AP1 families of transcription factors contains
members whose expression pattern is broad (36). Considering also that
all NF1 (AP1) isoforms can bind to identical DNA core sequences, it is
remarkable that these particular families of transcription factors
could mediate a cell-specific activity. Second, CE1 specific activity
in osteoblastic cells is the net result of a combination of
cell-specific absence of repression and of cell-specific activation.
Such combination allows a finer regulation than a simple "On/Off"
switching mechanism. Such fine-tuning could find its purpose when
Cbfa1 expression needs to be moderately and/or transiently
modified. For instance transient expression of NF1-A at specific
stage(s) of development could decrease Cbfa1 promoter
activity to limit its expression and thereby slow down the rate of
differentiation of osteochondro progenitors. Three lines of evidence
support such hypothesis. First, NF1-A is expressed transiently in 11.5 dpc limb buds (36), i.e. at a stage where proliferation of
osteochondro progenitors should be favored compared with their
differentiation (37), and therefore Cbfa1 expression should
be limited. Second, a negative NF1 cis-acting element has been
identified in the promoter of cartilage matrix protein
(CMP), a gene encoding an extracellular protein synthesized
by chondrocytes (31). Like Cbfa1, CMP is expressed in prehypertrophic
cells during chondrocyte differentiation (14, 38), suggesting that NF1-A could modulate the expression of several molecular determinants of this process. Lastly, the rare NF1-A deficient mice that survive beyond birth are runted and present cranio-facial abnormalities, two
phenotypes that disappear overtime (39). This observation would suggest
that NF1-A has a transient role during skeletogenesis. However, due to
the lethal neurological defect affecting these animals such a role will
have to await the generation and analysis of skeletal-specific
NF1-A-deficient mice to be established.
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