Human and Murine Osteocalcin Gene Expression: Conserved Tissue Restricted Expression and Divergent Responses to 1,25-Dihydroxyvitamin D3 in Vivo
Natalie A. Sims,
Christopher P. White,
Kate L. Sunn,
Gethin P. Thomas,
Melanie L. Drummond,
Nigel A. Morrison,
John A. Eisman and
Edith M. Gardiner
Bone and Mineral Research Program, Garvan Institute of Medical
Research, St. Vincents Hospital, Sydney, New South Wales,
Australia
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ABSTRACT
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Human and murine osteocalcin genes demonstrate
similar cell-specific expression patterns despite significant
differences in gene locus organization and sequence variations in
cis-acting regulatory elements. To investigate whether
differences in these regulatory regions result in an altered response
to 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3]
in vivo, we compared the response of the endogenous mouse
osteocalcin gene to a bacterial reporter gene directed by flanking
regions of the human osteocalcin gene in transgenic mice. Transgene
expression colocalized with endogenous osteocalcin expression in serial
sections, being detected in osteoblasts, osteocytes and hypertrophic
chondrocytes. In calvarial cell culture lysates from transgenic and
nontransgenic mice, the endogenous mouse osteocalcin gene did not
respond to 1,25-(OH)2D3
treatment. Despite this, transgene activity was significantly increased
in the same cells. Similarly, Northern blots of total cellular RNA and
in situ hybridization studies of transgenic animals
demonstrated a maximal increase in transgene expression at 6 h
after 1,25-(OH)2D3
injection (23.6 ± 3.6-fold) with a return to levels equivalent to
uninjected animals by 24 h (1.2 ± 0.1-fold). This increase
in transgene expression was also observed at 6 h after
1,25-(OH)2D3 treatment
in animals on a low calcium diet (25.2 ± 7.7-fold) as well as in
transgenic mice fed a vitamin D-deficient diet containing strontium
chloride to block endogenous
1,25-(OH)2D3 production
(7.5 ± 0.9-fold). In contrast to the increased transgene
expression levels, neither endogenous mouse osteocalcin mRNA levels nor
serum osteocalcin levels were significantly altered after
1,25-(OH)2D3 injection
in transgenic or nontransgenic mice, regardless of dietary
manipulations, supporting evidence for different mechanisms
regulating the response of human and mouse osteocalcin genes to
1,25-(OH)2D3. Although
the cis- and trans-acting mechanisms directing
cell-specific gene expression appear to be conserved in the mouse and
human osteocalcin genes, responsiveness to
1,25-(OH)2D3 is not.
The mouse osteocalcin genes do not respond to
1,25-(OH)2D3 treatment,
but the human osteocalcin-directed transgene is markedly up-regulated
under the same conditions and in the same cells. The divergent
responses of these homologous genes to
1,25-(OH)2D3 are
therefore likely to be due to differences in mouse and human
osteocalcin-regulatory sequences rather than to variation in the
complement of trans-acting factors present in mouse
osteoblastic cells. Increased understanding of these murine-human
differences in osteocalcin regulation may shed light on the function of
osteocalcin and its regulation by vitamin D in bone physiology.
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INTRODUCTION
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Osteocalcin is the most abundant noncollagenous protein expressed
in bone (1, 2), with its expression limited specifically to cells of
the osteoblast lineage, including mature osteoblasts, osteocytes, and
hypertrophic chondrocytes (3, 4, 5, 6). There is also some evidence for low
levels of osteocalcin expression in megakaryocytes, platelets (7), and
brain (8). Despite its well characterized specificity of expression,
the precise function of osteocalcin in bone remodeling is not clear.
The location of osteocalcin at bone-forming surfaces (9, 10) and the
increased bone mineralization observed in osteocalcin gene knockout
mice (11) and warfarin-treated rats (12, 13) supports a role for
osteocalcin in suppression of bone mineralization. Alternatively,
osteocalcin has been suggested to increase bone resorption through
osteoclast recruitment (10, 14).
Serum levels of osteocalcin increase in humans (15) and rats (16) after
injection with 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3], and increased osteocalcin
mRNA expression occurs in rat femurs 6 h after
1,25-(OH)2D3 treatment in vivo (17).
Expression of osteocalcin mRNA and protein is stimulated by
1,25-(OH)2D3 in rat osteoblastic cell lines
(18, 19, 20, 21, 22) and in human cultured primary osteoblasts (23, 24), as well as
in transfection studies of the human (25, 26) and rat (27, 28, 29)
osteocalcin genes.
Stimulation of gene transcription by
1,25-(OH)2D3 depends on binding of vitamin D
receptor/retinoid X receptor (VDR/RXR) heterodimers or VDR homodimers
to specific vitamin D-responsive elements (VDREs) within the human and
rat osteocalcin gene promoters (30, 31, 32). The organizations of the
human, rat, and mouse osteocalcin genes differ, however, with some rat
and all mouse strains studied, having a cluster of osteocalcin-related
genes rather than a single-copy gene as described in the human (18, 33, 34, 35). Despite these differences, the same pattern of tissue-specific
expression is shared by the three species, and the promoters of the two
mouse osteocalcin genes exhibit the same modular organization described
in the rat and human genes, including a VDRE consensus sequence at a
similar distance upstream of the transcription start site (33).
Cis-acting elements thought to direct tissue-specific and
hormone-responsive osteocalcin gene transcription differ, however,
between species (33, 34), and trans-acting factors that bind
to these regions are also likely to vary (34, 36, 37), leading to
potential variation in transcriptional control mechanisms. Supporting
this concept, recent studies have verified such a species-related
difference, showing the mouse osteocalcin gene to be down-regulated
rather than induced by 1,25-(OH)2D3 in
vivo and in vitro (38, 39). In each of these studies,
the inhibitory effect was demonstrated on the endogenous mouse
osteocalcin gene, and direct comparisons of rat and mouse
osteocalcin-regulatory activities were made in transfected rat and
mouse cell lines. In the present study, basal tissue-specific and
1,25-(OH)2D3-responsive regulation by the mouse
and human osteocalcin promoters were directly compared in transgenic
mice carrying a reporter transgene in an osteoblast-targeting vector
based on 5'- and 3'-sequences from the human osteocalcin gene
locus.
The human regulatory sequences conferred a spatially restricted
transgene expression pattern virtually identical to that of the
endogenous osteocalcin gene, confirming that the cis- and
trans-acting mechanisms directing the restricted pattern of
osteocalcin gene expression are conserved across species. However,
although the human osteocalcin-directed transgene was up-regulated by
1,25-(OH)2D3 treatment, endogenous mouse
osteocalcin gene expression was not induced by
1,25-(OH)2D3 treatment in transgenic or
nontransgenic mice, a confirmation of previous reports (38, 39).
Clearly, the vitamin D responsiveness of the osteocalcin gene does
differ between species, and this variation is not due simply to
differences in the cellular milieu in human and murine bone.
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RESULTS
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Transgene Expression Analyses
In the two sublines of OSCAT8 transgenic mice, transgene
expression was detectable only in RNA preparations from bone tissues
(Fig. 1A
), with no detectable expression
in any other tissue, including brain or kidney, in either subline and
no expression detectable in any nontransgenic tissues. Quantification
of transgene expression normalized to the 18S RNA signal indicated that
transgene expression in bones from the high copy number animals (OST2)
was 8-fold higher than in low copy number (OST1) animals. As the OST2
line carries approximately 60 transgene copies compared with the two
copies in OST1 mice, the OSCAT8 construct therefore does not confer
copy number-independent transgene expression.

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Figure 1. Restriction of CAT Expression to Bone in Two Mouse
Sublines Carrying the OSCAT Transgene
A, Northern blot analysis of transgene expression in transgenic (OST1,
OST2) and nontransgenic (FVB/N) mice. Bone (femur and calvaria), brain,
liver, kidney, muscle, heart, and lung were collected from sublines
carrying low (OST1) and high (OST2) numbers of copies of the OSCAT
transgene and from FVB/N mice. Total RNA was analyzed by Northern blots
probed for CAT (upper panel) and 18S RNAs (lower
panel). The doublet of CAT expression in the OST2 bone may be
due to alternative mRNA processing in the SV40 sequence included in the
OSCAT8 construct (70) or may result from retardation of the mRNA by the
18S ribosomal RNA. B, CAT enzyme activity in tissues from transgenic
and nontransgenic mice. Black bars represent activity
from OST2; white bars indicate activity from OST1; and
stippled bars depict activity from FVB/N tissues. Each
assay was performed in triplicate, and the results are the mean ±
SEM of two to six experiments.
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Measurement of chloramphenicol acetyl transferase (CAT) enzyme activity
in protein extracts from transgenic tissues was consistent with the
Northern blot results. In both OST1 and OST2 animals, substantial
transgene activity was detected in calvarial and femoral extracts but
not in extracts from other tissues, with greater activity in calvarial
preparations (Fig. 1B
). CAT activity levels in nonosseous tissues were
negligible and did not differ between OST and wild type mice. As in the
RNA analysis, greater transgene activity was seen in mice with higher
transgene copy number; activities in calvarial and femoral extracts
were 8- and 2-fold higher, respectively, in OST2 than in OST1 mice.
In femoral, tibial, calvarial, and vertebral sections from 8-week-old
OST2 mice, CAT protein was immunohistochemically detected in cuboidal
and flattened osteoblasts, osteocytes, and hypertrophic chondrocytes
(femurs shown in Fig. 2
, A-D). Positively
stained osteocytes were located close to bone-forming surfaces.
Osteoblastic transgene expression was detected on trabecular bone
surfaces and along endosteal and periosteal surfaces of cortical bone.
Trabecular CAT protein stained surface in the femoral metaphysis was
27% of the trabecular bone surface. CAT protein was not detectable by
immunohistochemistry in OST1 mice or in bones from nontransgenic mice
(Fig. 2E
).

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Figure 2. Cellular Localization of Transgene Expression by
Immunohistochemistry
In femurs of 8-week-old transgenic mice, CAT was detected: A, in both
cuboidal and flattened osteoblasts and osteocytes in trabeculae; B, in
cuboidal osteoblasts and osteocytes on endosteal surfaces, but not in
the marrow; C, in osteoblasts on the periosteal surface; and D, in
hypertrophic chondrocytes at the base of active chondrocyte stacks in
the growth plate. E, No CAT-specific staining was detected in
nontransgenic FVB/N bones by immunohistochemistry. Magnification:
400x. b, Bone; c, chondrocyte; m, marrow; ob, osteoblast; oy,
osteocyte; hc, hypertrophic chondrocyte.
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The pattern of transgene expression detected by in situ
hybridization reflected that observed by immunohistochemistry (Fig. 3
, A and B), with CAT mRNA detected in
flattened and cuboidal osteoblasts on both trabecular and cortical
surfaces, in osteocytes and in hypertrophic chondrocytes. Few
positively stained osteocytes, all of them immediately adjacent to
positively stained osteoblastic surfaces, were detected by in
situ hybridization. No specific transgene signal was detected by
immunohistochemistry or in situ hybridization in bones from
nontransgenic animals (Fig. 3
, E and F). Sections hybridized to sense
strand probes or to antisense probes, but without anti-digoxigenin
antibody, were similarly devoid of positive signal (Fig. 3
, C and D).
Transgene expression followed osteocalcin expression at the single-cell
level, shown by in situ hybridization on serial sections
(Fig. 3
, G and H). In no area did CAT expression overlap regions of
acid phosphatase activity in the same or adjacent sections (Fig. 3I
).
Osteocalcin mRNA was detectable in megakaryocytes of both transgenic
and nontransgenic mice by in situ hybridization in some
sections only, but transgene mRNA expression was not detected in
megakaryocytes by this technique, even after
1,25-(OH)2D3 treatment.

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Figure 3. Transgene Expression Pattern in Femurs of OST2 Mice
Transgene expression pattern by CAT immunohistochemistry (A) and
in situ hybridization (B) on sequential sections of
femurs of 8-week-old OST2 mice. Transgene expression was detected in
osteoblasts on the endosteal surface of the cortical bone (c)
and on trabecular surfaces (t) by both staining
techniques. No specific transgene staining was detected in marrow (m).
C, No CAT antibody, negative control for panel A; D, sense probe,
negative control for panel B; E, nontransgenic (FVB/N) femoral section
immunohistochemically stained for CAT protein; F, FVB/N femoral section
hybridized to antisense CAT probe. G-I, Colocalization of transgene and
osteocalcin expression by in situ hybridization in
serial OST2 femoral sections hybridized to CAT (G) and osteocalcin (H)
antisense probes. The CAT transgene and endogenous osteocalcin gene
were expressed in endosteal osteoblasts (ob) and osteocytes (oy) close
to the bone-forming surfaces. Variation in intensity of staining of
osteocytes in panels G and H may result from unequal partitioning of
the major portion of the osteocyte cell bodies in serial sections. I,
Adjacent section stained for osteoclasts (oc) by tartrate-resistant
acid phosphatase activity, with hematoxylin counterstain.
Magnification: 200x (A-F); 400x (G-I).
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1,25(OH)2D3
Response
Osteoblastic cultures from OST2 transgenic calvariae responded to
1,25-(OH)2D3 treatment with a significant
increase in transgene activity (P = 0.003 compared with
vehicle-treated OST2 cultures). In contrast to this elevation of
transgene expression by 1,25-(OH)2D3, there was
a slight but nonsignificant decrease in endogenous osteocalcin released
into cell culture supernatants in both OST2 (P = 0.08)
and FVB/N (P = 0.13) cultures (Fig. 4
).

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Figure 4. Increase of CAT Activity, but Not Osteocalcin
Release, in Primary Cell Cultures from Transgenic Mice Treated with
1,25-(OH)2D3
Calvarial osteoblastic cell cultures from transgenic (OST2) and
nontransgenic (FVB/N) mice were treated with 10-8
M 1,25-(OH)2D3 for 5 days. CAT
enzymatic activity was significantly increased in the OST2 culture
lysates after 1,25-(OH)2D3 treatment. In the
same OST2 cultures, and in parallel FVB/N control cultures, osteocalcin
protein content in the culture supernatants, corrected for DNA content,
was slightly decreased, but this decrease did not reach statistical
significance. *, P < 0.005. Values shown are the
mean ± SEM of eight OST2 and five FVB/N lysates.
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Consistent with calvarial culture results, a significant increase in
transgene mRNA levels after 1,25-(OH)2D3
treatment of OST1 and OST2 mice fed the high calcium (HighCa) diet was
detected by Northern blot. Transgene mRNA levels in bones reached
maximal increases of similar magnitude in both low (20.5 ± 9.7)
and high (17.0 ± 2.5) copy number mice at 6 h after
1,25-(OH)2D3 treatment. In contrast, the
endogenous osteocalcin mRNA level was not significantly changed by
1,25-(OH)2D3 treatment in either transgenic
line or in nontransgenic FVB/N mice (Fig. 5
, A and B). Baseline osteocalcin mRNA
levels were lower in uninjected OST1 and OST2 mice than in FVB/N mice,
with the following OC/18S mRNA ratios: FVB/N, 1.7± 0.3; OST1, 0.3
± 0.2; OST2, 0.5 ± 0.3, P = 0.045.

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Figure 5. Increased Transgene Expression in OST1 and OST2
Sublines Treated with 1,25-(OH)2D3 with Minimal
Effect on Osteocalcin (OC) Expression in Transgenic or Nontransgenic
Mice
A and B, OST1, OST2, and FVB/N mice were fed the high calcium diet and
given a single intraperitoneal injection of
1,25-(OH)2D3. Tissues were collected at 0, 6,
or 24 h after treatment for Northern blot analysis of total RNA
from bone (Bo), brain (Br), and kidney (Ki). A, A representative
Northern blot analysis of tissues from mice injected with
1,25-(OH)2D3 (+) or vehicle (-). Filter was
probed sequentially for CAT transgene, osteocalcin, and 18S
transcripts. B, Phosphorimage analysis of Northern blot signals. The
treated/control ratios of CAT and osteocalcin signals were quantitated
and calculated as described in Materials and Methods.
(Values are mean ± SEM; *, P <
0.05 vs. uninjected control value.) This experiment was
repeated three times. CE, OST2 and FVB/N mice were fed the low
calcium or SrCl2 diet for 10 days before injection with
1,25-(OH)2D3 or vehicle. Quantification of
Northern blot analyses of CAT (C) and osteocalcin (D) expression in
OST2 and osteocalcin (E) expression in FVB/N mice at the indicated
times after injection. Data are expressed as the ratios of normalized
signals of treated mice to uninjected mice, as described in
Materials and Methods [mean ± SEM; *,
P < 0.05 vs. vehicle value at the
same time point and vs. uninjected value
(To)]. This experiment was repeated five times for each
diet.
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The SrCl2 diet significantly reduced serum osteocalcin
levels in both transgenic and nontransgenic mice (P =
0.004). Levels in untreated FVB/N mice fed the SrCl2 diet
were 93.5 ± 11.7 compared with 125.7 ± 8.6 in low
calcium-fed (LowCa) mice. In OST2 mice, levels were 78.6 ± 7.8 on
SrCl2 and 103.8 ± 11.2 on LowCa. The lower level of
serum osteocalcin in OST2 mice compared with FVB/N was also
statistically significant (P = 0.048). This reduction
in serum osteocalcin in OST2 mice and the reduction in osteocalcin mRNA
levels in OST1 and OST2 mice described above may reflect competition
for transcriptional regulatory factors by the large number (40) of
integrated OSCAT8 transgene copies. Treatment of the mice with
1,25-(OH)2D3 did not significantly alter serum
osteocalcin levels at any time after hormone injection, regardless of
dietary manipulations or transgene status.
Similar CAT responses to 1,25-(OH)2D3 treatment
were seen in mice fed SrCl2 and LowCa diets. Transgene mRNA
levels were maximally increased 6 h after
1,25-(OH)2D3 treatment in both dietary groups,
remained significantly elevated at 9 h after treatment, and had
returned to uninjected control levels by 12 h (Fig. 5C
). The
increase in transgene expression at 6 h in
SrCl2-treated OST2 mice (6.6 ± 0.9-fold higher than
uninjected values) was considerably lower than that observed in mice on
the LowCa or HighCa diets treated with
1,25-(OH)2D3 (23.6 ± 3.6- and 25.2
± 7.7-fold, respectively). As previously noted for animals fed the
HighCa diet, osteocalcin mRNA levels in both transgenic and
nontransgenic mice fed LowCa and SrCl2 diets were not
altered by 1,25-(OH)2D3 treatment (Fig. 5
, D
and E).
Cellular responses to 1,25-(OH)2D3 treatment
were examined histologically. In situ hybridization analyses
indicated that in femoral sections from all OST2 mice, the spatial
distribution of transgene expression was unchanged in hormone-treated
animals, with strong staining in trabecular and endosteal osteoblasts,
in osteocytes close to bone-forming surfaces and in hypertrophic
chondrocytes (Fig. 6A
). With reduced CAT
probe concentration, only the most strongly transgene-expressing cells
were positively stained (Fig. 6B
, 0-h sample).

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Figure 6. Increased Transgene, but Not Osteocalcin,
Expression in 1,25-(OH)2D3-Treated OST2 and
Nontransgenic FVB/N Mice
Animals were treated with a single injection of
1,25-(OH)2D3 or vehicle, and in
situ hybridization was carried out on paraffin-embedded distal
femurs. Three experiments were performed in mice fed the high calcium
diet and five were performed in mice fed the low calcium or
SrCl2 diet, and five sections of each bone sample were
hybridized with CAT and osteocalcin probes. Representative sections for
each time point were selected for this figure. A, CAT in
situ hybridization, showing staining in trabecular and
endosteal osteoblasts, in osteocytes close to bone-forming surfaces and
in hypertrophic chondrocytes. B, CAT in situ
hybridization using a low concentration of probe (1.5 ng/slide) in
uninjected (0 h) and at 3, 6, 9, and 12 h post
1,25-(OH)2D3 treatment. The number of cells
expressing a high level of transgene was increased at 3, 6, and 9
h after treatment, with a maximum after 6 h. Note the low number
of stained cells in uninjected animals compared with panel A. C, CAT
expression in vehicle-treated animals was unchanged by
1,25-(OH)2D3 treatment (6 h posttreatment
shown). D, In situ hybridization for endogenous
osteocalcin expression, showing staining in the same cells as the
transgene (panel A). E, Expression of the endogenous mouse osteocalcin
gene using low levels of probe (0.75 ng/slide) in
1,25-(OH)2D3-treated animals. Endogenous mouse
osteocalcin expression was not increased by
1,25-(OH)2D3 treatment, with no alteration in
the number of positive cells or in staining intensity. F, Expression of
endogenous mouse osteocalcin mRNA in vehicle-treated animals was
unchanged (6 h posttreatment shown).
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At 6 h after 1,25-(OH)2D3 injection, the
number of cells positive for transgene mRNA expression using the low
probe concentration was dramatically increased in all three dietary
groups. Transgene expression was also increased at 3 and 9 h after
treatment, but the maximal level of staining consistently occurred at
6 h. Intense staining was seen in the same cell types and along
the same surfaces that stained positive using high CAT probe
concentration in uninjected animals (compare Fig. 6A
with 6B, 6-h
sample). At 12 (Fig. 6B
) and 24 (not shown) h after injection, the
number of strongly positive cells had returned to levels observed in
uninjected controls (Fig. 6A
) or in vehicle- treated animals (Fig. 6C
).
In OST1 mice (even using the high probe concentration) transgene
expression, which was below the level of detection in uninjected and
vehicle-treated mice, was apparent at 6 h after
1,25-(OH)2D3 treatment in osteoblasts in the
primary spongiosa and in some hypertrophic chondrocytes and was again
undetectable at 24 h after treatment (not shown).
The distribution of transgene expression mirrored osteocalcin
expression detected by in situ hybridization at the high
probe concentration (Fig. 6D
). Consistent with the Northern blot
results, expression of the endogenous mouse osteocalcin mRNA was not
increased by 1,25-(OH)2D3 treatment, with no
alteration in the number of positive cells or staining intensity,
regardless of transgene status or dietary manipulation (Fig. 6
, E and
F).
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DISCUSSION
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The osteoblast-targeting vector, OSCAT8, with 5'- and 3'-flanking
sequences from the human osteocalcin gene, directed reporter gene
expression specifically to osteoblastic lineages in transgenic mice.
The levels of transgene mRNA and transgene activity were high in bone,
and levels in nonosseous tissues were comparable to nontransgenic
levels. Brain and kidney tissue extracts yielded no detectable CAT
activity or RNA in the OSCAT8 mice. The histological pattern of
transgene expression closely followed osteocalcin expression and was
comparable to in situ hybridization patterns of osteocalcin
expression in rat femurs (5, 6, 41). Transgene and osteocalcin
expression were detected in osteoblasts, osteocytes, and hypertrophic
chondrocytes in the present study. Although some osteocalcin expression
was detectable in megakaryocytes (7), transgene expression was not
detected in bone marrow megakaryocytes in this study, even after
1,25-(OH)2D3 treatment. It is not clear whether
the previously reported low levels of osteocalcin gene expression in
megakaryocytes (7) and brain, intestine, and kidney (8) are derived
from the osteocalcin genes or from the osteocalcin-related genes
described in mice and rats. If osteocalcin expression does occur in
human megakaryocytes, data from the present study indicate either that
such expression is directed by regions of the osteocalcin promoter not
included in the 5'- and 3'-sequences in the OSCAT8 construct or that
the human sequences present in the transgene do not function in murine
megakaryocytes.
The pattern of transgene expression seen in the OST sublines is similar
to those reported in previous studies utilizing varying extents of
5'-flanking sequences from the human and rat osteocalcin genes, with
the exception that all previous reports have demonstrated ectopic
transgene expression in kidney and/or brain (42, 43, 44). Although the
present study suggests a regulatory role for the 3'-flanking sequence,
this possibility cannot be confirmed because there has been no direct
comparison of constructs with and without this 3'-sequence.
In situ hybridization analysis demonstrated that after
1,25-(OH)2D3 treatment, the spatial pattern of
the increased transgene expression was unchanged. Before hormone
treatment, although there was extensive transgene expression in
trabecular and endosteal osteoblasts as well as in osteocytes and
hypertrophic chondrocytes, only a few cells of each type exhibited a
high level of transgene expression. After
1,25-(OH)2D3 treatment, the number of
trabecular and cortical osteocytes and hypertrophic chondrocytes
expressing high levels of the transgene was increased, supporting a
function for vitamin D receptors in hypertrophic chondrocytes (45, 46)
and suggesting a presence of functional receptors in osteocytes.
1,25-(OH)2D3 regulation of collagen gene
products has been described in hypertrophic chondrocytes (40, 47), but
regulation of a noncollagenous protein in this cell type has not
previously been described.
Whereas the basal transgene expression pattern
closely followed endogenous osteocalcin gene expression in bone cells,
the hormone responsiveness of the human-derived transgene differed from
that of the endogenous gene. In both primary cell culture and whole
animal studies, expression of the human osteocalcin-directed transgene
was markedly increased by 1,25-(OH)2D3
treatment. In the same experiments and in nontransgenic controls,
however, hormone treatment did not significantly alter levels of mouse
osteocalcin protein in vitro, osteocalcin mRNA levels
in vivo, or serum osteocalcin levels over the time course
studied, indicating a species-related difference in osteocalcin
promoter responses to 1,25-(OH)2D3
treatment.
Other studies have also indicated a minimal response of the mouse
osteocalcin gene to 1,25-(OH)2D3 treatment.
Whereas rat osteocalcin gene expression increased in response to
1,25-(OH)2D3 treatment, in three of four murine
osteoblast-like osteosarcoma cell lines, expression of the mouse gene
was unchanged after 1,25-(OH)2D3 treatment, and
primary mouse calvarial osteoblast cultures had only a weak positive
response (18). Similarly, a recent study demonstrated that a human
osteocalcin locus transgene, including 4 kb of upstream sequence, the
coding region, and 15 kb of downstream sequence, stably incorporated in
the mouse genome, responded to 1,25-(OH)2D3
treatment with increased serum levels of human osteocalcin but no
change in endogenous mouse osteocalcin protein (48), as demonstrated in
the present study.
Other recent studies have reported a decrease in mouse osteocalcin
expression after 1,25-(OH)2D3 treatment. For
example, osteocalcin mRNA levels in mouse calvaria were reduced 6-fold
after 1,25-(OH)2D3 treatment in
vivo, and osteocalcin mRNA expression was halved in primary mouse
osteoblast cultures treated with 1,25-(OH)2D3
(38). Furthermore, gel mobility shift analysis indicated that the
VDRE-like sequence in the mouse osteocalcin genes does not mediate this
response to 1,25-(OH)2D3 (38); rather, indirect
regulation via decreased activity of the OSF2 transcription factor was
proposed (34). In another study, 1,25-(OH)2D3
caused a decrease in osteocalcin release into culture supernatant of
the murine osteoblast-like cell line MC3T3-E1, whereas in the same cell
line, a stably transfected rat osteocalcin promoter construct responded
positively to 1,25-(OH)2D3 treatment. Moreover,
in transient transfections, activity of a reporter construct bearing
the mouse osteocalcin VDRE fused to a minimal TATA-containing promoter
was repressed by 1,25-(OH)2D3 treatment,
whereas the activity of an analogous construct bearing the rat VDRE
sequence was stimulated by the hormone treatment (39). Contrary to the
previous report, however, this latter study also demonstrated that the
mouse osteocalcin VDRE is bound by a complex containing VDR and RXR
in gel mobility shift assays, a difference from the previously cited
work that may stem from variation in probe length in the two studies.
Despite this difference, the common finding in those published studies,
that 1,25-(OH)2D3 reduces mouse osteocalcin
expression to a significant extent, differs from those of the present
study. Whether these differences relate to duration of hormone
treatment, extent of mineralization of the primary osteoblast cultures
at the time of treatment, and/or variation in response between mouse
strains is not yet known, but the influence of these factors clearly
requires further investigation.
It is possible that the mouse osteocalcin gene shows no positive
response to 1,25-(OH)2D3 treatment because the
endogenous gene is maximally stimulated in the untreated state. This
interpretation would be consistent with the findings of Clemens and
colleagues (48) who reported higher serum levels of mouse osteocalcin
than human osteocalcin in their transgenic mouse model. However, even
though inhibition of vitamin D metabolism reduced serum osteocalcin
levels in the present study, 1,25-(OH)2D3 still
did not induce endogenous osteocalcin transcription. Interestingly, the
transgene response to 1,25-(OH)2D3 was reduced
in vitamin D-deficient mice. Since 1,25-(OH)2D3
may increase VDR transcription (49) or stabilize VDR protein (50) in
rat osteosarcoma cells, it may follow that VDR is less stable in
vitamin D deficiency, attenuating any transcriptional response to the
hormone.
Although osteocalcin and VDR do not appear to be required for embryonic
development in the mouse (11, 51), they clearly play important roles in
postnatal bone physiology. In mice lacking functional osteocalcin
genes, bone density is increased with apparently elevated osteoblast
activity, suggesting a role for osteocalcin in limiting bone formation
in the mouse (11). Similarly, growth retardation and widening of the
growth plate are observed in VDR knockout mice, although serum
osteocalcin levels are not altered (51). These alterations in bone
structure are similar to those seen in vitamin D- deficient states in
human (52, 53) and rat (54, 55, 56). Thus, although the interaction between
1,25-(OH)2D3 and bone is important for
postnatal bone growth, the present study suggests VDRE-mediated
up-regulation of osteocalcin is not required for this process in the
mouse. By extension, 1,25-(OH)2D3 regulation of
osteocalcin expression may not be involved in bone growth in humans.
Alternatively, the physiological role of VDRE-mediated osteocalcin
regulation may differ between species.
Given the murine-human difference in osteocalcin regulation by
1,25-(OH)2D3 described here, it is important
that an appropriate model be used to study the physiological regulation
of the human osteocalcin gene. The OSCAT8 transgenic mouse may
represent such a model, utilizing 5'- and 3'-human osteocalcin-flanking
regions to direct transgene expression and mimic the human osteocalcin
gene in both expression pattern and
1,25-(OH)2D3 regulation. In conjunction with
other reports, the present study provides strong evidence for a
species-related difference in the effect of
1,25-(OH)2D3 on VDRE-mediated regulation of
osteocalcin transcription in the mouse and human, with a
1,25-(OH)2D3-stimulated increase in the human
osteocalcin-directed transgene without a concomitant increase in
endogenous mouse osteocalcin transcription. This species difference in
1,25-(OH)2D3 responsiveness relates to sequence
variations in osteocalcin gene-regulatory regions rather than inherent
differences in the bone cellular milieu. Insights gained from further
investigation of these species-related differences may shed light on
the function of osteocalcin and its regulation by vitamin D in bone
physiology.
 |
MATERIALS AND METHODS
|
---|
DNA Constructs and Probes
The pOSCAT8 construct contained the CAT reporter gene flanked by
5'- and 3'-regions of the human osteocalcin gene in natural position
and orientation. The 3.8 kb of continuous 5'-flanking sequence,
including a known VDRE (25, 26), was generated by inserting a 3.5-kb
HindIII-SacI fragment of genomic DNA (26) into
the SacI site at position -344 of the human osteocalcin
gene in the pOSCAT1 construct (26). A 3.5-kb
BamHI-HindIII fragment from the human osteocalcin
gene including 108 bp of exon 4 and 3.4 kb of 3'-untranslated and
flanking DNA was inserted downstream of the SV40 polyadenylation
signal.
Southern blots were probed with a CAT-specific DNA probe (1.8 kb
BglII-BamHI fragment from the pCAT-promoter
vector (Promega, Madison, WI) or with a probe specific for the human
osteocalcin 3'-sequence (1.1 kb KpnI fragment from pOSCAT8
containing human osteocalcin 3'-flanking DNA). DNA slot blots were
probed for the mouse neurofilament gene (57) and subsequently with the
human osteocalcin 3'-probe. Northern blots were probed with random
primed CAT-specific DNA probe or osteocalcin-specific probe and
subsequently with a 32P-kinased oligonucleotide probe for
18S ribosomal RNA (5'-CGGCATGTATTAGCTCTAGAATTACCACAG-3').
For in situ hybridization, riboprobes were generated from
linearized pOC918 construct containing a rat osteocalcin cDNA insert (a
gift from Dr. S. E. Harris). For the CAT probes, a 1.7-kb
HindIII fragment of the pCAT-promoter vector was subcloned
into pGEM-II to form pGEMCAT6, which was linearized and transcribed to
generate the antisense strand CAT riboprobe. A reverse orientation
construct (pGEMCAT2) was similarly transcribed to generate the sense
strand riboprobe.
Generation of Transgenic Mice and Genomic DNA Analysis
Transgenic mice were generated by pronuclear injection of the
purified HindIII insert from pOSCAT8 into inbred FVB/N
embryos (58) and screened by Southern blot analysis of genomic DNA
prepared from tail biopsy. Upon back-crossing the founder animal to
FVB/N, two transgene insertions segregated. Inbred sublines (OST1 and
OST2) with stable transgene transmission patterns were generated by
back-crossing to FVB/N mice for two further generations before
interbreeding hemizygous siblings. Eight-week-old hemizygous transgenic
animals were bred from homozygous sires and nontransgenic FVB/N dams
for all studies. Nontransgenic FVB/N mice were specifically bred as
age-matched controls for these experiments. All experimental animals
and mice used to generate transgenic lines were bred at the Garvan
Institute in studies approved by the Institute Animal Experimentation
Ethics Committee.
Quantitative slot blot analysis was performed using 5 µg DNA and
probed with the mouse neurofilament probe and quantitated on a
Molecular Dynamics 445 SI PhosphorImager (Sunnyvale, CA). The filter
was stripped then reprobed with the transgene-specific 3'-osteocalcin
DNA probe and quantitated. Transgene copy number was calculated by
normalization to the neurofilament signal. In these two sublines of
OSCAT8 transgenic mice, quantification of the number of incorporated
copies of the transgene showed low and high copy number insertions,
with OST1 and OST2 containing 2 and 60 transgene copies per haploid
genome, respectively.
Primary Bone Cell Isolation and Culture
Osteoblast-like cells were derived from 3- to 6-day-old
FVB/N and OST2 mice as follows (59). Frontal and parietal bones were
removed and washed twice in PBS, and adherent soft tissue and sutures
were dissected away. Cleaned calvariae were transferred to culture
medium containing 30 mM HEPES. Bones were washed again and
minced into normal culture media (
-MEM) containing 10% FCS, 100
U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentomycin, 10
mM ß-glycerophosphate, 50 µg/ml ascorbic acid, 20
mM HEPES, 2 mM glutamine. Calvariae were washed
in PBS and transferred to 1 ml digest mix per five calvaria [1 mg/ml
collagenase (Boehringer-Mannheim, Mannheim, Germany), 0.05% trypsin,
0.02% EDTA in PBS] and stirred vigorously for 30 min at 37 C. Five
sequential digests were carried out; the last three were washed in PBS
and resuspended in fresh culture medium, pooled, aspirated repeatedly
through 19- and 21-gauge needles, and filtered through a 200-µm
polyester filter (Spectrum Industries, Houston, TX) to a single cell
suspension. Cells were seeded at a density of 104 cells per
cm2 into six-well plates and cultured in a humid atmosphere
with 5% CO2 at 37 C. Medium was changed after 3 days and
every 23 days thereafter. Mineralizing cultures were treated with
10-8 M 1,25-(OH)2D3
from day 29 and stopped on day 35; untreated controls were stopped on
day 35.
DNA, CAT, and Protein Assays
Cells were lysed in 500 µl 0.1% Triton-X100, 0.5
mM MgCl2 in 20 mM Tris, pH 10, and
stored at -20 C until assayed. The fluorometric DNA assay was adapted
from Rao and Otto (60) and Downs and Wilfinger (61). Duplicate samples
of 50 µl lysate were mixed with 200 ml of 1 µg/ml bisbenzimide in
water in clear polystyrene 96-well plates, allowed to stand for 5 min,
then read on a Fluoroskan II (Labsystems, Helsinki, Finland) with
excitation and emission settings at 355 nm and 460 nm, respectively.
DNA values were calculated using DeltaSoft analytical software
(Biometallics, Princeton, NJ).
For the CAT and protein assays the same cell lysates were freeze-thawed
three times to further release cellular proteins. A 1-h 65 C incubation
to inactivate nonspecific acetyl transferases was carried out. CAT was
assayed as described by Morrison et al. (26) based on the
nonchromatographic assay of Sleigh (62). Protein was assayed using the
Bio-Rad (Hercules, CA) protein assay based on the colorimetric method
of Bradford (63).
Osteocalcin levels in the culture medium were determined by RIA using
the method of Gundberg et al. (64) except sample sizes
assayed were 50 µl instead of 5 µl. The concentration of primary
antibody was altered accordingly to maintain the same total assay
volume. Primary antibody and osteocalcin standards were kindly provided
by Dr. C. Gundberg. Iodinated osteocalcin was purchased from Biomedical
Technologies, Inc (Stoughton, MA). Donkey anti-goat IgG secondary
antibody was purchased from Sigma (St. Louis, MO).
1,25(OH)2D3
Treatment in Vivo
Animals were maintained on standard laboratory chow (Barastoc,
Ridley Agri Products, Victoria, Australia) containing 0.94% calcium,
0.81% phosphorus, and 1800 IU vitamin D3/kg and supplied
with water ad libitum. Ten days before treatment, at 6.5
weeks of age, animals were allocated one of three diets: standard
laboratory chow (HighCa); semisynthetic AIN-93 diet (65) containing
0.1% calcium, 0.27% phosphorus, and 1800 IU vitamin D3/kg
(LowCa); or vitamin D metabolism-inhibiting diet containing 0.1%
calcium, 0.27% phosphorus, no vitamin D3, and 0.8%
strontium chloride (SrCl2) added to AIN-93. At 8 weeks of
age, animals were given a single intraperitoneal injection of
1,25-(OH)2D3 in 10% argon-saturated
isopropanol/90% propylene glycol at a dose rate of 2 µg/kg body
weight or an equivalent volume of vehicle (0.1 ml). At various times
after injection, animals were bled by cardiac puncture under
methoxyflurane anesthesia (Metofane, Pittman-Moore, Mundelein, IL) and
killed by cervical dislocation. Femurs, tibias, calvariae, brain,
kidney, muscle, heart, lung, spleen, and liver were collected at the
time of death and from uninjected mice at the time of injection. One
femur, taken for in situ hybridization, was immediately
fixed in 4% paraformaldehyde/PBS at 4 C overnight. Other tissues
collected were snap-frozen in liquid nitrogen and stored at -70 C
until RNA preparation. Serum was prepared from blood and stored at -70
C before serum osteocalcin measurement (64).
Initial experiments, repeated three times, included OST1, OST2, and
FVB/N animals fed only the HighCa diet. Tissues were collected at the
time of injection or at 6 or 24 h after injection. In subsequent
comparisons of OST2 and FVB/N mice fed the LowCa and SrCl2 diets,
tissues were collected at the time of injection or at 3, 6, 9, 12, or
24 h after injection. These experiments were repeated five times.
For all experiments, at each repetition, single animals were injected
with hormone or vehicle for each time point. Each experiment was
analyzed once by Northern blot.
Preparation and Analysis of RNA
Tissues were homogenized at 3000-5000 rpm for 15 sec in 4.5 ml
guanidine thiocyanate buffer (4 M guanidinium
isothiocyanate, 0.5% sarkosyl, and 0.1 M
ß-mercaptoethanol) using a Polytron (Brinkman, Westbury, NJ). Total
cellular RNA was isolated from homogenates by acid phenol-chloroform
extraction (66) followed by cesium chloride gradient centrifugation
(67). RNA (15 µg) was analyzed by Northern blot analysis on a nylon
filter (HybondN+, Amersham, Buckinghamshire, UK) and probed with the
random primed
-32P-CTP-labeled CAT probe followed, after
stripping, by the human osteocalcin probe, then the
-[32P]ATP 18S oligonucleotide probe. For each probe,
radioactive counts bound to the filter were quantitated by
phosphorimaging. For each sample, CAT and osteocalcin values were
normalized to the corresponding 18S signal. Treated/control ratios were
calculated by dividing the normalized CAT and osteocalcin values for
bone samples from 1,25-(OH)2D3-treated mice by
the corresponding normalized CAT and osteocalcin values for bones from
vehicle-treated mice at the same time points. For the
treated/T0 ratios, the normalized CAT and osteocalcin
values for the 1,25-(OH)2D3-treated mice were
divided by normalized CAT and osteocalcin values from uninjected
mice.
Histology
Skull, vertebrae, tibia, and femur samples were fixed in 4%
paraformaldehyde/PBS at 4 C. Skulls were bisected sagittally during
fixation. Femurs were trimmed and the distal third of each was used for
analysis. Specimens were decalcified for 4 days in 15% EDTA/0.5%
paraformaldehyde/PBS at 4 C and embedded in paraffin. Sections (5 µm)
were taken onto chrome alum-coated slides, dewaxed in Histoclear
(National Diagnostics, Atlanta, GA), and rehydrated in graded ethanols
followed by PBS before staining.
For immunohistochemistry, sections were blocked using 5% normal goat
serum and incubated for 1 h at room temperature with a rabbit
polyclonal anti-CAT antibody (5 Prime
3 Prime Inc., Paoli, PA)
diluted 1:100 in PBS containing 2% normal goat serum. Endogenous
peroxidase was blocked with 0.3% hydrogen peroxide, followed by a
30-min incubation with 0.3% biotinylated goat anti-rabbit IgG and an
avidin-biotinylated peroxidase complex (Vector Laboratories,
Burlingame, CA). Peroxidase was detected with a diaminobenzidine
staining kit (Vector Laboratories). For quantification of
immunohistochemical CAT staining, six femoral sections at 10-µm
intervals were analyzed from each of five 8-week-old transgenic mice.
CAT surface as a percentage of total trabecular bone surface was
determined in the metaphysis of each section using the Osteomeasure
system (Osteometrics Inc., Decatur, GA).
In situ hybridization was modified from Zhou and colleagues
(68). Rat osteocalcin and CAT antisense and sense probes were generated
as described above. Sections were incubated with 0.2 M HCl
for 20 min at room temperature, followed by digestion with 1 µg/ml
proteinase K in 0.1 M Tris buffer (pH 8.0) for 45 min at 37
C, followed by 2 mg/ml glycine in PBS for 5 min. The tissues were
refixed in 4% paraformol saline for 15 min before acetylation in
0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) for
10 min and rinsed in PBS between all pretreatments. Slides were stained
for 1 h at room temperature with Fast Red TR (Sigma) to eliminate
endogenous alkaline phosphatase staining and washed for 20 min in
absolute ethanol before rehydration in PBS. Prehybridization was
performed at room temperature for 30 min, followed by 2 h at 37 C
in hybridization buffer: 50% formamide, 5x NaCl-sodium citrate (SSC),
2% block reagent (Boehringer Mannheim) 0.1%
N-lauroyl-sarcosine, and 0.02% SDS. Digoxigenin-labeled
probes were applied in hybridization buffer at concentrations described
below before incubation for 1618 h at 42 C in a humidified chamber.
Slides were washed in 2x SSC, then 1x SSC, and finally 0.1x SSC,
each for 30 min at 37 C. Hybridized probe was detected using the
alkaline phosphatase-coupled anti-digoxigenin antibody method
(Boehringer Mannheim) with the addition of 1.2 mg/ml levamisole (Sigma)
in the final staining solution.
Osteocalcin and CAT mRNA transcripts were initially detected
using 7.5 ng and 15 ng probe/slide, respectively, for in
situ hybridization. However, with these excess probe
concentrations, the transgene signal was so strong that it was not
possible to detect the increase in transgene RNA that had been
documented using Northern blot analysis. Therefore, reduced probe
concentrations were empirically chosen based on hybridization to
control sections of uninjected OST2 femurs until a minimal level of
osteocalcin and CAT mRNA staining was observed, thus allowing detection
of even small changes in expression. Final concentrations used for
1,25-(OH)2D3 treatment experiments were 0.75
ng/slide and 1.5 ng/slide for osteocalcin and CAT, respectively.
Femoral sections were also stained for tartrate-resistant acid
phosphatase using a method modified from Parkinson et al.
(69) by inclusion of tartaric acid in the staining and prestaining
buffers with a hematoxylin counterstain. Some femoral sections stained
for CAT by immunohistochemistry were also stained for
tartrate-resistant acid phosphatase before immunohistochemical
staining. Sections were photographed using a Zeiss Axiophot microscope
(Carl Zeiss, Thornwood, NY) with differential interference contrast
optics at all magnifications shown.
Statistical Analysis
Treatment responses of mRNA expression and serum osteocalcin
were compared by one, two, and three-way ANOVA followed by Tukeys
post-hoc test. In all analyses, P < 0.05 was
considered significant. Histomorphometric measurements are expressed as
the mean ± SEM from five femoral sections from each
of six animals.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank A. Bourne and R. Enriquez for excellent
technical assistance, and the director and staff (Dr. J. Ferguson,
P. Gregory, T. Chaplin, L. Peters) of the Garvan Institute Biological
Testing Facility for consistently high quality animal care and
breeding. We also thank Dr. S. E. Harris (University of Texas
Health Sciences Center, San Antonio, TX) for the rat osteocalcin cDNA
clone, Dr. C. Gundberg (Yale University, New Haven, CT) for mouse
osteocalcin antibody, standards, and RIA method, E. Vasac (Centre for
Immunology, St. Vincents Hospital, Sydney, Australia) for sharing the
Fast Red TR technique, and Dr. K. W. Ng (St. Vincents Hospital,
Melbourne, Australia) for the use of the Osteomeasure Image Analysis
System.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Edith M. Gardiner, Bone and Mineral Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia.
This work was supported by NIH Grant 1RO1-AR43421 and by funding from
Aza Research Pty. Ltd.
Received for publication May 14, 1997.
Revision received July 22, 1997.
Accepted for publication July 23, 1997.
 |
REFERENCES
|
---|
-
Hauschka PV, Lian JB, Gallop PM 1975 Direct
identification of the calcium-binding amino acid
-carboxyglutamate
in mineralized tissue. Proc Natl Acad Sci USA 72:39253929[Abstract]
-
Price PA, Otsuka AA, Poser JW, Kristaponis J, Raman N 1976 Characterization of a
-carboxyglutamic acid-containing protein from
bone. Proc Natl Acad Sci USA 73:14471451[Abstract]
-
Lian JB, McKee MD, Todd AM, Gerstenfeld LC 1993 Induction of
bone-related proteins, osteocalcin and osteopontin, and their matrix
ultrastructural localization with development of chondrocyte
hypertrophy in vitro. J Cell Biochem 52:206219[Medline]
-
Pockwinse SM, Lawrence JB, Singer RH, Stein JL, Lian JB,
Stein GS 1993 Gene expression at single cell resolution associated with
development of the bone cell phenotype: ultrastructural and in
situ hybridization analysis. Bone 14:347352[Medline]
-
Nakase T, Takaoka K, Hirakawa K, Hirota S, Takemura T, Onoue
H, Takebayashi K, Kitamura Y, Nomura S 1994 Alterations in the
expression of osteonectin, osteopontin and osteocalcin mRNAs during the
development of skeletal tissues in vivo. Bone Miner 26:109122[Medline]
-
Ikeda T, Nomura S, Yamaguchi A, Suda T, Yoshiki S 1992 In situ hybridization of bone matrix proteins in
undecalcified adult rat bone sections. J Histochem Cytochem 40:10791088[Abstract/Free Full Text]
-
Theide MA, Smock SL, Petersen DN, Grasser WA, Thompson DD,
Nishimoto SK 1994 Presence of messenger ribonucleic acid encoding
osteocalcin, a marker of bone turnover, in bone marrow megakaryocytes
and peripheral blood platelets. Endocrinology 135:929937[Abstract]
-
Fleet JC, Hock JM 1994 Identification of osteocalcin mRNA in
nonosteoid tissue of rats and humans by reverse
transcription-polymerase chain reaction. J Bone Miner Res 9:15651573[Medline]
-
Weinreb M, Shinar D, Rodan GA 1990 Different pattern of
alkaline phosphatase, osteopontin, and osteocalcin expression in
developing rat bone visualized by in situ hybridization.
J Bone Miner Res 5:831842[Medline]
-
Roach H 1994 Why does bone matrix contain non-collagenous
proteins? The possible roles of osteocalcin, osteonectin, osteopontin
and bone sialoprotein in bone mineralisation and resorption. Cell Biol
Int 18:617628[CrossRef][Medline]
-
Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C,
Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G 1996 Increased bone formation in osteocalcin-deficient mice. Nature 382:44852[CrossRef][Medline]
-
Price PA, Williamson MK 1981 Effects of warfarin on bone.
Studies on the vitamin K-dependent protein of rat bone. J Biol
Chem 256:1275412759[Free Full Text]
-
Boivin G, Morel G, Lian JB, Anthoine-Terrier C, Dubois PM,
Meunier PJ 1990 Localisation of endogenous osteocalcin in neonatal rat
bone and its absence in articular cartilage: effect of warfarin
treatment. Virchows Arch [A] 417:505512
-
Glowacki J, Rey C, Glimcher MJ, Cox KA, Lian J 1991 A role for
osteocalcin in osteoclast differentiation. J Cell Biochem 45:292302[Medline]
-
Duda RJ, Kumar R, Nelson KI, Zinsmeister AR, Mann KG, Riggs BL 1987 1,25-dihydroxyvitamin D stimulation test for osteoblast function
in normal and osteoporotic postmenopausal women. J Clin Invest 79:12491253[Medline]
-
Price PA, Baukol SA 1981 1,25-dihydroxyvitamin D3 increases
serum levels of the vitamin K dependent bone protein. Biochem Biophys
Res Commun 99:928935[Medline]
-
Ikeda T, Kohno H, Yamamuro T, Kasai R, Ohta S, Okumura H,
Konishi J, Kikuchi H, Shigeno C 1992 The effect of active vitamin D3
analogs and dexamethasone on the expression of osteocalcin gene in rat
tibiae in vivo. Biochem Biophys Res Commun 189:12315[Medline]
-
Celeste AJ, Rosen V, Buecker 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]
-
Pan LC, Price PA 1987 Ligand-dependent regulation of the
1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells. J
Biol Chem 262:46704675[Abstract/Free Full Text]
-
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]
-
Yoon K, Rutledge SJC, Buenga RF, Rodan GA 1988 Characterization of the rat osteocalcin gene: stimulation of promoter
activity by 1,25-dihydroxyvitamin D3. Biochemistry 27:85218526[Medline]
-
Owen TA, Aronow MS, Barone LM, Bettencourt B, Stein GS, Lian
JB 1991 Pleiotropic effects of vitamin D on osteoblast gene expression
are related to the proliferative and differentiated state of the bone
cell phenotype: dependency upon basal levels of gene expression,
duration of exposure, and bone matrix competency in normal
rat osteoblast cultures. Endocrinology 128:14961504[Abstract]
-
Beresford JN, Gallagher JA, Poser JW, Russell RG 1984 Production of osteocalcin by human bone cells in vitro:
effects of 1,25(OH)2D3, 24,25 (OH)2D3, parathyroid hormone and
glucocorticoids. Metab Bone Dis Relat Res 5:229234[Medline]
-
Yamamoto T, Ecarot B, Glorieux FH 1991 In vivo
osteogenic activity of isolated human bone cells. J Bone Miner Res 6:4551[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]
-
Morrison NA, Shine J, Fragonas J-C, Verkest V, McMenemy
L, Eisman JA 1989 1,25-Dihydroxyvitamin D-responsive element and
glucocorticoid repression in the osteocalcin gene. Science 246:11581161[Medline]
-
Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM 1990 DNA
sequences in the rat osteocalcin gene that bind the
1,25-dihydroxyvitamin D3 receptor and confer responsiveness to
1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369373[Abstract]
-
Markose ER, Stein JL, Stein GS, Lian JB 1990 Vitamin
D-mediated modifications in protein-DNA interactions at two promoter
elements of the osteocalcin gene. Proc Natl Acad Sci USA 87:17011705[Abstract]
-
Terpening CM, Haussler CA, Jurutka PW, Galligan MA, Komm BS,
Haussler MR 1991 The vitamin D-responsive element in the rat bone Gla
protein is an imperfect direct repeat that cooperates with other
cis-elements in 1,25-dihydroxyvitamin D3-mediated
transcriptional activation. Mol Endocrinol 5:373385[Abstract]
-
Liao J, Ozono K, Sone T, McDonnell DP, Pike JW 1990 Vitamin D
receptor interaction with specific DNA requires a nuclear protein and
1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:97519755[Abstract]
-
Ross TK, Moss VE, Prahl JM, DeLuca HF 1992 A nuclear protein
essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its
response elements. Proc Natl Acad Sci USA 89:256260[Abstract]
-
MacDonald PN, Dowd DR, Nakajima S, Galligan MA, Reeder MC,
Haussler CA, Ozato K, Haussler MR 1993 Retinoid x receptors
stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin
D3-activated expression of the rat osteocalcin gene. Mol Cell Biol 13:59075917[Abstract]
-
Rahman S, Oberdorf A, Montecino M, Tanhauser SM, Lian JB,
Stein GS, Laipis PJ, Stein JL 1993 Multiple copies of the bone-specific
osteocalcin gene in mouse and rat. Endocrinology 133:30503053[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]
-
Desbois C, Hogue DA, Karsenty G 1994 The mouse osteocalcin
gene cluster contains three genes with two spatial and temporal
patterns of expression. J Biol Chem 269:11831190[Abstract/Free Full Text]
-
Towler DA, Bennett CD, Rodan GA 1994 Activity of the rat
osteocalcin basal promoter in osteoblastic cells is dependent upon
homeodomain and CP1 binding motifs. Mol Endocrinol 8:614624[Abstract]
-
Hoffmann HM, Catron KM, van Wijnen AJ, McCabe LR, Lian JB,
Stein GS, Stein JL 1994 Transcriptional control of the tissue-specific,
developmentally regulated osteocalcin gene requires a binding motif for
the Msx family of homeodomain proteins. Proc Natl Acad Sci USA 91:1288712891[Abstract/Free Full Text]
-
Zhang R, Ducy P, Karsenty G 1997 1,25-Dihydroxyvitamin
D3 inhibits osteocalcin expression in mouse through an
indirect mechanism. J Biol Chem 272:110116[Abstract/Free Full Text]
-
Lian J, Shalhoub V, Aslam F, Frenkel B, Green J, Hamrah M,
Stein G, Stein J 1997 Species-specific glucocorticoid and
1,25-dihydroxyvitamin D responsiveness in mouse MC3T3E1 osteoblasts:
dexamethasone inhibits osteoblast differentiation and vitamin D
downregulates osteocalcin gene expression. Endocrinology 138:21172127[Abstract/Free Full Text]
-
Gerstenfeld LC, Kelley CM, von Deck M, Lian J 1990 Effects of
1,25(OH)2D3 on induction of chondrocytes maturation in culture:
extracellular matrix gene expression and morphology. Endocrinology 126:15991609[Abstract]
-
Ikeda T, Nagai Y, Yamaguchi A, Yokose S, Yoshiki S 1995 Age-related reduction in bone matrix protein mRNA expression in rat
bone tissues: application of histomorphometry to in situ
hybridization. Bone 16:1723[CrossRef][Medline]
-
Kesterson RA, Stanley L, DeMayo F, Finegold M, Pike JW 1993 The human osteocalcin promoter directs bone-specific vitamin
D-regulatable gene expression in transgenic mice. Mol Endocrinol 7:462467[Abstract]
-
Baker AR, Hollingshead PG, Pitts-Meek S, Hansen S, Taylor R,
Stewart TA 1992 Osteoblast-specific expression of growth hormone
stimulates bone growth in transgenic mice. Mol Cell Biol 12:55415547[Abstract]
-
Frenkel B, Capparelli C, Van Auken M, Baran D, Bryan J, Stein
JL, Stein GS, Lian JB 1997 Activity of the osteocalcin promoter in
skeletal sites of transgenic mice and during osteoblast differentiation
in bone marrow-derived stromal cell cultures: effects of age and sex.
Endocrinology 138:21092116[Abstract/Free Full Text]
-
Balmain N, von Eichel B, Toury R, Belquasmi F,
Hauchecorne M, Klaus G, Mehls O, Ritz E 1995 Calbindin-D28K
and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and
mineralization induction in long-term primary cultures of rat
epiphyseal chondrocytes. Bone 17:3745[CrossRef][Medline]
-
Johnson JA, Grande JP, Roche PC, Kumar R 1996 Ontogeny of the
1,25-dihydroxyvitamin D3 receptor in fetal rat bone. J
Bone Miner Res 11:5661[Medline]
-
Schwartz Z, Schlader DL, Ramirez V, Kennedy MB, Boyan BD 1989 Effects of vitamin D metabolites on collagen production and cell
proliferation of growth zone and resting zone cartilage cells in
vitro. J Bone Miner Res 4:199204[Medline]
-
Clemens TL, Tang H, Maeda S, Kesterson RA, Pike JW, Gundberg C 1996 Analysis of serum osteocalcin in transgenic mice reveals major
differences in vitamin D responsiveness between human and mouse genes.
J Bone Miner Res 11 [Suppl I]:S425
-
Maenpaa P, Mahonen A, Pirskanen A 1991 Hormonal regulation of
vitamin D receptor levels and osteocalcin synthesis in human
osteosarcoma cells. Calcif Tissue Int 49:S8586
-
Arbour NC, Prahl JM, DeLuca HF 1993 Stabilization of the
vitamin D receptor in rat osteosarcoma cells through the action of
1,25-dihydroxyvitamin D3. Mol Endocrinol 7:130712[Abstract]
-
Yoshizawa T, Handa Y, Uematsu Y, Sekine K, Takeda S, Yoshihara
Y, Kawakami T, Sato H, Alioka K, Tanimoto K, Fukamizu A, Masushige S,
Matsumoto T, Kato S 1996 Disruption of the vitamin D receptor (VDR) in
the mouse. J Bone Miner Res 11 [Suppl 1]:S124
-
Cole DEC, Carpenter TO, Gundberg CM 1985 Serum osteocalcin
concentrations in children with metabolic bone disease. J Pediatr 106:770776[Medline]
-
Liberman UA 1996 Hereditary deficiencies in vitamin D action.
In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology.
Academic Press. San Diego, CA, pp 903916
-
Holtrop ME, Cox KA, Carnes DL, Holick MF 1986 Effects of serum
calcium and phosphorus on skeletal mineralization in vitamin
D-deficient rats. Am J Physiol 251:E234240
-
Parfitt AM, Mathews CHE, Brommage R, Jarnagin K, DeLuca HF 1984 Calcitriol but no other metabolite of vitamin D is essential for
normal bone growth and development in the rat. J Clin Invest 73:576586[Medline]
-
Lian JB, Carnes DL, Glimcher MJ 1987 Bone and serum
concentrations of osteocalcin as a function of 1,25-dihydroxyvitamin
D3 circulating levels in bone disorders in rats.
Endocrinology 120:21232130[Abstract]
-
Monteiro MJ, Hoffman PN, Gearhart JD, Cleveland DW 1990 Expression of NF-L in both neuronal and nonneuronal cells of transgenic
mice: increased neurofilament density in axons without affecting
caliber. J Cell Biol 111:15431557[Abstract]
-
Taketo M, Schroeder AC, Mobraaten LE, Gunning KB, Hanten G,
Fox RR, Roderick TH, Stewart CL, Lilly F, Hansen CT, Overbeek PA 1991 FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc
Natl Acad Sci USA 88:20652069[Abstract]
-
Wong G, Cohn D 1975 Target cells in bone for parahormone and
calcitonin are different: enrichment for each cell type by sequential
digestion of mouse calvaria and selective adhesion to polymeric
surfaces. Proc Natl Acad Sci USA 72:31673171[Abstract]
-
Rao J, Otto W 1992 Fluorometric DNA assay for cell growth
estimation. Anal Biochem 207:186192[Medline]
-
Downs T, Wilfinger W 1983 Fluorometric quantification of DNA
in cells and tissues. Anal Biochem 131:538547[Medline]
-
Sleigh MJ 1986 A non-chromatographic assay for expression of
the chloramphenicol acetyltransferase gene in eukaryotic cells. Anal
Biochem 156:251256[Medline]
-
Bradford M 1976 A rapid and sensitive method for the
quantiation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
-
Gundberg CM, Clough ME, Carpenter TO 1992 Development and
validation of a radioimmunoassay for mouse osteocalcin: paradoxical
response in the Hyp mouse. Endocrinology 130:19091915[Abstract]
-
Reeves P, Nielsen F, Fahey GJ 1993 AIN-93 purified diets
for laboratory rodents: final report of the American Institute of
Nutrition ad hoc writing committee on the reformulation of
the AIN-76A rodent diet. J Nutr 123:193951[Medline]
-
Chomczynski P, Sacchi N 1986 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef]
-
Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter W,
Goodman H 1977 Rat insulin genes: construction of plasmids containing
the coding sequences. Science 196:13131319[Medline]
-
Zhou H, Choong P, McCarthy R, Chou ST, Martin TJ, Ng KW 1994 In situ hybridization to show sequential expression of
osteoblast gene markers during bone formation in vivo.
J Bone Miner Res 9:14891499[Medline]
-
Parkinson IH, Fazzalari NL, Durbridge TC, Moore RJ 1991 Simplified approach to enzymatic identification of osteoclastic bone
resorption. J Histotech 14:8183
-
Huang MTF, Gorman CM 1990 The simian virus 40 small-t intron,
present in many common expression vectors leads to aberrant
splicing. Mol Cell Biol 10:18051810[Medline]