Stage-Specific Expression of Dlx-5 during Osteoblast Differentiation: Involvement in Regulation of Osteocalcin Gene Expression
H. M. Ryoo1,
H. M. Hoffmann,
T. Beumer,
B. Frenkel,
D. A. Towler,
G. S. Stein,
J. L. Stein,
A. J. van Wijnen and
J. B. Lian
Department of Cell Biology (H.M.R., H.M.H., T. B., B.F.,
G.S.S., J.L.S., A.J.W., J.B.L.), University of Massachusetts Medical
Center, Worcester, Massachusetts 01655,
Departments of
Medicine and Molecular Biology and Pharmacology (D.A.T.), Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT
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Two homeotic genes, Dlx and Msx, appear to
regulate development of mineralized tissues, including bone, cartilage,
and tooth. Expression of Msx-1 and Msx-2 has been studied during
development of the osteoblast phenotype, but the role of Dlx in this
context and in the regulation of bone-expressed genes is unknown. We
used targeted differential display to isolate homeotic genes of the Dlx
family that are expressed at defined stages of osteoblast
differentiation. These studies were carried out with fetal rat
calvarial cells that produce bone-like tissue in vitro. We
observed a mineralization stage-specific mRNA and cloned the
corresponding cDNA, which represents the rat homolog of Dlx-5. Northern
blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the
bone-specific osteocalcin genes exhibit similar up-regulated expression
during the mineralization period of osteoblast differentiation. This
expression pattern differs from that of Msx-2, which is found
predominantly in proliferating osteoblasts. Several approaches were
pursued to determine functional consequences of Dlx-5 expression on
osteocalcin transcription. Constitutive expression of Dlx-5 in ROS
17/2.8 cells decreased osteocalcin promoter activity in transient
assays, and conditional expression of Dlx-5 in stable cell lines
reduced endogenous mRNA levels. Consistent with this finding, antisense
inhibition of Dlx-5 increased osteocalcin gene transcription.
Osteocalcin promoter deletion analysis and binding of the in
vitro translation product of Dlx-5 demonstrated that repressor
activity was targeted to a single homeodomain-binding site, located in
OC-Box I (-99 to -76). These findings demonstrate that Dlx-5
represses osteocalcin gene transcription. However, the coupling of
increased Dlx-5 expression with progression of osteoblast
differentiation suggests an important role in promoting expression of
the mature bone cell phenotype.
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INTRODUCTION
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Bone formation occurs during embryonic development and postnatal
growth, but also during subsequent bone remodeling to support calcium
homeostasis and adaptation to physical forces. Osteogenesis is
additionally linked to osseous transplantation, implantation, or
skeletal tissue repair. This remodeling requires continuous
recruitment, proliferation, and differentiation of osteoprogenitor
cells. Like other connective tissue-forming cells, osteoprogenitor
cells originate from mesenchymal cells derived from the mesodermal germ
cell layer (1). Subsequently, osteoblast precursor cells develop into
mature osteocytic cells by progressing through a multistage
developmental sequence (2). An understanding of regulatory mechanisms
contributing to osteoblast differentiation is critical for gaining
insight into the pathogenesis of bone-related diseases, as well as for
the development of new treatment regimens.
Formation of the vertebrate body plan is controlled in part by
homeodomain transcription factors that regulate the temporal appearance
and location of preosseous tissues along the vertebral column (3, 4).
Thus, homeodomain proteins may directly mediate osteoblast
differentiation by selectively activating and/or repressing genes that
support development of skeletal tissues in vivo. Homeodomain
proteins contain a 60-amino acid segment, the homeobox, which
represents the DNA recognition domain (5, 6). Unlike the classic
homeobox (Hox) genes, which are clustered at four chromosomal loci and
specify anterior-posterior positional information along the vertebrate
body axis, the homeodomain superclass of genes encompasses many
atypical members. These genes include the Dlx and Msx genes, which have
a dispersed chromosomal distribution [reviewed by Gehring et
al., 1994 (7)].
Several lines of in vivo evidence support the concept that
the Dlx and Msx family of homeobox proteins may represent regulatory
genes that preferentially support skeletal tissue differentiation.
Expression of Dlx and Msx genes is primarily restricted to the
epithelial-mesenchymal interaction site during apical ectodermal ridge
(AER) development (8, 9, 10, 11). The presence of Dlx and Msx homeodomain
proteins during these transitions is critical for craniofacial
(12, 13, 14, 15), tooth (16, 17), brain, and neural development (18, 19).
Developmental studies in mouse (20), rat (19), and chicken limb bud (9)
have revealed that several Dlx family members are highly expressed in
cartilage and in developing endochondral and membranous bone.
Furthermore, results from genetic studies suggest that Dlx and Msx
genes are directly involved in bone morphogenesis (10, 11, 12, 21, 22, 23, 24).
Recent studies suggest the involvement of homeodomain proteins in
regulation of osteoblast development and bone tissue-specific gene
expression. For example, bone-specific expression of the osteocalcin
(OC) gene is controlled by a principal multipartite promoter element
(OC-box) that contains a homeodomain recognition motif (25, 26, 27).
Similarly, homeodomain motifs have been implicated in the regulation of
the collagen type I gene in osteoblasts (28, 29). Several studies have
suggested that Msx-2, which binds the OC-box homeodomain motif of the
OC gene, is a key regulator of OC gene expression (25, 27) and
development of the bone cell phenotype (21, 26). Although Dlx appears
to be important for skeletal formation, to date there is no direct
evidence for a role of Dlx in osteoblast differentiation or regulation
of osteoblast-restricted genes.
In this study we applied a homeobox-directed PCR approach that takes
advantages of both mRNA differential display (30) for sensitivity and
simplicity, and RNA finger printing (31) for reproducibility and
specificity. Using this method, we provide the first evidence for
differentiation-specific expression of a Dlx-5 homolog during rat
calvarial osteoblast differentiation. Our findings suggest that Dlx-5
may directly support expression of the mature bone cell phenotype, and
our results are consistent with the concept that multiple homeodomain
proteins may provide stringent developmental and tissue-specific
regulation of OC gene transcription.
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RESULTS
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Isolation of an Osteoblast Dlx-Related mRNA That Is Tissue Specific
and Expressed in a Developmental Stage-Specific Manner
Our goal in this study was to isolate Dlx-related
homeobox-containing genes that are expressed in a stage-specific manner
during osteoblast differentiation. For this purpose RNA was prepared
from primary cultures of calvarial-derived osteoblasts at two
developmental periods: early stage proliferating cells (day 2) that do
not express bone phenotypic genes and postproliferative cultures with
bone-like mineralized nodules (day 21) that express genes
characteristic of differentiated osteoblasts (2). Dlx-related sequences
in these RNA preparations were amplified using a homeobox-specific
5'-primer and a loosely conserved sequence of the 3'-end of the coding
region of Dlx as the 3'-primer (see Materials and Methods
for nucleotide sequences). This primer set provided high specificity
for the Dlx family of proteins, as well as reproducibility in the
representation of mRNAs by differential display.
Figure 1A
shows the differential display
pattern of mRNA from proliferating (P) and mineralized (M) rat
osteoblast (ROB), or proliferating (P) and confluent (C) ROS 17/2.8 rat
osteosarcoma cells (ROS). The large arrowhead indicates the
band of interest that is preferentially expressed in mature rat
osteoblast cultures having a mineralized matrix. The band is
constitutively expressed in ROS 17/2.8 cells, present at similar levels
in proliferating (day 2) and confluent (day 8) cells. This pattern of
expression was reproduced by Northern blot analysis of RNA harvested
from a ROB cell differentiation time course and confluent or
proliferating ROS 17/2.8 cells when probed with the isolated
reamplified mineralization-specific band from Fig. 1A
. This analysis
(Fig. 1B
) demonstrates that the Dlx isolate is very weakly expressed in
ROB proliferating cells (day 2 and day 6). Dlx expression was detected
after confluency (day 10) and continued to increase throughout the
experimental period. Equal levels of transcripts were found in the
proliferating and confluent ROS 17/2.8 cells. Notably, we observed that
there are two distinct transcripts in ROB cell RNA, as indicated by
arrowheads in Fig. 1B
, while only a single transcript is
present in ROS cells.

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Figure 1. Differential Display and Northern Expression of
Cytoplasmic RNA from Primary Cultured Rat Osteoblasts (ROB) during
in Vitro Differentiation and ROS 17/2.8 Rat Osteosarcoma
Cells (ROS) during Density Inhibition
A, Reverse transcription and PCR conditions are described in
Materials and Methods. The amplified product of interest
is indicated by the large arrow head. The sizes of bands
were calculated from a comigrating sequence reaction, and the
small arrows indicate position of sized markers. P,
Proliferating ROB and ROS at day 2; C, confluent ROS at day 8; M,
mineralized ROB at day 21. B, The reamplified product of the
differential displayed band was used as a probe for Northern
hybridization. Twenty five micrograms of DNase I-treated total cellular
RNA from ROB or ROS 17/2.8 cells harvested at different time points
were loaded on each lane. The hybridization of two distinct transcripts
was observed in the differentiated ROB cell and are indicated by the
arrowheads to the right of the figure.
The bands are designated Dlx-5 based on sequence analysis (see Fig. 2 ).
The blots were probed for 28S ribosomal RNA to demonstrate similar RNA
loading. The migration of the 18S ribosomal RNA is indicated by the
arrow to the left of the figure.
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The reamplified band of interest was cloned into the pCR II vector and
sequenced using Sp6 and T7 promoter primers. Sequence comparison of the
289-nucleotide clone with known sequences in data bases confirmed that
the clone is a rat Dlx gene. It is identical with sequences published
for rDlx (19) and rat Dlx-3 (32). Amino acid sequence comparison of our
isolate and homeodomain sequences of known Dlx-3 and Dlx-5 genes
indicates that our gene is the rat homolog of Dlx-5 (Fig. 2
). A 5'-primer that includes the
translation start site of Dlx-5 was designed to obtain a full-length
Dlx-5 coding region by RT-PCR of RNA from mineralized ROB cultures. The
resulting clone was confirmed by sequencing and was subsequently used
as a Dlx-5 cDNA. Northern analysis of RNA from a time course of
differentiating ROB cells showed the same pattern of Dlx-5 expression
when probed with this full length cloned cDNA (Fig. 3A
)
as the isolated differential display probe (shown in Fig. 1B
). Other
parameters of gene expression in this osteoblast differentiation time
course (Fig. 3A
), including histone H4 (H4) and OC, reflected a typical
osteoblast developmental sequence of gene expression (2). Msx-2 mRNA
levels are high in proliferating cells (day 2) and decrease after
confluency (day 7 to day 21), although an increase in the transcripts,
particularly the lower form, was observed from day 21 to day 28. The
developmental up-regulation of Dlx-5 expression appears to parallel OC
gene expression and osteoblast differentiation. Dlx-5 expression is not
observed postproliferatively in rat osteoblasts cultured in the absence
of both ascorbic acid and ß-glycerol phosphate, conditions that do
not support extracellular matrix mineralization (Fig. 3B
). Figure 3B
shows Dlx-5 expression is not detected in the nondifferentiated
osteoblasts that are maintained for the same time period (26 days) as
the mineralized cultures.

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Figure 2. Sequence Comparisons between the Homeodomains of
Our Isolate, Dlx-5, and Dlx-3
Our cloned cDNA fragment has an identical homeobox amino acid sequence
when compared with human and chicken Dlx-5 genes, rDlx and rat Dlx-3,
but differs 10% from Dlx-3 of mouse or zebra fish. Helix domains of
the homeobox are indicated.
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Figure 3. Developmental Expression of Dlx-5 during in
Vitro Osteoblast Differentiation
A, Total cellular RNA from ROB cells harvested at the indicated days
was examined by Northern analysis for representation of histone H4 (H4)
and OC gene expression (10µg/lane) to assess the extent of ROB differentiation, as well as Msx-2 (10 µg/lane) and Dlx-5 (25 µg/lane). Consistency of RNA loading is reflected by hybridization to 28S ribosomal RNA; a
representative blot (also used for Dlx-5) is shown. B, Northern blot
analysis of osteoblasts cultured for 26 days in the presence (+) or
absence (-) of ascorbic acid and ß-glycerophosphate to promote or
prevent differentiation, respectively. Note the absence of OC and Dlx-5
expression in nondifferentiated cells (-). Ethidium bromide staining
of the ribosomal RNA (EtBr) shows intactness of RNA and consistency in
loading (20 µg/lane). The agarose gels used here did not resolve the
two Dlx-5 transcripts.
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Tissue specificity of Dlx-5 expression was assessed by examination of
total cellular RNA from major organs and tissues of 3-month-old mice
(Fig. 4
). Northern blot analyses
indicated that Dlx-5 was expressed in bone tissue and was absent in all
the soft tissues we assayed. We also note that a single 1.5-kb
transcript is observed in the mouse tissues, similar to ROS 17/2.8
cells.

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Figure 4. Northern Analysis of Expression of Dlx-5 in
3-Month-Old Mouse Tissues
Twenty five micrograms of total cellular RNA from major organ and bone
tissues were separated on a 1% agarose/formaldehyde gel and
transferred overnight by capillary action onto Zetaprobe membrane. The
blot was hybridized with the Dlx-5 cDNA probe.
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Dlx-5 Selectively Represses OC Promoter Activity in Osseous
Cells
It is known that the homeodomain protein Msx-2, which is expressed
abundantly in proliferating osteoblasts, down-regulates OC
transcription and synthesis (27). To investigate whether Dlx-5 can also
regulate OC transcription, we examined the effect of forced expression
of Dlx-5 on OC promoter activity. A cytomegalovirus (CMV) promoter
containing plasmid expressing Dlx-5 was cotransfected with a plasmid
containing a reporter gene under the control of various promoters. As a
control, the promoter constructs were also transfected with a CMV
plasmid lacking the Dlx-5 gene. Dlx-5 does not significantly influence
activity of several eukaryotic promoters, including the thymidine
kinase, histone, osteopontin, and the dimerized SP1-binding site (2x
SP1) (Fig. 5A
). Dlx-5 suppressed OC
promoter activity in ROS 17/2.8 cells using both -1097
OC-choramphenicol acetyltransferase (CAT) (Fig. 5A
) and the -1050
OC-Luc (data not shown) chimeric constructs. We note that Dlx-5 also
inhibited SV40 promoter activity, further reflecting promoter
selectivity of Dlx-5.

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Figure 5. Regulation of Target Promoter Activity by Dlx-5
A, A construct expressing Dlx-5 driven by a CMV promoter (+ Dlx-5,
hatched bars) or lacking the Dlx-5 sequences (control,
solid bars) was cotransfected into ROS 17/2.8 cells
along with plasmids containing the CAT reporter gene fused to various
eukaryotic promoters: thymidine kinase (TK); histone H4 (H4);
osteopontin (OP); OC; a dimerized SP1 binding site (2XSP1); and the
SV40 promoter. Cells were assayed for CAT expression 48 h after
transfection. B, Activity of the -1097 OC promoter-CAT construct was
assayed in the presence (+Dlx, hatched bars) or absence
(-Dlx, solid bars) of a Dlx-5-expressing construct in
two cell lines (ROS 17/2.8 osteosarcoma cells and C2C12 myoblasts) as
well as primary rat osteoblasts. Promoter activity is reported as
percent conversion of chloramphenicol. C, Activity of reporter
construct containing 108 nucleotides of the OC proximal promoter and a
CBFA/AML-1 binding site was measured in IMR-90 cells. To activate the
promoter construct, 1 µg of a CMV construct expressing AML-1B was
cotransfected in the presence (hatched bars) of 4 µg
Dlx-5 expression construct or 4 µg vector alone, lacking Dlx-5
sequences (solid bars).
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To test the effect of increased Dlx-5 expression on OC promoter
activity in cells in which endogenous expression of Dlx-5 is lower than
in ROS 17/2.8 cells, transient transfection assays were performed in
several additional cell lines (Fig. 5B
). Dlx-5 expression was increased
by transient transfection of the CMV Dlx-5 construct, and control
reactions were cotransfected with the empty CMV vector, lacking the
Dlx-5 sequences. In proliferating ROB cells, a 2-fold reduction in
activity is observed in OC promoter activity. The OC promoter is
generally inactive in non-bone cell lines. Transient transfection
studies in C2C12 mouse myoblast cells indicate that background levels
of OC promoter activity are not altered by expression of Dlx-5,
demonstrating that Dlx-5 does not activate OC promoter activity in
non-bone cells. The OC promoter is also normally inactive in IMR-90
fibroblasts, but can be up-regulated by coexpression of the
CBFA/PEßP2
/AML factor (core binding factor
, also known as
polyoma enhancer binding protein and acute myelogenous leukemia
factor) as demonstrated by Banerjee et al. (33). When
activity is induced in IMR-90 cells by AML-1B expression, Dlx-5
coexpression down-regulates this AML-dependent level of OC promoter
activity (Fig. 5C
). Therefore, Dlx-5 consistently down-regulates OC
promoter activity in all cell lines tested.
The Dlx-5-Responsive Element Is Localized to OC-Box I
To locate the Dlx-5-responsive element in the OC promoter (-1050
to +32), we searched for possible homeodomain-binding sites. There are
four homeodomain core-binding sequences [(ATTA or TAAT located at -49
to -52, -86 to -89, -553 to -556, and -990 to -993, numbered
according to Lian et al. (34)]. Four OC promoter-luciferase
constructs, containing -1050, -637, -199, and -83 nucleotides or no
promoter sequences, were chosen to assay for Dlx-5 activity in
transient transfection assays in ROS 17/2.8 cells (Fig. 6A
). Each of these constructs
sequentially eliminates one of the four potential homeodomain-binding
sites. This promoter deletion analysis indicates that a
Dlx-5-responsive negative regulatory element is located between -199
and -83 nucleotides upstream from the transcription start site (Fig. 6A
). The conserved promoter element, OC-Box I, which contains a
functional homeodomain-binding site (25, 27), is located within this
sequence.

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Figure 6. Functional Analysis of Deletion Mutants of the OC
Promoter in Response to Forced or Antisense Expression of Dlx-5
A, A series of OC promoter-luciferase chimeric DNA constructs that
sequentially eliminate each of the four potential homeodomain motifs
were cotransfected with CMV/Dlx-5 (hatched bars) or a
control CMV plasmid without Dlx-5 sequences (solid bars)
into ROS 17/2.8 cells. Values are the mean of six independent
transfections. Standard deviation is indicated by lines
above each bar. Transfection efficiencies
were monitored by cotransfecting the OP-CAT plasmid. B, OC
promoter-luciferase chimeric DNA constructs were transiently
cotransfected with pUHD-30-anti-Dlx-5 expressing Dlx-5 antisense
sequences, or pUHD-30 without Dlx-5 antisense sequences into ROS 17/2.8
cells having a stably integrated tetracycline-responsive transactivator
(+Tet represents control activity when media contains 1 µg/ml
tetracycline). Dlx-5 antisense expression is activated in cells
transfected with antisense plasmid by withdrawal of tetracycline from
media (- Tet); this procedure had no effect in the absence of
antisense sequences on (-1050) OC-Luc. Solid column, +
tetracycline; hatched column, - tetracycline.
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To further confirm Dlx-5 suppressor activity on the OC gene and the
location of the active element, an antisense experimental approach was
carried out (Fig. 6B
). The OC promoter-deletion series of OC-Luc
constructs were cotransfected with the tetracycline-responsive Dlx
antisense expression plasmid into the tetracycline-regulated
transactivator (tTA) ROS 17/2.8 cell line (described in Materials
and Methods). The control vector (without Dlx-5 antisense
sequences) had no effect on the activity of the -1050 OC promoter.
Consistent with the overexpression results, all constructs containing
the OC-Box I (located at -99 to -76) were responsive to
overexpression of antisense Dlx-5. OC promoter activity was induced by
coexpression of antisense Dlx-5 (-Tet). No significant effect was
observed in the shorter OC-Luc construct (-83 OC-Luc), which lacks the
ATTA motif. These data suggest an active site for Dlx-5 interaction at
the OC-Box (-99 to -76) in the OC promoter.
Dlx-5 Functional Activity Is Mediated by the Homeodomain Sequence
within the OC-Box
To assay for binding specificity of Dlx-5 with OC-Box sequences,
gel mobility shift assays were performed (Fig. 7
). In vitro translation of
both wild type Dlx-5 and mutant Dlx-5, which lacks the homeobox,
produced the expected molecular masses (-35 kDa and -25 kDa,
respectively) as determined by SDS-PAGE and autoradiography (data not
shown). These in vitro translation products were incubated
with a labeled oligonucleotide containing OC-Box sequences -99 to -76
(WT, Table 1
). Nucleotide sequences of
wild type OC-Box and mutant OC-Box oligonucleotides that were used as
competitors are detailed in Table 1
. Dlx-5 protein binds strongly to
the OC-Box oligonucleotide (Fig. 7
). Products of a control in
vitro translation reaction without template and the
homeobox-deleted Dlx-5 protein showed no binding (Fig. 7
, lanes 1 and
2). Dlx protein-OC-Box sequence interactions in the presence of 50-fold
excess of wild type or mutant oligonucleotide competitors are shown
(Fig. 7
, lanes 412). Wild type OC-Box (WT), homeobox binding
consensus site (hbs), and mCC2 efficiently compete for Dlx-5 binding.
The mT-T, mAG, and mCC1 oligonucleotides show very low affinity for
Dlx-5, while the mAA, m8, and mutant homeobox consensus binding site
(mhbs) show no competition for Dlx-5 binding. These data confirm the
specificity of Dlx-5 interactions with the core ATTA homeodomain
binding site within the OC-Box.

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Figure 7. Binding Activity of in Vitro
Transcribed and Translated Dlx-5 with Wild Type OC-Box and Several
Mutants
Gel mobility shift assay of -32P end-labeled OC-Box
oligonucleotide bound with Dlx-5 synthesized by in vitro
translation. Lane 1 (no Dlx-5) shows binding of an in
vitro translation reaction that did not contain a template;
lane 2 (no Hom), binding activity of mutant Dlx-5 lacking the homeobox;
lane 3, WT, binding of in vitro translated WT Dlx-5;
lanes 412 show various effects of DNA competitors on Dlx-5
bind-ing including several mutant oligonucleotides described in
Table 1 .
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To further establish that this site within the OC-Box mediates
Dlx-5 responsiveness, OC promoter (-351 to +32) constructs containing
either wild type, mAA, or mAG mutant OC-Box sequences fused to the
luciferase reporter were cotransfected with Dlx-5 or a control plasmid
in ROS 17/2.8 cells. These mutations result in a significant decrease
in promoter activity (Fig. 8
). Forced
expression of Dlx-5 significantly suppressed wild type -351-Luc OC
promoter activity, but had no effect on activity of the constructs
containing the mutated OC-Box sequences (Fig. 8
). Thus mutations that
did not compete or competed weakly with wild type OC-Box sequences in
the gel mobility shift assays (Fig. 7
) were also not responsive to
Dlx-induced down-regulation of OC promoter activity. This finding
confirms that the homeodomain-binding site within the OC-Box is the
recognition sequence for functional Dlx-5 activity.

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Figure 8. Functional Analysis of Dlx-5 Activity on Wild Type
and OC-Box Promoter Mutations
Expression of (-1050) OC-Luc WT and the OC-Box mutant constructs in
ROS 17/2.8 cells in the presence (+ Dlx-5, hatched
columns) or absence (control, solid bars) of
Dlx-5 expressing plasmid. Cells were assayed for luciferase activity
48 h after transfection. Overexpression of Dlx-5 had no effect on
promoter activity of the OC-Box mutant constructs. Values are mean and
SD of six independent transfection experiments.
Transfection efficiencies were monitored by cotransfection of OP-CAT
and assaying CAT activity.
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Dlx-5 Regulates Endogenous Expression of the Bone-Specific
OC Gene
We have established that the OC gene represents a target for
Dlx-5 transcriptional regulation, which occurs via OC-Box I, a primary
tissue-specific promoter element (26, 35). To address directly whether
Dlx-5 can modulate endogenous OC gene expression, we created cell lines
conditionally expressing Dlx-5. ROS 17/2.8 cells expressing the
tetracycline regulated transactivator were stably transfected with a
Dlx-5 expression plasmid controlled by tetracycline. A cell line that
exhibited clear up-regulation of Dlx-5 by tetracycline was selected and
further studied. Endogenous expression of different genes was assayed
by Northern analysis in the presence or absence of tetracycline (Fig. 9
). Tetracycline levels were used that
have been shown not to influence the levels of OC biosynthesis in cells
lacking the Dlx-5 construct (data not shown). When tetracycline is
removed, expression of Dlx-5, fibronectin, and collagen type I is
up-regulated. Strikingly, elevation of Dlx-5 expression results in
down-regulation of OC expression. This finding supports our
observations in transiently transfected cells. For comparison, histone
H4, osteopontin, and alkaline phosphatase expression is not altered by
the tetracycline-related up-regulation of Dlx-5 expression.

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Figure 9. Effects of Dlx-5 Overexpression on Osteoblast
Proliferation and Differentiation Markers
ROS 17/2.8 cells containing the gene for the tetracycline-responsive
transactivator were stably transfected with a Dlx-5 expression plasmid
controlled by this transactivator. Stable cell lines with demonstrated
significant changes of Dlx-5 expression in the presence or absence of
tetracycline were selected by Northern analysis. Cells were maintained
in media containing tetracycline (TC+) or harvested 48 h after the
removal of tetracycline (TC-). Ten micrograms of total cellular RNA
were analyzed for the expression of OC, fibronectin (FN), collagen type
I (COL-I), histone H4 (H4), osteopontin (OP), alkaline phosphatase
(AP), and 18S ribosomal RNA (18S) as a loading control.
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DISCUSSION
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In this study we have characterized a differentially
displayed Dlx homeodomain gene, which is selectively up-regulated
during development of the osteoblast phenotype. The Dlx gene we
identified in differentiated osteoblasts was already reported as rDlx
(19) cloned from chondrosarcoma cells, and Dlx-3 in rat (32). However,
our comparison of amino acid similarities in the homeodomain, as well
as 5'- and 3'-flanking sequences indicates that rDlx, Dlx-3, and the
Dlx gene described here all represent the rat homolog of Dlx-5.
Dlx-5 Represses OC Gene Transcription
Our identification of a Dlx-5-responsive cis-acting
promoter element in the bone-specific OC gene provides the first
evidence for a Dlx-5 responsive target gene. We have presented multiple
converging lines of evidence that indicate that Dlx-5 functions to
repress OC gene transcription. For example, transient coexpression of
Dlx-5 decreases OC promoter activity, whereas antisense Dlx-5 mRNA
expression increases OC gene transcription. This down-regulatory effect
of Dlx-5 overexpression on OC promoter activity is observed in both
osseous (ROS 17/2.8) and nonosseous (IMR-90) cells and is also observed
in cells in which endogenous Dlx-5 mRNA levels are low or below the
level of detection (e.g. proliferating ROB and IMR-90
cells). Moreover, conditional expression of Dlx-5 in ROS 17/2.8 cells
results in repression of endogenous OC gene expression. Thus, our data
suggest that the OC gene represents a bona fide cellular target for
Dlx-5.
Our data show that the levels of Dlx-5 mRNA, and consequently its
inherent repressive function, are up-regulated when OC gene expression
is induced to maximal levels during osteoblast maturation in the
mineralization period. Induction of OC gene expression is determined in
part by a multiplicity of potent trans-activators that
mediate basal tissue-specific transcription (36). In addition,
increased OC gene expression appears to be the combined effect of
transcriptional up-regulation and mRNA stabilization (37). Therefore,
we suggest that Dlx-5 may attenuate this potentially hyperactive level
of OC gene expression in differentiated osteoblasts to permit
physiological control of OC biosynthesis. This function would maintain
a suboptimal tissue-specific basal transcription rate, which would
render the gene responsive to enhancement by other gene-regulatory
signaling mechanisms required for bone renewal (e.g. vitamin
D3). Furthermore, several lines of evidence indicate that
negative regulation of OC may be important for proper bone development
(38, 39, 40). However, apart from a role for Dlx-5 in regulating OC gene
transcription, it is plausible that other target genes exist in which
Dlx-5 may perhaps repress or activate gene transcription in conjunction
with other transcription factors and depending on promoter context.
We and others have previously shown that the Msx-2 homeodomain protein
is also a repressor of OC gene transcription (25, 26, 27). The results
presented here reveal that Dlx-5 and Msx-2 recognize the same
homeodomain motif located in the tissue-specific promoter element
OC-box I. Interestingly, Msx-2 is expressed at maximal levels in
proliferating osteoblasts, whereas maximal Dlx-5 expression is
restricted to differentiated osteoblasts. The stringent negative
regulation of OC expression by Msx-2 in the proliferation period and by
Dlx-5 later in differentiated osteoblasts is consistent with the
concept that OC biosynthesis is tightly controlled at specific stages
of osteoblast maturation to facilitate developmental modifications in
extracellular matrix composition, mineralization, and maintenance of
the bone cell phenotype.
Recently, Abate-Shen and colleagues (41) have shown that Dlx-2, which
recognizes the same DNA motif as Dlx-5, can activate transcription of
chimeric promoter constructs. Furthermore, Dlx-2 and Dlx-5 are each
capable of abrogating Msx-2-dependent repression by direct
protein/protein interactions, which results in mutual interference of
Msx and Dlx binding activity. These results may have important
ramifications for understanding the functional effects of modulations
in Msx-2 and Dlx-5 levels during osteoblast differentiation. For
example, our data show that during the postproliferative period, but
before the onset of extracellular matrix mineralization, there is a
brief temporal overlap in Dlx-5 and Msx-2 expression. At this time,
Dlx-5 and Msx-2 may form heterodimers and mutually negate repressive
transcriptional effects, which could result in transient derepression
of OC gene transcription coinciding with induction of OC gene
expression.
Dlx-5 Expression and Osteoblast Differentiation
The rat Dlx-5 gene is expressed in developing cartilage, discrete
neuronal tissues, and teeth (19). The mouse homolog of the gene is also
expressed in all developing skeletal elements (20), with Dlx-5
expression proceeding along the mineralization front during long bone
growth. Similarly, the chick homolog of Dlx-5 plays an important role
in limb bud development and cartilage differentiation (9). These data
suggest that Dlx-5 genes have important and evolutionary conserved
roles in tissue development of cartilage and bone. Our findings provide
an important demonstration that Dlx-5 expression correlates with
osteoblast differentiation and suggest Dlx-5 may be involved in
maturation of the bone cell phenotype. We show that maximal expression
of Dlx-5 occurs in the final stages of osteoblast differentiation
in vitro when the extracellular matrix mineralizes. This
pattern of Dlx-5 expression may reflect a general role of Dlx-5 in
lineage commitment and progression of osteoblast differentiation.
Of interest, the runt domain containing CBFA/AML factors
were shown to regulate bone tissue-specific expression of the OC gene
(42, 43, 44, 45, 46). More recently the CBFA1/AML-3 transcription factor was shown
to be abundantly expressed in bone and less in thymus, but no other
soft tissues (42, 45), and is a key regulator of bone formation in the
developing embryo (46, 47, 48) and osteoblast differentiation in
vitro (42). CBFA1/AML-3 appears slightly later in the formation of
the mouse skeleton (912 days) (46, 47, 48), followed by the homeodomain
proteins, Msx-2 and Dlx-5 (11, 19, 41, 49, 50, 51). Both Dlx-5 and Msx-2
are coexpressed in similar zones during early embryological development
(e.g. the AER) at approximately 8.5 days (8, 10), and their
expression is prominent along the anterior margin of the limb bud
mesenchyme. CBFA1/AML-3 expression peaks at 12.5 days in mesenchymal
condensations of developing bone structure (46, 47) before the first
ossification center at 14.5 days. Dlx-5, Msx-2, and CBFA1/AML-3 may
control osteogenesis analogous to the regulators of myogenesis
(e.g. MyoD, myf-5, myogenin, and MRF4) (52, 53) and
adipocyte differentiation (e.g. PPAR
and
C/EBPs)(54, 55, 56). Each of these sets of factors together control lineage
determination and/or execution of the final differentiation program.
Msx, Dlx, and AML genes may provide a cascade of factors that
contribute to the initial formation and development of the skeleton and
to osteoblast differentiation and maturation during postnatal bone
formation. Thus, Dlx-5 appears to be a component of the combinatorial
mechanism that controls formation and differentiation of skeletal
tissues and may contribute to the progression of osteoblast
differentiation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Tissue Isolation
Rat osteoblasts (ROB) were prepared from calvaria of 21-day
fetal rats as described by Owen et al. (57). Briefly, ROB
cells were plated at a density of 6.5 x 105 cells per
100-mm dish and grown in MEM (GIBCO, Grand Island, NY) supplemented
with 10% FCS. Osteoblast mineralization was enhanced by the addition
of 50 mg/ml ascorbic acid and 10 mM ß-glycerophosphate in
medium on day 5 after plating. Osteoblast-like rat osteosarcoma cells,
ROS 17/2.8 (58), were cultured at a density of 4.0 x
105 cells per 100-mm dish in F-12 medium (GIBCO)
supplemented with 5% FCS. C2C12 mouse myoblasts were plated for
transfection at 4 x 105 cells per 100-mm disk in DMEM
(GIBCO) supplemented with 10% FCS. IMR-90 rat fibroblasts were plated
for transfection at 6 x 105 cells per 100-mm dish in
Basal Medium Eagle (BME, GIBCO) supplemented with 10% FCS. Soft
tissues and bone were harvested from a 3-month-old male mouse and
stored at -70 C. Calvaria and long bone were cleaned of periosteum and
marrow.
RNA Isolation and Purification
Total cellular RNA was extracted with TriZol (GIBCO/BRL,
Gaithersburg, MD) (59) according to the manufacturers instructions.
To extract cytoplasmic RNA, harvested cells were resuspended in lysis
buffer (50 mM Tris-Cl, pH 8.0, containing 100
mM NaCl, 5 mM MgCl2, and 0.5%
Nonidet P-40), incubated for 5 min on ice, and centrifuged at
15,000 x g for 2 min at 4 C. RNA was extracted from
the supernatant with TriZol. Frozen tissues were ground and resuspended
in TriZol solution to extract the RNA. Extracted RNA (100 µg) was
incubated for 30 min at 37 C with 20 U of RNasin (Promega, Madison, WI)
and 20 U of RNase free DNase I (Promega, Madison, WI) in 10
mM Tris-Cl, pH 8.3, 50 mM KCl, 1.5
mM MgCl2. Samples were phenol/chloroform
extracted, chloroform extracted, and then ethanol precipitated. RNA was
resuspended in DEPC (diethylpyrocarbonate)-treated water. The RNA
integrity was assessed by the 28S/18S ribosomal RNA ratio after
electrophoresis in 1% agarose/5.5% formaldehyde gels.
Differential Display
Differential display analysis was carried out according to Ref.
30 with modifications as described by Zhao et al.) (60).
Briefly, RNA was prepared from ROB cells harvested on day 2
(proliferating cultures) and day 21 (mineralized cultures). Total RNA
(300 ng) was reverse transcribed in a 30-µl reaction mixture with 300
U of superscript murine Moloney leukemia virus reverse transcriptase
(GIBCO/BRL) and 100 U of RNasin in the presence of 2.5 mM
of random hexamer and 20 mM deoxynucleoside triphosphate
(dNTP) for 60 min at 40 C. Control reactions were performed in the
absence of reverse transcriptase. Two microliters of the reverse
transcription product were amplified with the GeneAmp kit (Perkin-Elmer
Cetus, Norwalk, CT) in the presence of 0.5 µM 3'-primer
and 5'-primer with 2 µM dNTPs and 0.5 µl
35S-
dATP (NEN, Boston, MA). To detect Dlx family
members, a 5'-primer (5'-ANCNCAGGTSAAAATCTGG-3') was designed from very
highly conserved homeobox sequences, and 3'-primers were designed from
the loosely conserved 3'-end of Dlx coding regions
(5'-GGCAGGTGGGAATTGATTGA-3'; D3). The buffer, MgCl2, and
Taq polymerase concentrations were as suggested by the
manufacturer (Perkin-Elmer Cetus). The temperature profile was as
follows: one cycle of 94 C for 1 min, 42 C for 4 min, and 72 C for 1
min, followed by 35 cycles as follows: 94 C for 1 min, 60 C for 2 min,
and 72 C for 1 min, and a final 5-min elongation at 72 C. Amplified
cDNAs were separated on a 6% 29:1 polyacrylamide denaturating gel. The
gel was dried and exposed for 2448 h to BioMax film (Kodak,
Rochester, NY). The cDNA bands of interest were excised and eluted with
100 µl of Tris-EDTA. The eluted cDNA was ethanol precipitated. One
half of the recovered cDNA was reamplified under the same PCR
conditions as the first PCR reaction in the absence of isotope and
increased dNTP concentrations to 20 µM. Ten of 50 µl of
the PCR product were electrophoresed in a 1% agarose gel to estimate
molecular weight and concentration of cDNA in the PCR product. The
remaining samples were stored at minus]20 C for screening and cloning.
The PCR product was cloned into the pCR II vector using the TA-cloning
system (Invitrogen, San Diego, CA). The cloned cDNA insert was
sequenced with Sequenase Version 2.0 (USB, Cleveland, OH). The
nucleotide sequences obtained were compared with known sequences by
searching GenBank and EMBL databases (March 1996) with the Fasta
program (Genetic Computer Group, Madison, WI).
Northern Blot Analysis
Total cellular RNA (2030 µg) was separated on a 1%
agarose/5.5% formaldehyde gel and transferred to Zetaprobe membrane
(Bio-Rad, Melville, NY) using 20x NaCl-sodium citrate (SSC) buffer.
RNA was cross-linked to filters by UV irradiation for 1 min and stored
until use. DNA probes, either PCR product or cloned cDNA of Dlx-5 (this
paper), human histone H4 cDNA (pFO 002) (61), rat OC (62), and rat
Msx-2 (25) were labeled with
-[32P]dCTP (3,000
Ci/mmol; NEN, Boston, MA) using the random primer technique (63). The
blot was prehybridized in 50% formamide, 5 x SSPE (0.18
M NaCl, 0.01 M NaH2PO4,
0.001 M Na2EDTA, pH 7.7), 5 x Denhardts
solution, 0.1% SDS, and 100 µg/ml salmon sperm DNA at 42 C for
3 h. For hybridization, 106 cpm/ml of heat denatured
radioactive DNA probe was added and incubated at 42 C overnight. After
hybridization, the blots were washed three times in 2 x SSC/0.1%
SDS at room temperature for 15 min each, twice in 0.1 x SSC/0.1%
SDS at room temperature for 20 min each, and twice in 0.1x SSC/0.1%
SDS at 42 C for 20 min each. Blots were exposed to Kodak XAR film at
-70 C with intensifying screens.
Plasmids
Complementary DNA containing 95% of Dlx-5 coding sequences,
including the translation start site, was obtained by RT-PCR and cloned
into the pCR II vector (Invitrogen, San Diego, CA). Dlx-5 sequences
were then subcloned into pcDNA I/Amp (Invitrogen, San Diego, CA) using
the XbaI/HindIII restriction sites and designated
pDlx-5. For expression in eukaryotic cells, Dlx-5 sequences were
removed from pDlx-5 by EcoRI digestion and placed in the
pUHD103 vector, which contains the tTA responsive element (64). This
clone is designated pUHD103/Dlx-5. Homeobox-deleted Dlx-5 was
produced by PCR amplification of part of the Dlx-5 clone, as previously
described by Zarlegna et al. (65), and designated
pDlx-5-Del. Clones were confirmed by restriction digestion and
sequencing.
OC promoter deletion constructs in the luciferase vector, -1050
OC-Luc, -637 OC-Luc, -199 OC-Luc, and -83 OC-Luc, were constructed
as previously described by Towler et al. (27). OC
promoter/OC-Box mutant constructs in the pGL2 luciferase vector were
subcloned from previously constructed promoter mutants (25) as follows:
Promoter fragments were removed by BglII/HindIII
(-351 to +32 of OC promoter) or XhoI/HindIII
(-1097 to +32 of OC promoter) digestions and placed into pGL2-luc
(Promega, Madison, WI). The plasmids used for tTA stable transfection,
pUHD 151, pUHD 133, pUHD 103, and pSV2Neo, were the kind gift of
Dr. Gossen (64). RSV-luc and CMV-luc reporter plasmids contain the
luciferase gene (66) in the pGL2 vector (Promega). 205 H4CAT is
described by Ramsey-Ewing et al. (67) as F0108CAT and
contains 205 nucleotides of the H4 proximal promoter. TKCAT is pBLCAT2,
as described by Luckow and Schütz (68). The SV40CAT is described
by Gorman et al. (69) as pSV2CAT. Construction of the VDRE
tetramer construct is described in Blanco et al. (70). The
osteopontin (OP)-CAT chimeric gene construct pOPCAT is described in
Ref. 71 and contains 776 nucleotides of the proximal osteopontin
promoter. The AML-1B expression construct has been described by Meyers
et al. (72), and construction of the AML/108CAT reporter
gene plasmid (pTGRECAT) has been documented by Banerjee et
al. (73).
Stable Transfection of Tetracycline-Regulated Transactivator
Stable transfection of tTA into ROS 17/2.8 rat osteosarcoma
cells was done via the calcium phosphate coprecipitation method (74).
Briefly, ROS 17/2.8 cells were plated at a density of 0.65 x
106 cells per 100-mm plate 24 h before transfection.
pUHD 151 plasmid (18.6 µg) was cotransfected with 1.4 µg pSV2Neo.
G418-resistant colonies were selected by adding 150 µg/ml of G418
into the media for 23 weeks. Success of stable transfection of tTA,
pUHD 151, was screened by transient transfection of pUHD 133, which
contains a tTA-responsive element controlling luciferase. Clone 316
showed 20-fold induction of luciferase activity when tetracycline (1
µg/ml) was removed and was therefore chosen for further
experiments.
Clone 316 was subject to second round stable transfection with
pUHD103 Dlx-5 and a Tk-hygromycin vector (75) using the calcium
phsophate coprecipitation method (74). The transfected cells were
maintained for 2 to 3 weeks in selection medium containing F12 (GIBCO,
BRL, Grand Island, NY), 5% FCS, 150 µg/ml G418, 200 µg/ml
hygromycin B (Calbiochem, La Jolla, CA), and 1 µg/ml tetracycline.
Viable colonies were subcultured under the same conditions and used for
experiments and screened by Northern analysis for
tetracyclin-controlled regulation of Dlx-5 expression. To determine the
effect of Dlx-5 overexpression on OC gene expression, cells were
cultured at a density of 6 x 105 cells per 100-mm
plate and maintained in selection medium. Seven days after plating,
Dlx-5 expression was induced by removal of tetracycline from the medium
48 h before harvesting.
Transfection Assays
Cells were plated at a density of 46 x 105
cells per 100-mm plate for transient transfection experiments. ROS
17/2.8, ROB, C2C12, and tTA stably transfected ROS 17/2.8 cells were
transfected by the diethylaminoethyl-dextran method (74). IMR-90 cells
were transfected by the HEPES/calcium phosphate method (74) with a
1-min 10% dimethylsulfoxide shock. The total amount of exogenous DNA
was maintained at 20 µg/plate consisting of 2 µg luciferase
construct, 8 µg CAT construct, 4 µg of a Dlx construct, and 6 µg
Salmon sperm DNA. The IMR-90 cells were transfected with 1 µg AML-1B
expression plasmid and 5 µg Salmon sperm DNA. All plasmid DNA was
prepared using Qiagen Maxi Kits (Qiagen Inc., Chatsworth, CA) and
checked for supercoiled structure on 1% agarose/0.045 M
Trizma base/0.045 M boric acid/1.25 mM EDTA
(TBE) gels. Plasmids of similar quality were used for comparison of
relative expression in each experiment. Cells were harvested 48 h
after transfection.
Luciferase and CAT Assays
Luciferase activity was determined using the luciferase assay
system (Promega, Madison, WI). The cell pellets were treated with 1x
reporter lysis buffer (0.25 M Tris-HCl, pH 8.0, 0.1%
Triton X-100, Promega, Madison, WI), and luminescence was measured on a
Monolite 2010 (Analytical Luminescence Laboratory, San Diego, CA). CAT
activity was determined as previously described (74). The samples were
incubated with 0.25 µCi (1 Ci = 37 gigabecquerels) of
[14C]chloramphenicol (Dupont, Boston, MA) for 412 h,
extracted with ethyl acetate, and separated by chromatography. Results
were evaluated using a ß-scope 603 blot analyzer from Betagen
(Mountain View, CA).
In Vitro Transcription and Translation of Dlx5
Protein
Plasmids containing wild type Dlx-5 and homeobox-deleted Dlx-5
sequences in the pCR II vector were linearized by restriction digest
and used as templates for in vitro transcription with the
Sp6 promoter. These transcripts were used for translation of protein
using the TNT TM Coupled Reticulocyte lysate system (Promega, Madison,
WI).
Gel Mobility Shift Assay
Wild type OC-Box oligonucleotide was end labeled with
32P
-dATP by using T4 polynucleotide kinase. The probe
and mutant oligonucleotide used as competitors are in Table 1
. Gel
mobility shift assays were performed by binding in vitro
transcribed and translated Dlx-5 protein (5 µl) to a labeled,
double-strand DNA probe in the presence or absence of 50-fold molar
excess of competitor for 10 min at room temperature. The binding
reaction mixtures contained 10 mM Tris-HCl, pH 7.5, 50
mM NaCl, 5% glycerol, 5% sucrose, 0.2 mM
EDTA, 7.4 mM MgCl2, 500 µg BSA per ml, 0.1%
Nonidet P-40, 50 µg poly(deoxyinosinic-deoxycytidylic)acid per ml,
and 10 mM dithiothreitol. Protein-DNA complexes were
separated at 4 C on a 6.5% polyacrylamide gel containing 0.5x TBE
buffer.
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank L. Buffone, J. Green, and C. Capparelli for
technical support, and Judy Rask for manuscript preparation.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jane B. Lian, Ph.D., Department of Cell Biology, University of Massachusetts Medical Center, 55 Lake Avenue, North Worcester, Massachusetts 01655.
This publication was made possible by NIH Grants AR-33920 and AR-39588.
The contents are solely the responsibility of the authors and do not
necessarily represent the official views of the NIH.
1 Present address: Department of Biochemistry, School of Dentistry,
Kyungpook National University, Taegu, Korea, 700422. 
Received for publication January 13, 1997.
Revision received June 30, 1997.
Accepted for publication July 26, 1997.
 |
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