Ascorbic Acid-Dependent Activation of the Osteocalcin Promoter in MC3T3-E1 Preosteoblasts: Requirement for Collagen Matrix Synthesis and the Presence of an Intact OSE2 Sequence
Guozhi Xiao,
Yingqi Cui,
Patricia Ducy,
Gerard Karsenty and
Renny T. Franceschi
Department of Periodontics, Prevention, and Geriatrics (G.X.,
Y.C., R.T.F) School of Dentistry and Department of Biological
Chemistry School of Medicine University of Michigan Ann
Arbor, Michigan 48109-1078, and Department of Molecular Genetics
(P.D., G.K.) University of Texas/MD Anderson Cancer Research
Institute Houston, Texas 77030
 |
ABSTRACT
|
---|
Osteocalcin is a hormonally regulated
calcium-binding protein made almost exclusively by osteoblasts. In
normal cells, osteocalcin expression requires ascorbic acid (AA), an
essential cofactor for osteoblast differentiation both in
vivo and in vitro. To determine the mechanism of this
regulation, subclones of MC3T3-E1 preosteoblasts were transiently
transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter
driving expression of firefly luciferase. AA stimulated luciferase
activity 20-fold after 45 days. This response was stereospecific to
L-ascorbic acid and was only detected in
MC3T3-E1 subclones showing strong AA induction of the endogenous
osteocalcin gene. Similar results were also obtained in MC3T3-E1 cells
stably transfected with the osteocalcin promoter. A specific inhibitor
of collagen synthesis, 3,4-dehydroproline, blocked AA-dependent
induction of promoter activity, indicating that regulation of the
osteocalcin gene requires collagen matrix synthesis. Deletion analysis
of the mOG2 promoter identified an essential region for AA
responsiveness between -147 and -116 bp. This region contains a
single copy of the previously described osteoblast-specific element,
OSE2. Deletion and mutation of OSE2 in DNA transfection assays
established the requirement for this element in the AA response.
Furthermore, DNA-binding assays revealed that MC3T3-E1 cells contain
OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to
OSE2 is up-regulated by AA treatment. Taken collectively, our results
indicate that an intact OSE2 sequence is required for the induction of
osteocalcin expression by AA.
 |
INTRODUCTION
|
---|
Ascorbic acid (AA, reduced vitamin C) is essential for the
formation of bone (1) and other connective tissues and necessary for
the in vitro differentiation of osteoblasts and other
mesenchyme-derived cell types including adipocytes, myoblasts,
chondrocytes, and odontoblasts [for review, see Franceschi, 1992
(2)]. In vivo autoradiographic studies demonstrated
that radiolabeled vitamin C, when injected systemically, accumulates at
sites of active bone formation (3). To facilitate this process,
osteoblasts contain a specific Na+-dependent AA transport
system that is essential for the intracellular accumulation of vitamin
C and cellular responsiveness (4, 5). In primary cultures of
osteoblast-like cells (6) or nontransformed osteoblast-like cell lines
such as murine MC3T3-E1 cells (7, 8, 9), AA stimulates procollagen
hydroxylation, processing, and fibril assembly followed by a dramatic
induction of specific genes associated with the osteoblast phenotype
including those encoding osteocalcin, alkaline phosphatase, bone
sialoprotein, and the PTH/PTH-related protein receptor. Actions of AA
on bone require collagen matrix synthesis. Identical dose-response
relationships are obtained for the stimulation of collagen matrix
formation by AA and induction of osteoblast markers. Furthermore,
induction of gene expression by AA is specifically and reversibly
blocked by inhibitors of collagen matrix formation or purified
bacterial collagenase (7, 8). Thus, studies with AA strongly support a
model in which expression of the osteoblast phenotype is profoundly
affected by the extracellular matrix (ECM).
To begin exploring mechanisms involved in AA/collagen matrix-dependent
regulation of osteoblast-specific gene expression, we have focused on
the gene for osteocalcin, a
-carboxylated calcium-binding protein
whose expression is largely restricted to the osteoblasts of bone and
the odontoblasts and cementoblasts of teeth (10). Osteocalcin
expression is regulated by a number of calcitropic hormones, including
calcitriol and glucocorticoids (11, 12, 13). In the mouse, two osteocalcin
genes (mOG1, mOG2) and a third osteocalcin-related gene (ORG) form a
contiguous 23-kb gene cluster (14). mOG1 and mOG2 are highly homologous
and expressed only in bone whereas ORG is expressed only in kidney.
Like the osteocalcin gene in human (15) and rat (12), mOG1 and mOG2
each contain four exons and a proximal promoter region containing
canonical TATA and CCAAT box sequences. Two additional regions,
designated OSE1 and OSE2, that are required for the selective
expression of this gene in osteoblast-like osteosarcoma cells have been
identified in the promoter region of mOG2 (16). Both regions bind
nuclear factors from osteosarcoma and primary osteoblast cultures that
are not detected in other tissues. OSF2, the nuclear factor that binds
to OSE2, was recently shown to be immunologically and functionally
related to the polyomavirus enhancer core binding protein
/acute
myeloid leukemia (PEBP
/AML) family of transcription factors (17, 18). In the present study, we show that mOG2 promoter activity is
up-regulated by AA and that this response requires collagen matrix
synthesis and the presence in the mOG2 promoter of an intact OSE2
sequence.
 |
RESULTS
|
---|
Analysis of MC3T3-E1 Subclones for AA Responsiveness and OG2
Promoter Induction
MC3T3-E1 cells, like other osteoblast cells lines (19), are
phenotypically heterogeneous (i.e. only a fraction of cells
exhibit osteoblast characteristics). To obtain a highly responsive cell
population, MC3T3-E1 cells were subcloned by limiting dilution, and
approximately 50 clones were isolated. RNA was prepared from individual
clonal lines that were grown for 12 days in the presence or absence of
AA. Northern blots were probed for two osteoblast-related mRNAs,
osteocalcin and bone sialoprotein. Representative clones with high and
low responsiveness to AA are shown in Fig. 1
, A and B.
Clones 4, 14, and 26 expressed osteocalcin and bone sialoprotein mRNAs
at high levels whereas clones 17 and 24 had low to undetectable levels
of message. Lipofectamine was used to transiently transfect each clone
with p1.3OG2-luc, a plasmid containing 1.3 kb of the proximal OG2
promoter driving expression of firefly luciferase, and pSV2CAT. As
shown in Fig. 1C
, AA most dramatically stimulated luciferase activity
in clones exhibiting high induction of the endogenous osteocalcin gene.
The parent MC3T3-E1 cell population used for cloning showed only a
modest induction, which may explain our initial difficulty in detecting
AA stimulation of this promoter before subclones were examined. The
unrelated cytomegalovirus (CMV) promoter was not induced by AA
treatment nor was the pSV2CAT used for normalization of transfections
(result not shown). Highly responsive clone 4 cells were used in all
subsequent transient transfection experiments. Because important
cis-acting elements have been described in the promoter of
the mOG2 gene, we focused the remainder of our analysis on the role of
AA in osteocalcin expression.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 1. AA Induction of p1.3OG2-luc Expression in MC3T3-E1
Subclones
A and B, Expression of endogenous osteoblast-related mRNAs. MC3T3-E1
subclones isolated as described in Materials and Methods
or the parent cell line were cultured for 5 days in the presence (+) or
absence (-) of 50 µg/ml AA. For each group, total RNA was extracted
for Northern blot hybridization using cDNA probes for osteocalcin mRNA
(OCN), bone sialoprotein mRNA (BSP), and 18S rRNA (for normalization).
Panel B shows normalized values for osteocalcin mRNA as determined by
imaging of hybridized blots. C, Normalized luciferase activity.
MC3T3-E1 cells or subclones were cotransfected with p1.3OG2-luc
reporter plasmid (0.5 µg) and pSV2CAT (0.5 µg) and treated as
described in panel A. A separate group of subclone 4 cells was
transfected with pcDNA3-luc, which uses the CMV promoter to drive
luciferase expression (4/CMVluc). After 5 days, cells were harvested
and assayed for luciferase and CAT activities. In all cases, luciferase
was normalized to CAT activity, which did not vary by more than 50%
between samples. Experiments were repeated a minimum of two times.
Error bars indicate SD of the mean of three separate
transfections.
|
|
Time Course and Specificity of mOG2 Induction by AA
Several days of AA treatment are required for induction of
endogenous osteocalcin (7). Consistent with this observation, 4 to 5
days were required for AA to increase luciferase activity in cells
either transiently or stably transfected with OG2-luc (Figs. 2A
and 3C
). Endogenous osteocalcin mRNA
was also induced in both transient and stable clones (Fig. 2A
, inset, and Fig. 3
, A and B). Because the 5-day period
required for detection of the AA response is relatively long for a
transient transfection experiment, two control studies were carried out
to exclude differences in plasmid retention or luciferase stability
between control and AA-treated cells as possible explanations for
results. Plasmid DNAs were measured by slot blot hybridization using
aliquots of total cellular DNA isolated 5 days after transfection (Fig. 2B
). For both p1.3OG2-luc and pSV2CAT, the relative level of plasmid in
AA vs. control cells was approximately 1.3. The half-life of
luciferase enzyme activity in AA and control cells was nearly the same
(
18 h; results not shown). Neither result can explain the 15- to
20-fold induction of luciferase activity seen with AA
treatment.

View larger version (34K):
[in this window]
[in a new window]
|
Figure 2. Characterization of AA Induction of p1.3OG2-luc in
Transient Transfections
A, Time course. MC3T3-E1 subclone 4 cells were cotransfected with
p1.3OG2-luc/pSV2CAT (2.0 µg each) for 4 h. After transfection,
the cells are cultured for different times (1, 2, 3, 4, and 5 days) in
the presence (closed circles) or absence (open
circles) of AA. Inset, Endogenous osteocalcin
mRNA. A separate set of identically transfected cells were used to
measure endogenous osteocalcin mRNA by Northern blot hybridization
after 5 days with (5AA) or without (5C) AA. B, Measurement of
transfected plasmid levels. Cells were transfected as in panel A and
cultured with or without AA for 5 days. Total cellular DNA was
isolated, and DNA equivalent to the amount of cell extract used for
luciferase assays was either blotted directly or diluted as indicated
before being probed for p1.3OG2-luc (Luciferase) or pSV2CAT (CAT).
Imaging of blots gave the following ratios for each plasmid in
AA-treated/control cells: p1.3OG2-luc, 1.3; pSV2CAT, 1.33. C,
Stereospecificity of AA induction. MC3T3-E1 subclone 4 cells were
transfected as described in Fig. 1C . Cells were then grown for 5 more
days in control medium containing increasing concentrations of
L-AA (open circles) or
D-isoascorbic acid (closed circles). Values
are mean ± SD of triplicate transfections.
|
|
The use of a cell line containing stably integrated p1.3OG2-luc allowed
a more detailed analysis of the relationship between activation of the
transfected promoter and endogenous osteocalcin and bone sialoprotein
mRNAs during a long-term (21-day) culture period (Fig. 3
). In control
cultures, luciferase activity and levels of both mRNAs remained low at
all times. As was seen with transient transfections, AA consistently
stimulated luciferase activity after 46 days. Activity continued to
increase to day 21. Endogenous osteocalcin mRNA was first detected
after 68 days and generally paralleled luciferase activity for the
duration of the experiment. Bone sialoprotein mRNA followed a
similar time course.
A Na+-dependent AA transporter is required for MC3T3-E1
cells to respond to vitamin C (5). This transporter is stereospecific
in that it allows cells to preferentially accumulate L-AA
vs. D-isoascorbic acid (D-IAA),
thereby explaining known differences in the biological activity of
these two isomers both in vivo and in cell culture (5, 20).
Marked stereoselectivity for extracellular L-AA relative to
D-IAA was also seen when AA-dependent OG2 promoter activity
was measured after 5 days in culture. As shown in Fig. 2C
, half-maximal
stimulation of the OG2 promoter was achieved with 6 µM
L-AA vs. 30 µM for
D-IAA, a 5-fold difference. This stereoselectivity is
similar to that previously reported for AA transport in the same cell
line (5).
A Specific Collagen Matrix Synthesis Inhibitor, 3,4-Dehydroproline,
Blocks AA-Dependent Induction of Promoter Activity
In an earlier study, we showed that 3,4-dehydroproline
(3,4-DHP) blocked AA-dependent induction of endogenous osteocalcin mRNA
(7, 8). Effects of this and other inhibitors were reversible and could
not be explained by nonspecific toxicity. As shown in Fig. 4
, 3
, 4
-DHP (500 µM) used under identical
conditions to those of our earlier work almost completely blocked
AA-dependent induction of the mOG2 promoter if the inhibitor was added
to cells at the beginning of the experiment (compare 5AA with 5AA +
DHP). In contrast, if cells were treated with AA for 2 days before
addition of inhibitor and promoter activity was measured after 3 more
days, only partial inhibition was seen (compare 5AA + DHP with 2AA/3AA
+ DHP). As a further control to eliminate the possibility that 3,4-DHP
was toxic to cells, one group of cultures was pretreated with inhibitor
in the absence of AA for 2 days. AA was then added for an additional 5
days. OG2 activity was induced to the same extent as the 5-day AA
sample that had not been previously exposed to inhibitor (compare
2DHP/5AA with 5AA).

View larger version (38K):
[in this window]
[in a new window]
|
Figure 4. Effect of a Collagen Synthesis Inhibitor on
Induction of p1.3OG2-luc by AA
After transient transfections, MC3T3-E1 subclone 4 cells were grown for
5 days under the following conditions: control medium (5C), control
medium containing 500 µM 3,4-DHP (5DHP), AA-containing
medium (5AA), AA-containing medium plus 3,4-DHP (5AA+DHP), 2 days in
control medium followed by 3 days in control medium plus 3,4-DHP
(2C/3DHP), or 2 days in AA-containing medium followed by 3 days in AA
medium plus 3,4-DHP (2AA/3AA+DHP). A separate group of cells was
pretreated with 3,4-DHP for 2 days, washed twice to remove inhibitor,
transfected, and then grown for 5 more days in AA-containing medium
(2DHP/5AA).
|
|
Taken together, the above results establish that AA increases
osteocalcin expression by stimulating the 1.3-kb mOG2 promoter, that
this response is preferentially stimulated by the L-isomer
of AA, and that the AA response requires collagen matrix synthesis.
Localization of AA-Responsive Regions in the mOG2 Promoter
Because important cis-acting elements have been
described in the promoter of the mOG2 gene, we focused the remainder of
our analysis on the role of this promoter in AA-inducible osteocalcin
expression. Several deletion mutants of the mOG2 promoter have been
described and shown to be active in osteoblastic cell lines (16). These
constructs were transiently transfected into subclone 4 MC3T3-E1 cells
and assayed after 5 days in culture (Fig. 5
). AA
stimulated promoter activity in -1.3, -0.657, and -0.147 kb
constructs, but did not affect the -34 to +13 basal promoter. Although
the magnitude of AA stimulation declined between -0.657 and -0.147
kb, it is clear from these results that an AA-responsive element is
present in the -0.147 mOG2 promoter, a region shown earlier to contain
two osteoblast-specific cis-acting elements (16). For this
reason, subsequent analysis focused on the -0.147 promoter region.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 5. AA Responsiveness of mOG2 Promoter Deletion Mutants
MC3T3-E1 subclone 4 cells were transiently transfected with p1.3OG2-luc
or the indicated deletions plus pSV2CAT and grown for 5 days in the
presence (+) or absence (-) of AA. For each construct, mean luciferase
activity for control samples was set at unity and the fold-stimulation
in AA-treated samples was determined. *, Ratio (AA/C) is
significantly different from unity at P < 0.001
|
|
AA Treatment Increases Binding of Nuclear Proteins to OSE2
The above described functional analysis of the OG2 promoter
indicates that the -147 to -34 bp promoter region contains sufficient
sequence information for AA responsiveness. Methylation protection
assays (not shown) indicated that subclone 4 MC3T3-E1 cell nuclear
extracts contain factors capable of binding to the three sequence
elements previously described in ROS17/2.8 cells (16); the OSE1 region
(-74 to -47 bp), the OCE1 region (-111 to -83 bp), which contains
an E box, and the OSE2 region (-146 to -132 bp). OSE1 and OSE2
elements are both required for osteoblast-specific promoter
activity.
Results of gel retardation assays using OSE1, OSE2, and OCE1-specific
double-stranded oligonucleotides and increasing amounts of nuclear
extracts from control or AA-treated subclone 4 cells are shown in Fig. 6
. As shown in panel B, AA treatment had virtually no
effect on the binding of nuclear proteins to OSE1 and OCE1. In
contrast, AA treatment of cells dramatically increased binding of
nuclear proteins to OSE2 (panel A). Imaging of several gel retardation
experiments indicated a mean increase of 5-fold with AA treatment. To
demonstrate the specificity of the OSE2 binding observed, competition
experiments were performed. The shifted species generated by AA-treated
nuclear extracts were the result of sequence-specific interactions in
that they were disrupted by a 25- to 100-fold molar excess of OSE2
oligonucleotide, but not affected by the same oligonucleotide
containing two point mutations in the OSE2 core sequence. Similar
results (not shown) were obtained with control nuclear extracts.
Thus, these results show that AA treatment of cells increases binding
of nuclear factors to OSE2.

View larger version (61K):
[in this window]
[in a new window]
|
Figure 6. Effect of AA Treatment on the Binding of Nuclear
Extracts to Osteoblast-Specific Promoter Elements
A, Gel retardation assays using an OSE2 oligonucleotide. Nuclear
extracts were prepared from MC3T3-E1 subclone 4 cells grown for 6 days
in control or AA-containing medium. The indicated amounts of extracts
(expressed as DNA equivalents) were incubated with end-labeled OSE2
double-stranded oligonucleotide alone or in the presence of competitors
and analyzed by electrophoresis on 4% polyacrylamide gels as described
in Materials and Methods. Lane 1, labeled OSE2 without
nuclear extract; lanes 2 and 4, OSE2 with nuclear extract from control
cells; lanes 3 and 5, OSE2 with nuclear extract from AA-treated cells;
lanes 612, labeled OSE2 incubated with 2 µg nuclear extract from
AA-treated cells alone (lane 6) or with the indicated molar excess of
unlabeled wild type (OSE2 Wt) or mutated OSE2 (OSE2 Mut). B, Gel
retardation assays using OSE1 and OCE1 oligonucleotides. Gel
retardation assays were performed as in panel A except that labeled
OSE1 (lanes 15) or OCE1 (lanes 610) double-stranded
oligonucleotides were used. Lanes 1 and 6, Labeled oligonucleotide in
the absence of nuclear extract; lanes 2, 3, 7, and 8, labeled
oligonucleotides incubated with control nuclear extracts; lanes 4, 5,
9, and 10, labeled oligonucleotides incubated with nuclear extracts
from AA-treated cells.
|
|
A Wild Type OSE2 Is Required for AA Action
If OSE2 is indeed required for the activation of mOG2 by AA,
mutation or deletion of this element should abolish the AA response.
Furthermore, OSE2 fused to a minimal promoter should also respond to
vitamin. As predicted, a two-nucleotide substitution in the OSE2 core
element of the -147 to +13 promoter region abolished the AA response
(Fig. 7
). In addition, deletion analysis revealed a
major loss in AA responsiveness between -147 and -116, the region of
the proximal promoter containing OSE2. Finally, fusion of six copies of
OSE2 to the -34 to +13 minimal promoter, which by itself is not
induced by AA, restored responsiveness while a similar construct
containing mutated OSE2 was inactive.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 7. Functional Analysis of the Role of OSE2 in AA
Responsiveness
MC3T3-E1 subclone 4 cells were transfected with the following plasmids:
p147-luc, p147-luc containing two-point mutations in OSE2
(p147mut-luc), p116-luc, the -34/+13 minimal promoter (p34-luc),
minimal promoter to which six copies of either wild type OSE2
(OSE2/34-luc) or mutated OSE2 (OSE2mt/34-luc) sequence had been fused
in sense orientation, p657-luc, or p657-luc containing two-point
mutations in OSE2 (p657mut-luc). Cells were grown for 6 days in the
presence (+) or absence (-) of AA. The fold increase in luciferase
activity with AA treatment is indicated for each group. *,
Ratio (AA/C) is significantly different from unity at
P < 0.001. All other groups were not significantly
different at P < 0.05.
|
|
To examine the degree to which the OSE2 sequence is responsible for AA
responsiveness in the context of a larger promoter fragment that may
contain other 5'-regulatory sequences, an OSE2 mutation was introduced
into p657-luc. This construct and the largest promoter construct
examined, p1316-luc, have equivalent activity (Fig. 5
). Mutation of
OSE2 in p657-luc also abolished AA induction (Fig. 7
).
Taken together, our results demonstrate that AA acts through an OSE2
sequence located in the first 147 bp of the mOG2 promoter. This is
consistent with the known osteoblast-specific activity of OSE2 and
ability of AA to stimulate osteoblast-specific gene expression.
 |
DISCUSSION
|
---|
Treatment of mouse MC3T3-E1 preosteoblast cells or primary
osteoblasts with AA initiates the formation of a collagenous ECM and
synthesis of several osteoblast-related proteins including osteocalcin,
alkaline phosphatase, bone sialoprotein, and the PTH/PTH-related
protein receptor. In scurvy, bone formation and levels of
osteoblast-related proteins are greatly reduced relative to vitamin
C-sufficient animals (1), suggesting that similar defects in osteoblast
differentiation are associated with AA deficiency in vivo.
With the long-term goal of understanding the molecular mechanism of
osteoblast differentiation and bone formation as well as the specific
roles of AA and the ECM in this process, we have examined the
osteocalcin gene that contains the best-characterized
osteoblast-related promoter activity. Our studies demonstrate: 1) that
the mOG2 promoter is induced by AA and that this induction requires
collagen matrix synthesis, 2) that this effect can be reproduced with a
short 147-bp promoter, and 3) that an OSE2 sequence in the 147-bp
promoter appears to be the target of AA action. Our results do not
exclude the possibility that other sequences elsewhere in the
promoter may be responsive to AA, although such sequences clearly
require OSE2 for activity. This study represents the first
demonstration of vitamin C having stimulatory actions on a specific
gene promoter through a defined cis-acting element.
Our initial attempts to show induction of the mOG2 promoter in MC3T3-E1
cells produced inconsistent results. Because these cells, like other
osteoblast-related cell lines, are phenotypically heterogeneous
(i.e. only a fraction of cells exhibit osteoblast
characteristics), subclones were isolated with high and low osteoblast
differentiation potential. AA most dramatically stimulated OG2-luc
activity in subclones exhibiting high induction of the endogenous
osteocalcin gene. The parent MC3T3-E1 cell population used for
subcloning showed only a modest induction of OG2-luc, which explains
our initial difficulty in detecting activation of this promoter before
subclones were examined. Similarly, strong AA induction of the OG2
promoter was only observed in stably transfected subclones having high
endogenous osteoblast-specific gene expression (result not shown).
Inducible and uninducible subclones of MC3T3-E1 cells may prove to be
powerful experimental tools for the study of transcriptional regulation
of osteoblast differentiation and bone formation.
Nontransformed bone cells, such as MC3T3-E1 preosteoblasts and primary
osteoblasts, differ significantly from other osteoblast cell culture
models, such as ROS17/2.8 osteosarcoma cells, in that the former
require AA and collagen matrix synthesis before they will express
osteoblast marker proteins such as osteocalcin. In contrast, ROS17/2.8
cells appear to be terminally differentiated, expressing high levels of
osteocalcin in the absence of AA (21). Interestingly, in preliminary
studies (G. Xiao and R. T. Franceschi, unpublished) we observed that
ROS17/2.8 cells do not assemble substantial amounts of ECM after AA
treatment. Furthermore, AA does not increase osteocalcin mRNA or
stimulate p1.3OG2-luc in ROS17/2.8 cells. This suggests either that an
important level of regulation has been lost in this osteosarcoma cell
line or that ROS17/2.8 cells have progressed beyond the point in the
osteoblast lineage where ECM synthesis is required for phenotypic
expression.
Production of type I collagen is one of the earliest events associated
with osteoblastic differentiation. Studies examining effects of AA on
this process generally concluded that actions of this vitamin require
collagen matrix formation (7, 8, 9, 22). Induction of osteoblast markers
by AA can be blocked by collagen synthesis inhibitors or digestion of
the ECM with purified collagenase (8). The present data indicate that a
specific inhibitor of collagen matrix synthesis, 3,4-DHP, also blocks
AA-dependent induction of promoter activity. As was found for AA
induction of endogenous osteocalcin expression, this inhibitor was
fully effective only if added simultaneously with AA; pretreatment of
cells with AA for 2 days before inhibitor addition led to partial
induction of mOG2 promoter activity as would be expected if some
minimal amount of collagenous ECM had to accumulate around cells before
induction of the promoter could commence. The marked time interval of 4
to 5 days between AA addition to cells and the earliest induction of
mOG2 activity is also consistent with a model in which ECM accumulation
was a prerequisite for promoter induction. Thus, transcriptional
regulation of the osteocalcin gene promoter requires synthesis of a
collagenous ECM. One interpretation of these results is that the ECM
produced by osteoblasts interacts with cells possibly via integrins or
other cell-surface receptors to initiate signaling cascades that
ultimately up-regulate and/or activate transcription factors necessary
for osteoblast-specific gene expression and differentiation. Of
interest in this regard is the recent report by Takeuchi and co-workers
showing that a collagen synthesis inhibitor
(L-azetidine-2-carboxylic acid) and an anti-
2ß1
integrin antibody could block AA-induced alkaline phosphatase activity
in MC3T3-E1 cells and that a Asp-Glu-Gly-Ala peptide that interferes
with the binding of collagen to an
2ß1 integrin also inhibits
alkaline phosphatase induction and the differentiation-dependent
down-regulation of the transforming growth factor-ß receptor (23).
Further studies will be required to test this model, although there are
many examples of matrix-integrin interactions being required for
tissue-specific gene expression (24). The present results emphasize the
importance of ECM synthesis to the overall tissue-specific expression
of the osteocalcin gene. Our finding that AA induces promoter activity
approximately 20-fold indicates that at least 95% of total promoter
activity is ECM-dependent.
Experiments described in this report show that an OSE2 sequence
contained in the first 147 bp of the mOG2 promoter is necessary for AA
induction. Mutation or deletion of this element abolished activity.
Furthermore, OSE2 oligomers in the absence of other mOG2 promoter
elements were able to confer AA responsiveness to a -34 minimal mOG2
promoter construct. The stronger effect of AA when using longer mOG2
promoter constructs may be explained by the presence of cryptic
AA-responsive elements elsewhere in the promoter. However, these
upstream sequences clearly require the downstream OSE2 because a point
mutation in this element abolished the AA responsiveness of a -657
promoter construct. Gel retardation assays using an OSE2-containing
oligonucleotide and nuclear extracts from control and AA-treated cells
detected a sequence-specific shifted species in MC3T3-E1 subclone 4
cells. Furthermore, this component was dramatically increased in
AA-treated cell nuclear extracts. OSF2, the osteoblast-related nuclear
protein(s) binding to OSE2 has not yet been isolated. However, OSE2
contains a consensus sequence for the PEBP
/AML family of
transcription factors, and one member of this family, AML-1B, can
specifically bind OSE2 and activate plasmids containing this element.
Furthermore, OSF2 is immunologically related to, but distinct from, the
known AML proteins (17, 18). Once a cDNA and antibodies for OSF2 become
available, it will be important to determine whether its expression and
activity are regulated by AA. The general applicability of our findings
to the control of osteoblast-specific gene expression by AA and ECM
will require analysis of other promoters showing a bone-selective
pattern of expression.
 |
MATERIALS AND METHODS
|
---|
Reagents
Tissue culture medium and FBS were obtained from HyClone (Logan,
UT). D-Threo[dichloroacetyl-1-14C[]
chloramphenicol and
-[32P[]-dCTP (3000 Ci/mmol) were
purchased from Amersham (Arlington Heights, IL). Chloramphenicol,
2,6,10,14-tetramethylpentadecane, n-butyryl coenzyme A,
3,4-dehydro-L-proline, AA, and D-isoascorbic
acid were obtained from Sigma Chemical Co. (St. Louis. MO). All other
chemicals were of analytic grade.
RNA Analysis
Total RNA was isolated from cell layers as described by
Chomczynski and Sacchi (25). Aliquots of total RNA were fractionated on
0.8% agarose-formaldehyde gels and blotted onto nitrocellulose paper
as described by Thomas (26). The mouse cDNA probes used for
hybridization were obtained from the following sources: osteocalcin
from Dr. John Wozney (Genetics Institute, Boston, MA) (15) and bone
sialoprotein (27) from Dr. Marion Young (National Institute of Dental
Research, Bethesda, MD). All cDNA inserts were excised from plasmid DNA
with the appropriate restriction enzymes and purified by agarose gel
electrophoresis before labeling with
-[32P[]dCTP
using a random primer kit (Boehringer-Mannheim, Indianapolis, IN).
Hybridizations were performed as previously described using an Autoblot
hybridization oven (Bellco Glass, Vineland, NJ) (8) and quantitatively
scanned using an InstantImager (model A2024, Packard Instrument Co,
Downers Grove, IL). All values were normalized for RNA loading by
probing blots with cDNA to 18S rRNA (28).
DNA Constructions
All inserts containing mOG2 promoter regions were cloned in the
p4Luc promoterless luciferase expression vector as previously described
(16). p147mut-luc and p657mut-luc, which both contain a 2-bp
substitution mutation in OSE2 at positions -134 and -133
(CCAAGAACA), were generated from p147-luc and p657-luc by
PCR amplification (29). pCMV-luc, a generous gift from Dr. Jeffrey
Bonadio (University of Michigan Medical School, Ann Arbor, MI), was
constructed by inserting 1.9 kb of firefly luciferase cDNA into pcDNA3
(Invitrogen, San Diego, CA). pSV2CAT was obtained from Promega
(Madison, WI).
Cell Cultures
MC3T3-E1 cells, a generous gift from Dr. M. Kumegawa (Josai
Dental University, Sakado, Japan), were cultured in AA-free
-modified Eagles medium containing 10% FCS as previously
described (7). MC3T3-E1 subclones were obtained by limiting dilution
and evaluated for differentiation potential by growing for 10 days in
AA-containing medium and measuring mRNA levels for osteocalcin and bone
sialoprotein by Northern blot hybridization. Three highly responsive
clones (nos. 4, 14, and 26) and two poorly responsive clones (nos. 17
and 24) were selected for the studies described.
Transfections
MC3T3-E1 cells were transfected using LipofectAMINE reagent
according to the manufactures protocol (GIBCO BRL, Gaithersburg, MD).
Briefly, cells were plated on 35 mm-diameter dishes at a density of
25,000 cells/cm2 and fed 24 h later. After an
additional 24 h, cells were transfected with 0.5 µg each of
p1.3OG2-luc and pSV2CAT. Four hours later, DNA was removed and cells
were fed with 10% FBS-
-modified Eagles medium containing the
indicated additions. Cells were then fed daily until harvest.
Luciferase activity was measured using a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, Ann Arbor, MI) and reagents and
protocols provided by Promega (Madison, WI). After luciferase assays,
cell extracts were heated at 65 C for 15 min for chloramphenicol
acetyltransferase (CAT) assays (30). All transfections were conducted
in triplicate and values are reported as means ± SD.
Cellular plasmid levels were determined by dot blot hybridization of
total cellular DNA using 32P-labeled luciferase and CAT
cDNAs as probes (31). To measure the stability of luciferase protein,
cells previously transfected with p1.3OG2-luc and pSV2CAT were grown in
the presence or absence of AA for 5 days, washed, and treated with
cyclohexamide (2.5 µg/ml). Luciferase and CAT activities were
measured after culturing with cyclohexamide for increasing times (0,
12, 24, and 72 h). Stable transfectants of MC3T3-E1 cells were
established as follows: p1.3OG2-luc was linearized with
BamHI and cotransfected with pcDNA3 at a DNA ratio of 5:1.
After 24 h, cells were transferred to medium containing 400
µg/ml G418 and selected for 14 days. Single cell clones were then
isolated by limiting dilution and tested for AA induction of both
luciferase activity and endogenous osteoblast marker mRNAs.
Preparation of Nuclear Extracts and Gel Retardation Assays
To ensure that clean nuclei were obtained from AA-treated cells,
which contain large amount of collagenous matrix, nuclei were pelleted
through 2 M sucrose two times (30,000 x g,
45 min) before preparation of nuclear extracts according to the method
of Dignam et al. (32). The DNA remaining in the pelleted
fraction after preparation of extracts was measured using the method of
Schneider (33). Amounts of nuclear extract added to each gel
retardation assay are expressed in DNA equivalents. For gel retardation
assays, double-stranded oligonucleotides containing wild type OSE1,
OCE1, or OSE2 sequence (16) were labeled with
[
-32P]ATP and T4 polynucleotide kinase, filled in with
the Klenow fragment of DNA polymerase I, and purified on an acrylamide
gel. Approximately 5 fmol of probe were added to the indicated amounts
of nuclear extracts in 15 µl of a buffer containing 10% glycerol, 50
mM Tris-HCl (pH 7.5), 50 mM NaCl, 2
mM EDTA, 1 mM dithiothreitol, and 2.5 µg each
of leupeptin and pepstatin per ml.
Poly(deoxyinosinic-deoxycytidylic)acid·poly(deoxyinosinic-deoxycytidylic)acid
(1.0 µg) was used as nonspecific competitor. After incubation at 4 C
for 30 min, samples were subjected to electrophoresis on a 4%
polyacrylamide gel in Tris/Glycine buffer [50 mM Tris-HCl
(pH 8.5), 380 mM glycine, 2 mM EDTA, 0.2
mM ß-mercaptoethanol] at 160 V for 100 min in a 4 C cold
room. The gels were dried and autoradiographed. Competition studies to
assess the specificity of nuclear factor binding used unlabeled wild
type OSE2 oligonucleotide and the following mutant (16):
GATCCGCTGCAATCACCAAGAACAGCA
GCGACGTTAGTGGTTCTTGTCGTCTAG
Statistical Analysis
All transfection data are reported as means ±
SD based on triplicate independent cell cultures from a
representative experiment. All experiments were repeated at least
twice, and qualitatively identical results were obtained. Tukey-Kramer
multiple comparisons test was used to assess statistical significance
between samples.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Mr. M. Douglas Benson for figure
preparation and helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Renny T. Franceschi, Department of Periodontics, Prevention, and Geriatrics, School of Dentistry, University of Michigan, 1011 North University Avenue, Ann Arbor, Michigan 48109-1078.
This work was supported by NIH Grants DK-35317 and DE-11723 (to R.T.F.)
and DE-AR11290 (to G.K.), Basic Research Award IFY920871 from the
March of Dimes Foundation (to G.K.), GCRC Grant M01-RR00042, and the
Michigan Multipurpose Arthritis Center Grant AR20557.
Received for publication February 3, 1997.
Accepted for publication April 10, 1997.
 |
REFERENCES
|
---|
-
Togari A, Arai M, Nakagawa S, Banno A, Aoki M,
Matsumoto S 1995 Alteration of bone status with ascorbic acid
deficiency in ODS (osteogenic disorder Shionogi) rats. Jpn J
Pharmacol 68:255261[Medline]
-
Franceschi RT 1992 The role of ascorbic acid in mesenchymal
differentiation. Nutr Rev 50:6570[Medline]
-
Hammarstrom L 1966 Autoradiographic studies on the
distribution of 14C-labeled ascorbic acid and
dehydroascorbic acid. Acta Physiol Scand 70:184
-
Wilson JX, Dixon SJ 1989 High-affinity sodium-dependent
uptake of ascorbic acid by rat osteoblasts. J Membr Biol 111:8391[Medline]
-
Franceschi RT, Wilson JX, Dixon SJ 1995 Requirement for
Na+-dependent ascorbic acid transport in osteoblast
function. Am J Physiol 268:C1430C1439
-
Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L,
Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS 1990 Progressive development of the rat osteoblast phenotype in
vitro: reciprocal relationships in expression of genes associated
with osteoblast proliferation and differentiation during formation of
the bone extracellular matrix. J Cell Physiol 143:420430[Medline]
-
Franceschi RT, Iyer BS 1992 Relationship between collagen
synthesis and expression of the osteoblast phenotype in MC3T3E1
cells. J Bone Miner Res 7:235246[Medline]
-
Franceschi RT, Iyer BS, Cui Y 1994 Effects of ascorbic acid
on collagen matrix formation and osteoblast differentiation in murine
MC3T3E1 cells. J Bone Miner Res 9:843854[Medline]
-
McCauley LK, Koh AJ, Beecher CA, Cui Y, Rosol TJ, Franceschi
RT 1996 The PTH/PTHrP receptor is temporally regulated during
osteoblast differentiation and is associated with collagen synthesis. J
Cell Biochem 61:638647[CrossRef][Medline]
-
McKee M, Glimcher M, Nanci A 1992 High-resolution
immunolocalization of osteopontin and osteocalcin in bone and cartilage
during endochondral ossification in the chicken tibia. Anat Rec 234:479492[Medline]
-
Kerner SA, Scott RA, Pike JW 1989 Sequence elements in the
human osteocalcin gene confer basal activation and inducible response
to hormonal vitamin D3. Proc Natl Acad Sci USA 86:44554459[Abstract]
-
Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, Collart
D, Zambetti G, Stein G 1989 Structure of the rat osteocalcin gene and
regulation of vitamin D-dependent expression. Proc Natl Acad Sci USA 86:11431147[Abstract]
-
Morrison NA, Shine J, Fragonas JC, Verkest V, McMenemy ML,
Eisman JA 1989 1,25-dihydroxyvitamin D-responsive element and
glucocorticoid repression in the osteocalcin gene. Science 246:11581161[Medline]
-
Desbois C, Hogue DA, Karsenty G 1994 The mouse osteocalcin
gene cluster contains three genes with two separate spatial and
temporal patterns of expression. J Biol Chem 269:11831190[Abstract/Free Full Text]
-
Celeste AJ, Rosen V, Bueker JL, Kriz R, Wang EA, Wozney JM 1986 Isolation of the human gene for bone gla protein utilizing mouse
and rat cDNA clones. EMBO J 5:18851890[Abstract]
-
Ducy P, Karsenty G 1995 Two distinct osteoblast-specific
cis-acting elements control expression of a mouse osteocalcin gene. Mol
Cell Biol 15:18581869[Abstract]
-
Geoffroy V, Ducy P, Karsenty G 1995 A PEBP2/AML-1-related
factor increases osteocalcin promoter activity through its binding to
an osteoblast-specific cis-acting element. J Biol Chem 270:3097330979[Abstract/Free Full Text]
-
Banerjee C, Hiebert SW, Stein JL, Lian JB, Stein GS 1996 An
AML-1 consensus sequence binds an osteoblast-specific complex and
transcriptionally activates the osteocalcin gene. Proc Natl Acad Sci
USA 93:49684973[Abstract/Free Full Text]
-
Grigoriadis AE, Petkovich PM, Ber R, Aubin JE, Heersche JNM 1985 Subclone heterogeneity in a clonally-derived osteoblast-like cell
line. Bone 6:249256[Medline]
-
Goldman HM, Gould BS, Munro HN 1981 The antiscorbutic action
of L-ascorbic acid and D-isoascorbic acid (erythorbic acid) in guinea
pig. Am J Clin Nutr 34:2433[Abstract]
-
Bortell R, Owen TA, Shalhoub V, Heinrichs A, Aronow MA,
Rochette-Egly C, Lutz Y, Stein JL, Lian JB, Stein GS 1993 Constitutive
transcription of the osteocalcin gene in osteosarcoma cells is
reflected by altered protein-DNA interactions at promoter regulatory
elements. Proc Natl Acad Sci USA 90:23002304[Abstract]
-
Aronow MA, Gerstenfeld LC, Owen TA, Tassinari MS, Stein GS,
Lian JB 1990 Factors that promote progressive development of the
osteoblast phenotype in cultured fetal rat calvaria cells. J Cell
Physiol 143:213221[Medline]
-
Takeuchi Y, Nakayama K, Matsumoto T 1996 Differentiation and
cell surface expression of transforming growth factor-ß receptors are
regulated by interaction with matrix collagen in murine osteoblastic
cells. J Biol Chem 271:39383944[Abstract/Free Full Text]
-
Roskelley CD, Srebrow A, Bissell MJ 1995 A heirarchy of
ECM-mediated signalling regulates tissue-specific gene expression. Curr
Opin Cell Biol 7:736747[CrossRef][Medline]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Thomas PS 1980 Hybridization of denatured RNA and small DNA
fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:52015205[Abstract]
-
Young MF, Ibaraki K, Kerr JM, Lyu MS, Kozak CA 1994 Murine
bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic
mapping. Mamm Genome 5:108111[Medline]
-
Renkawitz R, Gerbi SA, Glatzer KH 1979 Ribosomal DNA of fly
Sciara coprophila has a very small and homogeneous repeat unit. Mol Gen
Genet 173:113[Medline]
-
Zhang R, Ducy P, Karsenty G 1997 1,25(OH)2D3 inhibits osteocalcin expression in
mouse through an indirect mechanism. J Biol Chem 272:110116[Abstract/Free Full Text]
-
Kingston RE, Sheen J 1995 Isotopic methods for assaying
chloramphenicol acetyltransferase. In: Ausubel F, Brent R, Kingston R,
Moore D, Seidman J, Smith J, Struhl K (eds) Current Protocols in
Molecular Biology, vol 1. Wiley, New York, pp 9.7.19.7.5
-
Murphy JM, Chrysogelos SA 1995 Direct measurement of
transfection efficiency in transient transfection assays. BioTechniques 18:967969[Medline]
-
Dignam JD, Martin PL, Shastry S, Roeder RG 1983 Eucaryotic
gene transcription with purified components. Methods Enzymol 101:582598[Medline]
-
Schneider WC 1957 Determination of nucleic acids in tissues
by pentose analysis. Methods Enzymol 3:680684