From the
Department of Oral Biology, School of
Dentistry, University of Missouri, Kansas City, Missouri 64108, the
¶Department of Pathology, University of
Rochester, Rochester, New York 14642, and the
||Department of Cellular and Structural Biology,
University of Texas Health Science Center, San Antonio, Texas 78229-3900
Received for publication, December 3, 2002 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Bone morphogenetic proteins (BMPs)1 play an important role in skeletal development (35). At the early stage of bone development, BMP-2 and BMP-4 are expressed in specific areas of the limb bud3 and misexpression of noggin, a glycoprotein that blocks actions of BMP-2 and BMP-4, prior to the onset of chondrogenesis leads to the total absence of skeletal elements (6), suggesting that BMP signaling is absolutely required for prechondrogenic condensations. At the late stage, BMP-2, expressed in the growth plate of long bone, accelerates longitudinal bone growth by stimulating growth plate chondrocyte proliferation and hypertrophy (7, 8).
To understand the regulatory mechanism of BMP-2 gene, we have
previously characterized the BMP-2 promoter and identified control
elements in osteoblasts and chondrocytes in BMP-2 gene
(9). BMP-2 autoregulates its
own expression through its proximal promoter
(10) in a similar fashion that
the decapentaplegic (dpp) gene is
regulated in drosophila. dpp is a drosophila homologue of
BMP-2. In drosophila, expression of dpp is regulated by
dorsal, a homologue of mammalian NF-B
(11,
12). In chick embryo,
overexpression of a mutated form of I-
B
protein, which blocks
NF-
B activation, leads to abnormality in limb bud development, and
changes in downstream signals include BMP-4
(13). These findings suggest
that NF-
B may participate in the regulatory process of the
BMP-2 gene.
NF-B is a group of transcription factors including five members,
p65, c-Rel, RelB, p50, and p52. They form homodimers or heterodimers to
regulate expression of a variety of genes and are involved in many biological
functions (14). It has been
demonstrated previously that NF-
B family members p50 and p52 are
absolutely required for osteoclast development and p50/p52 double knock-out
(dKO) mice failed to form osteoclasts and developed severe osteopetrosis
(15).
The role of NF-Bin BMP-2 gene expression and functions of
NF-
B in other bone cells, such as chondrocytes, have not been reported.
In the present studies, we have examined the regulatory mechanism of
BMP-2 by NF-
B in chondrocytes in vitro and in
vivo. We searched the 2712/+165 region of BMP-2 gene and
found two putative NF-
B response elements. Using different experimental
approaches, we demonstrate that these NF-
B response elements are
functional in chondrocytes and NF-
B positively regulates BMP-2
gene transcription through these response elements. We also show that in
growth plate chondrocytes of 2-week-old NF-
B p50/p52 dKO mice,
BMP-2 mRNA expression is significantly decreased. Corresponding to a
decreased expression of BMP-2, chondrocyte proliferation is also
significantly reduced in growth plate chondrocytes of p50/p52 dKO mice. These
results suggest that expression of BMP-2 may be controlled, at least
in part, by NF-
B in chondrocytes, which may have important impacts in
late stage chondrogenesis.
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EXPERIMENTAL PROCEDURES |
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LacZ StainingMale transgenic animals containing the BMP-2 promoter-LacZ transgene were crossed to CB6F1 females and embryos were collected at appropriate times, fixed, and stained overnight as described by Lazik et al. (16). Embryos were embedded in plastic and sectioned at 15 µm and viewed using Zeiss Axioplot microscope under dark-field illumination (16).
Cell Culture and Luciferase AssayTMC-23 cells were cultured
with Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10%
fetal calf serum. Cells were plated in 24-well plates at density of 2 x
104 cells/well. 24 h later, cells were transfected with BMP-2
promoter constructs (2712/+165/pGL3 and 150/+165/pGL3) or
pNF-B-luc reporter and
-galactosidase with either empty vector
(pCMX) or mutant I-
B
expression plasmids
(mI-
B
/pCMX and I-
B
Ub/pCMX)
(13) using LipofactAMINE Plus
reagents (Invitrogen). 48 h after transfection, cells were lysed with lysis
buffer, and luciferase activities were measured using a luciferase assay kit
(Promega, Madison, WI). The luciferase activities were normalized by the
-galactosidase activities.
Site-specific mutations were created in the intact promoter by using PCR-directed mutagenesis (Stratagene, La Jolla, CA) on a construct containing a 2.7-kb BMP-2 promoter (2712/+165) or BMP-2 minimal promoter (150/+165) upstream of a luciferase reporter gene. The designed mutations were verified by sequencing.
Electrophoretic Mobility Shift Assay (EMSA)Nuclear extract
was prepared from TMC-23 cells. Double-stranded oligonucleotides corresponding
to consensus NF-B response element were prepared and used in
competition assays. NRE-1 and NRE-2 binding sites
(Table I) were end-labeled and
purified as described previously
(17). Five fmol of labeled
oligonucleotide was incubated with 10 µg TMC-23 cell nuclear extract. The
incubation mix for nuclear extract binding assay consisted of binding buffer
and 2 µg/ml poly(dI-dC). Incubation took place at room temperature for 5
min. Supershift experiments were carried out as described above, except that
the TMC-23 nuclear extract was preincubated for 10 min at room temperature
with antibodies against p50, p52, and p65 in binding buffer prior to their
incubation with the labeled oligonucleotide.
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p50/p52 dKO MiceHeterozygous NF-B p50 and
p52 knock-out mice (p50+/ and
p52+/) were obtained from Dr. Ulrich
Siebenlist (15). NF-
B
p50 and p52 homozygous knock-out mice
(p50/ and
p52/) were generated by
breeding p50+/ and
p52+/ mice, respectively and
NF-
B p50/p52 dKO mice
(p50//p52/)
were generated by inter-crossing NF-
B
p50+/ and
p52/ mice. The mice were
genotyped by PCR as described previously
(15).
In Situ HybridizationBone samples were fixed overnight in
fresh 10% paraformaldehyde in RNase-free phosphate-buffered saline,
transferred to ethanol series to 100%, followed by methanol and xylene, and
finally embedded in wax. 10 µm sections were placed on microscope slides,
dewaxed, refixed, and utilized for in situ hybridization. A 1.0-kb
mouse BMP-2 RNA probe was prepared and hybridized to appropriate
sections as described by Wilkinson and Nieto
(18) and Shamim et
al. (19). To analyze
changes in message levels of BMP-2 and BMP-6 in NF-B
p50/p52 dKO mice, in situ hybridization slides were viewed by the
Nikon E400 microscope that is linked to a color video monitor, and images were
captured. The message levels of BMP-2 and BMP-6 were
quantitated using a software Image-Pro Plus, version 3.0 (Media Cybernetics,
Silver Spring, MD).
BrdUrd LabelingBrdUrd labaling was performed using Zymed Laboratories Inc. (South San Francisco, CA) BrdUrd labeling reagent and BrdUrd staining kit according to manufacturer's protocols (Zymed Laboratories Inc.). Briefly, BrdUrd was injected intraperitonally into mice 24 h before the animals were sacrificed. Bone tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. 34-µm thick sections were cut and placed on polylysine-coated slides. Slides were dried in a 60 °C oven for overnight and deparaffinized in 2 changes of xylene for 5 min each and then rehydrated in a series of graded alcohol. Slides were stained for BrdUrd according to manufacturer's protocols. Streptavidin-peroxidase was used as a signal generator for the BrdUrd system, and deaminobenzidine in the presence of hydrogen peroxide was used as a chromogen, staining BrdUrd-incorporated nuclei dark brown.
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RESULTS |
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To determine whether NF-B regulates BMP-2 gene
transcription in chondrocytes, we analyzed the sequence of the
2712/+165 region of BMP-2 gene. Two putative NF-
B
response elements were found and are designated as NRE (NF-
B
response element)-1 and NRE-2. The NRE-1 is located in
the 5'-flanking region and the NRE-2 is located in Exon 1 of
BMP-2 gene (Table I
and Fig. 2a). To
determine whether these NF-
B response elements are functional in
chondrocytes, we co-transfected two different mutant mouse I-
B
expression plasmids with BMP-2 promoter (2712/+165)-luciferase
reporter construct into TMC-23 cells, a clonal chondrocyte cell line
(20). In one mutant I-
B
construct, the inducible phosphorylation sites (Ser32 and
Ser36) and constitutive phosphorylation site at its carboxyl
terminus were substituted with alanine residues (mI-
B
), and the
other mutant was generated by substituting three lysine residues at the
ubiquitination sites (I-
B
Ub). These mutant I-
B
proteins retain their ability to bind with NF-
B proteins and cannot be
degraded properly and therefore block NF-
B activity in vitro
and in vivo (13).
Transfection of mutant I-
B
expression plasmids inhibited
BMP-2 promoter activity
50%
(Fig. 2b), indicating
that NF-
B response elements in BMP-2 gene serve as positive
regulatory elements for BMP-2 gene transcription. To confirm that
these mutant I-
B
expression plasmids do inhibit NF-
B
activity in chondrocytes, we co-transfected them with pNF-
B-luc
reporter construct (Promega), which contains six copies of NF-
B
consensus response elements, into TMC-23 cells. Transfection of mutant
I-
B
expression plasmids significantly inhibited luciferase
activity of the NF-
B reporter construct (data not shown).
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To confirm that NF-B transcription factors are transcriptional
activators for BMP-2 gene, we transfected BMP-2 promoter luciferase
construct into TMC-23 cells and treated cells with NF-
B inducer
TNF-
and NF-
B inhibitor pyrrolidine dithiocarbamate (PDTC).
TNF-
induces phosphorylation of I-
B
and causes
dissociation of I-
B
·NF-
B complex, resulting in
nuclear translocation of NF-
B. Treatment with TNF-
(50200
ng/ml) for 24 h caused a dose-dependent increase in BMP-2 promoter
activity in TMC-23 cells (Fig.
2c). In contrast, treatment with PDTC (0.251
µM) for 24 h significantly inhibited BMP-2 promoter
activity in a dose-dependent manner in the same cells
(Fig. 2c).
To determine the function of NRE-1 in BMP-2 gene transcription, we
mutated 3 nucleotides in this response element
(Table I) and found that
BMP-2 promoter activity was decreased over 30% when NRE-1 site is
mutated (Fig. 2b).
Cotransfection of mutant I-B
expression plasmids with the 2.7-kb
BMP-2 promoter, in which three nucleotides at NRE-1 site were
mutated, caused reduction in BMP-2 promoter activity in TMC-23 cells
(Fig. 2b), suggesting
that NRE-2 site located at Exon 1 may also serve as a positive regulatory
element. We then examined the effects of mutant I-
B
on
BMP-2 minimal promoter (150/+165). The luciferase activity of
BMP-2 minimal promoter is about 30% lower than that of larger
BMP-2 promoter (2712/+165) in TMC-23 cells (data not shown).
Transfection of mutant I-
B
expression plasmids with
BMP-2 minimal promoter (150/+165) suppressed the activity of
BMP-2 minimal promoter in TMC-23 cells
(Fig. 2d). Mutation of
NF-
B response element NRE-2 in this region completely abolished the
inhibitory effect of mutant I-
B
(Fig. 2d). When both
NRE-1 and NRE-2 sites of BMP-2 promoter (2712/+165) were
mutated, the promoter activity was reduced about 50%. Mutant I-
B
had no significant effects on this mutant BMP-2 promoter
(Fig. 2e). Taken
together, these results suggest that both NRE-1 and NRE-2 sites in
BMP-2 gene are functional and NF-
B transcription factors
positively regulate BMP-2 gene transcription.
To examine the binding of NF-B to its response element in
BMP-2 gene, we performed EMSA. The nuclear proteins were extracted
from TMC-23 cell nuclear preparations. Labeled oligonucleotides containing
NRE-1 and NRE-2 sequences of the BMP-2 gene
(Table I) were incubated with
TMC-23 cell nuclear extract, leading to the formation of a protein-DNA complex
that migrated at the same location as that formed upon incubation of TMC-23
cell nuclear extract with labeled oligonucleotides containing NF-
B
consensus sequence (Table I and
Fig. 3a). Addition of
different amounts of unlabeled oligonucleotides containing NF-
B
consensus element inhibited formation of this protein-DNA complex in a
dose-dependent manner (Fig.
2a). Addition of unlabeled oligonucleotides containing
Sp1 element had no significant effects on NF-
B binding to NRE-1
response element (data not shown). To show that NF-
B was part of this
protein-DNA complex, supershift experiments were performed using antibodies
against different NF-
B subunits, p50, p52, and p65. The incubation of
TMC-23 nuclear extract with antibodies against p50 and p65 prior to the
addition of labeled oligonucleotides led to the formation of a slower mobility
complex. This complex was specific, as it was not observed when using an
antibody against p52 (Fig.
3b).
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To determine whether NF-B regulates BMP-2 gene expression
in chondrocytes in vivo, we examined BMP-2 mRNA expression
in 2-week-old NF-
B knock-out (KO) mice and their wild-type littermates
by in situ hybridization (n = 3 in each group and three
non-consecutive sections per mouse were analyzed). In wild-type mice,
BMP-2 mRNA was predominantly expressed in the lower portion of
proliferating zone, the junction between the proliferating and hypertrophic
zone and the upper layer of hypertrophic zone of growth plate. BMP-2
was also highly expressed in articular chondrocytes
(Fig. 4a, upper
panel). In NF-
B p50 KO or p52 KO mice, no significant changes in
BMP-2 mRNA expression were observed (data not shown). In contrast, in
NF-
B p50/p52 dKO mice, BMP-2 mRNA expression was significantly
reduced over 80% in growth plate chondrocytes
(Fig. 4, a and
c). In articular chondrocytes, BMP-2 expression
remains unchanged (Fig.
4a, lower panel). The effect of NF-
B on
BMP-2 expression seems specific, since expression of BMP-6
mRNA in growth plate chondrocytes was not significantly changed in p50/p52 dKO
mice (Fig 4, b and
c).
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It has been reported that BMP-2 stimulates chondrocyte proliferation
(7,
8). To investigate whether
decreased BMP-2 expression in NF-B p50/p52 dKO mice causes
reduction in chondrocyte proliferation, we monitored BrdUrd incorporation in
growth plate chondrocytes in these mice. The numbers of BrdUrd-positive cells
were decreased over 65% in proliferating chondrocytes in dKO mice
(Fig. 5, a and
b). These results suggest that reduced BMP-2
expression in growth plate chondrocytes in dKO mice has important biological
impacts on growth plate chondrogenesis.
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DISCUSSION |
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NF-B is a family of transcription factors, which are composed of
five subunits, p65, c-Rel, RelB, p50, and p52, among which p50 and p52 are two
highly homologous members and are the most frequent partners among family
members (14). In our gel shift
assay, we only detect binding of p50 and p65 to NRE-1 site of BMP-2
gene but not p52 in chondrocyte TMC-23 cells. Consistent with these
observations, expression of p50 and p65, but not p52, proteins was detected by
Western blot in TMC-23 cells (data not shown). Previously it has been shown
that p50/p52 dKO mice are impaired in the development of osteoclasts and B
cells, suggesting that these two subunits of NF-
B play indispensable
role in osteoclast and B cell development
(15). In the present studies,
defects in BMP-2 gene expression at growth plate chondrocyte area
were also observed in p50/p52 dKO mice, suggesting that these two factors,
which may be produced by different subpopulations of chondrocytes, play a
redundant role in regulating BMP-2 gene expression.
The major function of BMP-2 in the growth plat of postnatal mice is to
control longitudinal bone growth by stimulating chondrocyte proliferation and
hypertrophy (7,
8). In the present studies, we
have demonstrated that NF-B positively regulates BMP-2 gene
transcription, and in NF-
B p50/p52 dKO mice, expression of the
BMP-2 message in the growth plate chondrocytes is dramatically
reduced, which leads to a significant decrease in chondrocyte proliferation
(67%). Consistent with these findings, NF-
B p50/p52 dKO mice showed
retarded growth and shortened long bones
(15). Decreases in
BMP-2 expression and chondrocyte proliferation in NF-
B p50/p52
dKO mice may at least partially contribute to this phenotype.
We have found that in p50/p52 dKO mice, the hypertrophic zone of the growth
plate was expanded, indicating the normal process of chondrocyte hypertrophy
was disrupted. Since MMP-9 produced by osteoclasts plays a key role in
regulation of cell apoptosis of hypertrophic chondrocytes
(22), and dKO mice have no
osteoclasts, it is difficult to evaluate the contribution of BMP-2 in
chondrocyte hypertrophy under this circumstance. To dissect the direct effect
of NF-B on chondrocyte hypertrophy from its indirect effect through
osteoclasts, tissue- or cell-specific approaches are required, such as
generating transgenic mice in which NF-
B signaling is disrupted
specifically in chondrocytes. Nevertheless, reduction in cell proliferation in
dKO mice suggests that NF-
B-regulated BMP-2 expression does
play an important role in chondrocyte function.
In addition to direct regulation of BMP-2 gene transcription,
NF-B may also regulate BMP-2 gene expression through indirect
mechanisms and other transcription factors may also play a role in
BMP-2 gene transcription in growth plate chondrocytes. The summation
of these transcriptional inputs determines the spatial and temporal expression
pattern of BMP-2 gene in growth plate chondrocytes in
vivo.
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FOOTNOTES |
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These authors contributed equally to this work.
** Present address: Southwest Foundation for Biomedical Research, San Antonio,
TX 78227.
To whom correspondence should be addressed: Dept. of Cellular and Structural
Biology, University of Texas Health Science Center, 7703 Floyd Curl Dr., Mail
code 7762, San Antonio, TX 78229-3900. Tel.: 210-614-0770 (ext. 239); Fax:
210-614-0797; E-mail:
chend1{at}uthscsa.edu.
1 The abbreviations used are: BMP, bone morphogenetic protein; KO, knock-out;
dKO, double KO; dpc, days postcoitum; BrdUrd, 5-bromo-2'-deoxyuridine;
NRE, NF-B response element; PDTC,
pyrrolidine dithiocarbamate; TNF, tumor necrosis factor; EMSA, electrophoretic
mobility shift assay.
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ACKNOWLEDGMENTS |
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REFERENCES |
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