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Department of Cell Biology and Cancer Center, University of Massachusetts Medical Center, Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to authors at the Department of Cell Biology and Cancer Center, University of Massachusetts Medical Center, Worcester, Massachusetts 01655-0106.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In transgenic mice, 1.7 kb of the rat (18) and 3.9 kb of the human (19, 20, 21) osteocalcin promoter have been shown to target reporter gene expression to the skeleton. The present study was designed to 1) further define minimal sequence requirements for osteocalcin transcription in transgenic mice; 2) characterize osteocalcin promoter activity in vivo as a function of age, sex, and skeletal site; and 3) address osteocalcin promoter activity as a function of osteoblast differentiation in bone marrow-derived cell cultures. Our results demonstrate that as little as 0.35 kb of the rat osteocalcin proximal promoter, which excludes the distal DHS and the VDRE, can facilitate bone-specific expression. However, neither this nor longer promoter sequences, up to 1.7 kb, can confer site of integration-independent expression in transgenic mice. Detailed studies with a transgenic lineage expressing the 1.7-kb osteocalcin promoter-reporter construct at high levels demonstrate that osteocalcin transcription in vivo is dependent on age and sex and is correlated between distant skeletal sites (femurs, calvariae, and lumbar vertebrae). During osteoblast differentiation in transgenic bone marrow-derived stromal cell cultures, the activity of this 1.7-kb promoter is dependent on the developmental stage and is independent of the sex and age of the donor animal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice
Plasmids were digested and the osteocalcin-CAT fusion genes were
electrophoretically separated from vector sequences, electroeluted, and
purified by cation exchange chromatography (Plasmid Mini Kit, Qiagen,
Chatsworth, CA). Microinjection of fertilized oocytes from C57Bl/6XSJL
mice (Jackson Laboratories, Bar Harbor, ME) and transfer of
microinjected embryos into Swiss-Webster pseudopregnant recipient mice
(Taconic Services, Germantown, NY) were performed in the Transgenic
Animal Core Facility of the University of Massachusetts Medical Center.
Animals were maintained and used in accordance with the Federal Animal
Welfare Act and the NIH Guide for the Care and Use of Laboratory
Animals.
Southern blot analysis
Integration of the transgenes was assessed in EcoRI-
or HincII-digested tail DNA (10 µg) by Southern analysis
using as probe a rat osteocalcin HincII/SacI
(-531/-309) fragment. Animals carrying the shortest promoter
construct (-348OCCAT3) were identified using as probe a rat
osteocalcin BglII/HindIII (-348/+24) fragment
(the HindIII site at position +24 was artificially
introduced to prepare all the chimeric constructs). In addition to
hybridizing to the transgenes in the positive mice, these probes
hybridize with the highly homologous mouse osteocalcin promoter
sequences. Thus, transgene copy number could be directly determined
based on the intensity of the band representing the transgene compared
to those of the bands representing the three endogenous mouse
osteocalcin genes (24).
Bone marrow stromal cell cultures
Mice were killed by cervical dislocation, and the femurs were
aseptically removed. The epiphyses and growth plates were dissected,
and the diaphyseal marrow was flushed in cold culture medium (
MEM)
supplemented with 20% heat-inactivated FBS (Atlanta Biologicals,
Norcross, GA) and 10-8 M menadione sodium
bisulfate (Sigma Chemical Co., St. Louis, MO). Cells were centrifuged
for 10 min at 1000 x g, resuspended in fresh culture
medium, filtered through a 100-mesh steel screen, and seeded onto 35-mm
six-well plates at 107 cells/well. Ascorbic acid (50
µg/ml) and ß-glycerophosphate (10 mM) were added
24 h later and were present throughout the culture period. Medium
was changed on day 4 and every 48 h thereafter. Secreted
osteocalcin was determined by RIA, as previously described (25), using
radioiodinated mouse osteocalcin from Biomedical Technologies
(Stoughton, MA) and antimouse osteocalcin antiserum provided by Dr.
Caren Gundberg (Yale University, New Haven, CT).
CAT assays
Tissue samples (50–250 mg wet weight) were dissected, frozen in
liquid nitrogen, and stored at -70 C. Each sample was homogenized in
0.7–1.4 ml 0.25 M Tris-Cl buffer (pH 7.5) and placed in a
dry ice-ethanol bath. All samples were then subjected to three cycles
of freezing and thawing, followed by 15-min incubation at 65 C to
inactivate acetyl coenzyme A (CoA)-consuming activity (26). Insoluble
tissue components were removed by 10 min of centrifugation (15,000
x g) at room temperature, and the supernatant was stored at
-70 C. Cultured cells were rinsed twice and collected in cold PBS,
pelleted, and lysed in 100 µl reporter lysis buffer (Promega). CAT
assays were performed, as previously described (27), in the presence of
0.1 mM (57 mCi/mmol) [14C]chloramphenicol
(New England Nuclear, Boston, MA) and 1 mM acetyl CoA
(Sigma). The duration of incubation with these reagents and the amount
of cell or tissue extracts were adjusted to comply with linearity
requirements. Samples with low CAT activity were assayed for 12 h,
and two additional acetyl CoA doses were added at 2 and 4 h. The
substrate and products were extracted, separated by TLC, and
quantitated by direct counting using a Betascope analyzer (Betagen,
Waltham, MA). CAT activity was calculated as the percent conversion
corrected for protein. Differences between CAT activities in duplicate
samples did not exceed 15%.
Statistical analyses
One transgenic line (SR62, carrying the 1.7-kb promoter
construct) was expanded, and CAT values from three skeletal sites of
multiple F1 progenies were analyzed using the SPSS statistical software
package (SPSS, Chicago, IL). The data were first transformed by natural
logarithms to better approach a normal distribution. The effects of sex
and age on osteocalcin promoter activity in vivo were
evaluated for each skeletal site separately by univariate analyses of
covariance or for all skeletal sites simultaneously by multivariate
analysis of covariance. Correlation between activities at the various
skeletal sites was estimated by principal component analysis, in which
the independent proportion of the total variability that is correlated
between these sites is completely partitioned into the first principal
component (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, each of these four constructs
exhibited CAT expression in bone in at least one transgenic line.
However, in most of the lines there was no detectable expression in any
tissue examined. CAT activity in bone was detectable in only one of
five lines carrying the 1.7-kb promoter construct, one of two lines
carrying the 1.1-kb promoter construct, two of five lines carrying the
0.72-kb promoter construct, and one of six lines carrying the 0.35-kb
promoter construct. Thus, as little as 0.35 kb of the rat osteocalcin
promoter can direct tissue-specific expression in vivo.
However, neither this nor longer promoter fragments up to 1.7 kb
suffice for site of integration-independent expression. The low
frequency of expressing lines does not allow statistically significant
comparisons among expression levels of the constructs used in this
study.
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shows a representative CAT assay of a line SR62 progeny,
carrying the 1.7-kb promoter construct. This line, which exhibited the
highest CAT levels, was chosen for additional experiments presented
here. Similar to the study of Kesterson et al. (19), a low
level of ectopic osteocalcin promoter activity in lineage SR62 was
detected in the brain (
1% of the bone level). Lack of ectopic
expression with the shorter constructs (Fig. 1A) could have resulted
from the sensitivity limit of the assay and should not be interpreted
as improved tissue specificity.
Variability of osteocalcin promoter activity in vivo
Assay of CAT activity in multiple F1 progenies of lineage SR62,
highly expressing the 1.7-kb osteocalcin promoter construct, indicated
a wide range of expression levels. Figure 2 shows
differences of up to 100-fold among five 6-month-old females. To
establish whether these differences reflected inconsistent sample
processing or variability in the promoter activity in vivo,
we examined the correlation between CAT activity measured in both
femurs of each of these transgenic animals. The results (Fig. 2)
demonstrate very good correlation between the activities measured in
the left and right corresponding femurs (r2 = 0.997),
indicating that the differences observed among individual animals do
occur in vivo. Because CAT is relatively stable in mammalian
cells (t1/2 = 50 h) (29), the variability among
individual mice is unlikely to reflect differences in handling the
animals immediately before or during killing. Also, there was no
correlation between osteocalcin promoter activity and the time of day
at which the animals were killed. Thus, the activity of the osteocalcin
promoter is significantly variable among individual transgenic mice at
a given time point.
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), calvariae (Fig. 3B), and
lumbar vertebrae (Fig. 3C) of the F1 progeny of lineage SR62, carrying
the 1.7-kb promoter construct. Again, the data demonstrate high
variability among individual animals in all three skeletal sites and in
all of the age and sex groups. Five-week-old animals exhibit the
highest CAT levels in all skeletal sites. By 3 months of age, promoter
activity declines, but significant levels are retained, reflecting bone
turnover in the adult mice. The effect of age on CAT activity was
highly significant (P < 0.0004, P <
0.001, and P < 0.01 for femurs, calvariae, and
vertebrae, respectively). Unexpectedly, we also observed higher
promoter activity in females than in males. The gender effect was
highly significant (P < 0.003) when tested
simultaneously in all skeletal sites. Analysis of the gender effect in
each skeletal site separately indicated a highly significant difference
in vertebrae (P < 0.001) and femurs (P
< 0.005). The calvariae exhibited the same trend, although a
statistically significant difference was not achieved
(P = 0.056). Focusing on the 6–7 month age group, the
ratio between the mean activities in females and males was 9-fold for
the femurs (mean ± SEM, 16 ± 18 vs.
1.7 ± 2.5; P < 0.004), 10-fold for the calvariae
(62 ± 116 vs. 6.1 ± 8.6; P <
0.04), and 7-fold for the lumbar vertebrae (40 ± 36
vs. 5.4 ± 7.9; P < 0.001).
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) is likely to reflect fluctuations that occur in
vivo in either the cellular composition or the promoter activity
per osteoblast in the examined tissues. These fluctuations could occur
independently in the various skeletal sites. Alternatively, there could
be a mechanism coordinating osteocalcin promoter activity among femur,
calvarium, and vertebrae. To address these possibilities, we examined
the correlations between osteocalcin promoter activity in pairs of
skeletal sites, focusing initially on the 6–7 month age group. As
shown in Fig. 4, there is positive correlation between
any two given sites in both females (top three panels) and
males (bottom three panels), i.e. high promoter
activity in any given skeletal site of a given animal is generally
associated with high activity in the other sites. The best correlations
were between the calvariae and the femurs of the females
(r2 = 0.88) and between the vertebrae and the calvariae of
the males (r2 = 0.96). To examine the interrelationships
among the three skeletal sites simultaneously, the data were reduced to
a single value by principal component analysis (see Materials and
Methods). The results indicated that a single large component of
the data accounted for 70% of the total variation, and that this
factor was heavily loaded for all three skeletal sites. Therefore, a
common underlying (latent) variable simultaneously affects osteocalcin
promoter activity in distant skeletal sites.
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We initially established optimal conditions for osteoblast
differentiation in bone marrow cultures derived from the transgenic
mice. In particular, we addressed the requirement for glucocorticoids,
as these are essential for osteoblast differentiation in rat (30, 31, 32)
and human (34) bone marrow cultures. However, consistent with previous
studies of osteoblast proliferation and differentiation in mouse
calvarial (36, 37)- and bone marrow (33)-derived cell cultures, we
found that osteoblast differentiation in marrow stromal cultures from
SR62 transgenic mice occurred in the absence of glucocorticoids (Fig. 5). Significant activity of the osteocalcin promoter was
demonstrable on day 14 of culture by both CAT assay of the cells (Fig. 5A, left panel) and RIA of medium samples (right
panel). Furthermore, these parameters were strongly inhibited by
10-8 M dexamethasone (Fig. 5A), a
concentration that is optimal for promotion of osteoblast
differentiation in rat and human marrow-derived cultures. TGF-ß1
(0.05 ng/ml), recently suggested to play a role in osteoblast
differentiation in mouse bone marrow-derived cultures (38), did not
affect any of the parameters evaluated in this study (not shown).
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). As shown in Fig. 5C, the activity of the 1.7-kb
osteocalcin promoter, assessed by CAT assay, was undetectable in the
early cultures (day 4, proliferative period). In contrast, cells
collected on both days 10 and 14 from cultures with nodules (see Fig. 5B) exhibited high CAT activity (Fig. 5C), reflecting development of
the osteoblast phenotype. Thus, the -1727/+24 osteocalcin gene
sequences direct the correct developmental expression of osteocalcin
promoter activity during osteoblast differentiation in bone
marrow-derived stromal cell cultures.
Assays of CAT activity in bone marrow stromal cell cultures obtained
from individual mice showed minimal variations in the level of promoter
activity (Fig. 5, C and D). This contrasts with the high variability
observed in vivo among individual transgenic animals (Fig. 3). Also, unlike the sex- and age-dependent promoter activity observed
in vivo (Fig. 3), osteocalcin promoter activity in the bone
marrow-derived cultures was not dependent on either sex or age when
expressed on a protein basis (Fig. 5D). However, higher cellularity in
the cultures derived from the younger animals resulted in higher CAT
activity per well (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3-subunit gene (41), the -239/+55 sequence of the
olfactory marker protein gene (42), the -236/+64 sequence of the POMC
gene (43), the -350/+64 sequence of the 2(I) collagen
gene (44), and the -426/+28 sequence of the probasin gene (45). In the
present study we report bone-specific CAT expression in a transgenic
line (Bgl46) carrying five copies of the CAT gene under the control of
the -348/+24 osteocalcin promoter sequence. Expression of this short
promoter may be attributed to the ability of this osteocalcin gene
segment to form a DNase I-hypersensitive site (nucleotides -70/-170),
detected only in cells expressing osteocalcin (12, 14). Potentially
contributing to the bone-specific expression of the -348/+24 promoter
construct are elements that bind osteoblast-restricted transcription
factor complexes, including OC box II/osteocalcin-specific element-2
(nucleotide -130) (5, 10, 15, 16, 17), OC box I (nucleotide -100) (6, 7, 8, 9),
and osteocalcin-specific element 1 (nucleotide -60) (10).
Significantly, expression of the -348/+24 osteocalcin promoter segment
in the Bgl46 transgenic mice occurs in the absence of the distal,
VDRE-containing, DHS that resides between -400 and -600 (13). Thus,
our results demonstrate that the -348/+24 osteocalcin basal promoter
can express autonomously in vivo, albeit at low levels.
Compared to some other short mammalian promoters (40, 41, 42, 43, 44, 45), proximal
osteocalcin promoter sequences as well as longer segments up to 1.7 kb
appear very sensitive to negative regulation by host sequences at the
sites of integration in transgenic mice, resulting in undetectable
expression in most of the transgenic lines generated. Elements
insulating the gene from these negative effects and/or far distant
enhancer elements must reside upstream of position -1727 and/or
downstream of position +24. Also excluded from the -1727/+24 rat
osteocalcin promoter are sequences that inhibit transcriptional
activity in the brain. Interestingly, both a human 3.9-kb osteocalcin
promoter (19) and a mouse 0.4-kb 2(I) collagen promoter
(44), which are expressed in osseous tissues, exhibit ectopic activity
in the brain. Inhibition of osteocalcin promoter activity in the brain
may be mediated by 3'-flanking sequences (21), which were not included
in our study.
The expression level of the 1.7-kb rat osteocalcin promoter construct
in lineage SR62 was 100-fold higher than the levels observed with
the 1.1-, 0.72-, and 0.35-kb promoter deletion constructs (lines Z4,
Nco8, Nco17, and Bgl46). However, more expressing lines will have to be
generated to provide a statistically significant comparison between the
1.7-kb and the shorter promoter constructs. The -1.7/-1.1-kb sequence
contains three A · T alternating tracts (86%, 94%, and 88% AT
richness within 44-, 49-, and 25-bp stretches, respectively) as well as
B1 and B2 repetitive sequences. Based on the roles of AT tracts and
repetitive sequences in gene/nuclear matrix interactions, recently
reviewed by Boulikas (46), we speculate that this promoter domain may
enhance transcriptional activity by facilitating interactions of
osteocalcin promoter elements with protein components of the nuclear
matrix (47).
Using the SR62 transgenic line, expressing the 1.7-kb rat osteocalcin promoter construct, and consistent with a previous preliminary report (20), we observed a decline in osteocalcin promoter activity as a function of age, possibly contributing to the age-related decrease in mouse circulating osteocalcin levels reported by Gundberg et al. (25). However, our study also revealed severalfold higher promoter activity in females than in age-matched males. This may reflect higher osteocalcin promoter activity per osteoblast due to sex-related differences in the representation or activity of transcription factors and/or increased bone-forming surfaces in the females. Interestingly, neither the sex- and age-related differences nor the interanimal variations (discussed below) occur in osteoblasts cultured from bone marrow of individual mice, implicating extracellular signals, rather than intrinsic cellular parameters, in the differences observed in vivo.
Osteocalcin promoter activity exhibits high variability among sex- and age-matched animals, which is not attributable to intraassay variations. Analysis of the variance in three skeletal sites demonstrated that promoter activity in the femurs, calvariae, and lumbar vertebrae are coupled. This finding suggests a common regulatory mechanism that strongly influences osteocalcin transcription in distant osseous sites. The nature of this mechanism, possibly a humoral factor(s), and to what extent it contributes to the sex-related differences remain to be explored. In any case, our results suggest fundamental regulatory mechanisms common to osteoblasts in skeletal sites that exhibit distinct histological, biochemical, and hormone-responsive properties (48, 49, 50).
Developmental control of the osteocalcin promoter, i.e. absence of transcriptional activity in osteoprogenitor cells and during the early proliferative stages, has been well documented in calvaria-derived osteoblast cultures (reviewed in Ref.1). The activity of the 1.7-kb rat osteocalcin promoter in marrow-derived stromal cell cultures seems to follow this developmental pattern. Notably, this promoter sequence lacks osteocalcin intragenic silencer sequences previously suggested by us and others to play a role in preventing premature osteocalcin transcription. Despite some compelling evidence supporting this idea (51, 52, 53), several observations appear inconsistent with a role for the osteocalcin silencer in the developmental control of this gene: 1) potent silencing is mediated by osteocalcin intragenic sequences in both transiently (53, 54) and stably (our unpublished data) transfected ROS 17/2.8 cells, which represent a mature osteoblastic phenotype; and 2) the 1.7-kb osteocalcin promoter construct, lacking the intragenic negative regulatory sequences, is silent during the early stages of osteoblast differentiation in marrow-derived stromal cell cultures.
In summary, we have used transgenic mice to characterize osteocalcin promoter activity in vivo. We demonstrate bone-specific transcription driven by rat osteocalcin promoter sequences ranging from 0.35 kb (lacking the VDRE) to 1.7 kb. Additional studies are required to identify sequences that confer 1) site of integration-independent expression and 2) elimination of the low expression observed in the brain. Detailed examination of the SR62 transgenic line, which expresses the 1.7-kb promoter construct at relatively high levels, demonstrated 1) an age-related decrease in promoter activity, 2) higher activity in females than in males, 3) linkage of promoter activities among distant skeletal sites, and 4) correct developmental control of osteocalcin promoter activity during osteoblast differentiation in stromal cell cultures derived from bone marrow of transgenic animals.
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Acknowledgments |
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Footnotes |
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Received November 7, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 subunit gene are required for
brain expression in transgenic mice. Nucleic Acids Res 22:4748–47552(I) collagen promoter.
Studies in transgenic mice and in tissue culture cells. J Biol
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