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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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). 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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). 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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). 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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). 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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). 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 4–12). 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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). 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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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (9–12 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RNA Isolation and Purification
Total cellular RNA was extracted with TriZol (GIBCO/BRL,
Gaithersburg, MD) (59) according to the manufacturer’s 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 24–48 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 (20–30 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 Denhardt’s
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
pUHD10–3 vector, which contains the tTA responsive element (64). This
clone is designated pUHD10–3/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 15–1, pUHD 13–3, pUHD 10–3, 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 15–1 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 2–3 weeks. Success of stable transfection of tTA,
pUHD 15–1, was screened by transient transfection of pUHD 13–3, 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 pUHD10–3 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 4–6 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 4–12 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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, 700–422. 
Received for publication January 13, 1997. Revision received June 30, 1997. Accepted for publication July 26, 1997.
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