| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Articles |
University of Massachusetts Medical Center, Department of Cell Biology and Cancer Center, Worcester, Massachusetts 01655-0106
Address all correspondence and requests for reprints to authors at the Department of Cell Biology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655-0106.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
The mouse MC3T3-E1 cell line undergoes a normal developmental sequence of osteoblast differentiation in association with formation of a bone-like mineralized extracellular matrix (ECM) (13, 14, 15) similar to rat calvarial-derived osteoblasts (16) and glucocorticoid inducted rat marrow stromal cells (17). We examined the effects of the glucocorticoid Dex in regulating osteoblast maturation in this mouse cell line for several reasons. Differences in responsiveness of mouse and rat calvarial derived osteoblasts to glucocorticoid effects on cell growth and regulation of vitamin D receptor (VDR) levels (18, 19, 20) as a function of cell density were reported over a decade ago before the knowledge that mouse osteoblasts develop a mineralized ECM in vitro. We experimentally addressed the biological question as to whether glucocorticoids promote accelerated differentiation of MC3T3-E1 cells similar to fetal rat calvarial derived osteoblasts. Additionally, as a cell line, MC3T3-E1 osteoblasts permit investigation of the regulated expression of stably integrated promoter-reporter constructs during osteoblast maturation in a mineralizing ECM. However, selection of cell lines with stably integrated transgenes requires many population doublings, and the ability of these cells to exhibit phenotypic properties and produce a mineralized ECM is potentially limited to early passaged cells (21). Thus, we explored conditions for Dex to maximize complete differentiation of these cells and biosynthesis of a mineralized matrix.
Here we report that subclones of MC3T3-E1 cells with stably integrated transgenes differentiate. More importantly, our studies reveal differences in responsiveness to Dex, related to ECM mineralization and gene expression, that are distinct from results with rat or human osteoblasts (4, 22). MC3T3-E1 cells also exhibit selective hormone responsiveness in gene expression related to pre- and postmineralization stages, consistent with observations in other cell systems (23, 24). However, a murine species-specific property is the lack of transcriptional enhancement of OC by 1,25-(OH)2D3. In all other species examined (including human, rat, and chicken) OC gene expression is enhanced by vitamin D treatment in vitro (reviewed in 25).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
-DMEM medium with 10% FCS. After expansion of the
cells, experiments were initiated, after passage 15, by plating at
0.4 x 106/100 mm2 plate or 0.08 x
106/24 mm well. Beginning on day 2, cells were fed every
other day with a differentiation medium that included 10 mM
ß-glycerophosphate and 50 µg/ml ascorbic acid in the -DMEM-10%
FCS stock medium. Mineralization was highly dependent on serum lots,
but passage number (up to passage 35 was examined) was not a
contributing factor. Cultures were viable for over 60 days. When
screening serum lots, we observed that measurements of secreted OC in
the 48 h-conditioned media paralleled the extent of mineralization of
the ECM, as reported for rat calvarial cultures (16). OC secretion was
quantitated by RIA (26) using species-specific mouse OC antisera (a
kind gift from Dr. Caren Gundberg, Department of Orthopaedics, Yale
University, New Haven, CT). Iodinated mouse OC was purchased from
Collaborative Research (Stoughton, MA). For acute treatments, hormones
were added with fresh culture media on a scheduled feeding day and
cells harvested 24 h later; for chronic treatments, hormones were
added with fresh medium at each feeding. Dex (Sigma Biochemical, St.
Louis, MO) and 1,25-(OH)2D3 (a gift from Milan
Uskokovic, Hoffmann-La Roche, Nutley, NJ) were added to cultures in
ethanol to generate a 0.001% final concentration of vehicle. For
alkaline phosphatase (AP) histochemical staining, the Sigma reagents
(napthal AS-MX phosphatase and Fast Red TR salt) were used as
previously described (16). Mineral deposition was monitored by both
Alizarin red staining (40 mM, pH4.2, on 70% ethanol fixed
cell layers) for calcium and the von Kossa stain on 2.5%
paraformaldehyde fixed cell layers (3% silver nitrate) for phosphate
detection.
Plasmid constructs
PLd40-Luc, which contains the 40-bp
H-2Ld promoter (27) in the pGL2 basic luciferase plasmid
(Promega), was used as a heterologous promoter construct.
Double-stranded 29-bp oligonucleotides, containing the rat osteocalcin
(rOC) vitamin D response element (VDRE) (nucleotide -519 to -491) or
the mouse osteocalcin (mOC) VDRE (nucleotide -468 to -440), were
cloned into the SmaI site of the PLd40-Luc
plasmid. Constructs were validated by restriction digests and
dideoxy-sequencing to confirm sequences and orientations of
oligonucleotides cloned.
Transient transfections and Luciferase assays
MC3T3-E1 cells, plated at a density of 0.5 x
106 cells per 100 mm plate, were used for transient
transfections by the calcium phosphate coprecipitation (27). Total DNA
was maintained at 20 µg/plate and consisted of 10 µg test DNA and
10 µg salmon sperm DNA. After overnight incubation with the DNA,
cells were fed with or without 10-8 M
1,25-(OH)2D3 and harvested 48 h later.
Cells were transfected, with n = 6 per treatment group. Cells were
lysed in 100 µl reporter lysis buffer (Promega), and luciferase
activity was measured with a luminometer (27).
Stable transfection and CAT assays
A -1727-nt OC promoter-CAT reporter gene (-1727 OCCAT) (28)
was assayed in stably transfected cells to provide a basis for
comparison between activity of the endogenous OC gene in the murine
MC3T3-E1 cells and the rat OC promoter activity. MC3T3-E1 and ROS
17/2.8 cells were cotransfected in 35-mm wells with 5 µg p-1727 OCCAT
and 0.4 µg pCEP4 (Invitrogen Corp., San Diego, CA) encoding
Hygromycin B phosphotransferase, as previously described (29).
Nontransfected cells were killed with 55 u/ml (ROS 17/2.8) or 250 u/ml
(MC3T3-E1) Hygromycin B (Calbiochem, La Jolla, CA), and 
15 resistant
colonies were pooled and expanded for 3–5 passages. To evaluate
promoter activity, MC3T3-E1 cells were grown under the differentiation
protocol described above. ROS 17/2.8 cells were maintained in Ham’s
F-12 medium (Life Technologies, Grand Island, NY), supplemented with
5% FCS (Atlanta Biological, Norcross, GA). Cells obtained from
confluent 35-mm wells were lysed in reporter lysis buffer (Promega,
Madison, WI) and assayed for CAT activity, as previously described
(29). The samples were incubated with 0.25 µCi (1 Ci - 37 GBq) of
14C-chloramphenicol (Dupont, Boston, MA) for 4–12 h. After
ethyl acetate extraction, the samples were separated by chromatography
on Whatman TLC plates (Fisher Scientific, Malvern, PA). Radioactivity
associated with the substrate and the products was quantitated using a
Betascope 603 blot analyzer from Betagen (Waltham, MA). CAT activity
was calculated as percent conversion and corrected for protein.
RNA isolation and hybridizations
Total cellular RNA was isolated from frozen cell pellets, stored
at -70 C, by the procedure of Chirgwin et al. (30).
Quantitation and intactness of RNA and the details of the Northern
analyses used in these studies have been detailed elsewhere (4). The
gene plasmids of histone, or the human 18S ribosomal gene (4), or the
cDNAs of rat type I collagen (31), rat osteopontin (OP) (32), rat OC
(33), the rat VDR (34), or the rat glucocorticoid receptor (GR) (35)
were labeled, as previously described, and used as probes (4).
Quantitation of the messenger RNAs (mRNAs) was done by image analysis
(GDS2000 Gel Documentation System, Ultra-Violet Products Inc., San
Gabriel, CA) of short autoradiographic exposures. The values for each
transcript were normalized to 18S ribosomal RNA.
Gel mobility shift assays
Nuclear extracts were prepared, as previously described (36),
from confluent ROS 17/2.8 cells treated with vitamin D for 24 h.
Gel-purified oligonucleotides (Table 1
) were end-labeled
with (
32P)ATP and used for protein/DNA binding reactions
in the presence or absence of a 100-fold molar excess of competitor
DNA. DNA binding reactions contained 5 µg protein mixture in a final
KCl concentration of 50 mM, 1 mM DTT, and 1.5
µg of the nonspecific competitor DNA, poly(dI-dC)•poly(dI-dC), and
were incubated for 20 min at room temperature. Antibodies were
incubated with the binding reactions for 1 h on ice before
addition of probe. Immunoreactivity of gel shift complexes was assessed
with monoclonal VDR antibody (no. F2A10, kindly provided by Dr. H. F.
DeLuca, University of Wisconsin, Madison, WI).
|
, ß,) recognizes
the D/E region of RXR; this antibody cross-reacts with RXR, RXRß,
and RXR. Antibodies 4RX3A2, 16RX3E8, and 12RX2D3 specifically
recognize RXR, RXRß, and RXR, respectively. To control for band
specificity, antibodies were incubated with labeled oligonucleotide
alone. A nonspecific polyclonal anti human glucocorticoid receptor
antibody was used as an additional control. Protein/DNA complexes were
separated in a 4% polyacrylamide gel (acrylamide:bisacrylamide =
40:1) in 1.0 x TBE electrophoresis buffer (0.09 M
Tris, 0.09 M boric acid, 2 mM EDTA). Gels were
dried and exposed to x-ray film.
Biochemical assays for DNA, protein, and AP activity
DNA was assayed after extraction of the cell layers in 1N
perchloric acid, followed by DNA hydrolysis for 30 min at 80 C. The DNA
was quantitated by fluorometric assay using diaminobenzoic acid reagent
(37). Total protein was estimated in solubilized cell layers by the
Bradford reagent (38). AP enzyme activity was quantitated in cell
layers dissolved in 0.1 M glycine, 0.001 M
MgCl2, 0.1% Triton X-100, pH 10.5. Enzyme activity was
assayed by the method of Lowry et al. (39) using
p-nitrophenol phosphate as substrate (Sigma). Absorbance at 410
nM was measured with a Beckman DU-70 spectrophotometer
(Beckman Instruments, Inc., Fullerton, CA) to determine the amount of
p-nitrophenol produced.
Statistical analysis
Results are given as means ± SEM. Student’s
t test and, when appropriate, two-factor ANOVA were used to
examine the individual effects and interaction between vitamin D and
glucocorticoid treatments. The statistical analyses were done using
MacIntosh STATview software (Abacus Concepts, Berkeley, CA).
P < 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
). Dex
had significant stimulatory effects on cell growth when chronic
treatment was initiated at each period, reflected by an increased total
DNA content (Fig. 1A), except at the higher concentration
(10-7 M) initiated on day 4. Protein
normalized to DNA was not significantly affected (Fig. 1B). However, AP
enzyme activity/µg DNA (Fig. 1C) was reduced to 30–50% of control
at both 10-7 and 10-8 M Dex
concentrations (P < .001). The day 30 levels of
secreted OC revealed a marked inhibition of OC synthesis at both doses
and at all times of treatment initiation (Fig. 1D). Addition of Dex at
10-7 or 10-8 M on day 4
significantly (P < .001, 10-7
M; P < 0.05, 10-8
M) decreased calcium accumulation to 25–75% of control
(Fig. 1E). Addition of Dex on day 7 or day 13 had no significant effect
on calcium accumulation.
|
). In addition, the pattern of AP and mineral staining
differed from the uniform appearance in control cultures. Dex treatment
resulted in punctate staining with small islands of intensely stained
cells, and internodular areas of AP negative cells remained clearly
distinct. When Dex treatment was initiated on day 13, no significant
differences in distribution of AP from control cultures were
observed.
|
). Cellular representation of mRNAs on day 21
demonstrates that the 10-7 M Dex initiated on
days 4 and 7 suppressed OC and OP expression, but no significant
changes were evident at the 10-8 M dose. By
day 30, when ECM mineralization is completed, OC and OP mRNA levels are
further up-regulated in untreated control cultures. Notably, OC
expression remains suppressed at 10-7 M and is
lower than control at 10-8 M Dex. However, OP
mRNA levels on day 30 are only slightly lower than the control level.
Dex increased histone transcripts on day 30, consistent with
stimulation of cell growth (see Fig. 1A
).
|
) as an indication of gene expression associated with
formation of the ECM. Levels of glucocorticoid and VDR mRNAs (Fig. 4)
also were monitored because of the well-documented influences of the
cognate ligands on expression of their receptors.
|
).
However, in postproliferative osteoblasts (day 20), H4 mRNA levels were
up-regulated approximately 2-fold by Dex. This increase over control
persisted in the presence of combined vitamin D and Dex treatment (D +
Dex group, day 20). Collagen type I mRNA was represented at similar
levels and modestly decreased by vitamin D at both stages of
maturation. Dex had a greater inhibitory effect on expression of type I
collagen, particularly on day 20. With combined treatment of both
hormones, a dramatic reduction of collagen mRNA levels occurred,
particularly on day 6 (to 6% of control values). OC expression was completely inhibited by Dex in the day 6 cultures. In contrast to the vitamin D-mediated up-regulation of OC that occurs in rat, human, chicken, and bovine cells, a 50% decrease in OC mRNA is observed in the mouse MC3T3-E1 cell cultures at both early and late culture stages. The combination of vitamin D and Dex further lowered OC mRNA, suggesting an additive effect of the two hormones. The observed inhibition of OC gene expression by vitamin D and Dex is selective and does not reflect a general metabolic inhibition, as these responses were not observed when OP expression was examined. Vitamin D up-regulated OP mRNA levels 5-fold on day 6 and 2.2-fold on day 20. Dex had no significant effect on mRNA levels on day 6; but in mature osteoblasts (day 20), a slight decrease was observed. The combination of vitamin D with Dex did not significantly affect the vitamin D-stimulated levels.
Interestingly, control levels of glucocorticoid receptor mRNA were decreased, whereas VDR mRNA levels did not change between day 6 and day 20 MC3T3-E1 cultures, compared with relative increases in basal OC and OP levels from day 6–20. Dex down-regulated GR mRNA levels, and 1,25-(OH)2D3 had a modest inhibitory effect on GR mRNA. VDR mRNA was down-regulated by vitamin D and up-regulated by Dex, only on day 6. Dex on day 20 decreased VDR mRNA levels. However, the combination of D + Dex resulted in a dramatic increase in VDR mRNA levels on both days (3- and 6-fold, days 6 and 20, respectively).
These observations show selective effects of Dex on histone and OP, and vitamin D on VDR mRNA levels, as a function of the stage of osteoblast differentiation. The OC, GR, and collagen genes exhibit the same general trends on day 6 and day 20, but the extent of the changes depends on the stage of differentiation.
Negative control of vitamin D on mouse OC gene expression
There are three copies of the mouse OC gene (40). Only the two
nearly identical copies are expressed selectively in bone and contain a
VDRE. In the present studies (shown in Fig. 4), we determined that in
MC3T3-E1 cells, mouse OC mRNA levels are decreased to 50% of control
levels in response to 1,25-(OH)2D3. Therefore,
in a separate experiment, we examined OC synthesis in mouse MC3T3-E1
cultures (Fig. 5) after a 24-h treatment with hormones.
In confluent cultures (day 11), vitamin D inhibited OC synthesis to
25% of control levels and, in mineralizing cultures (day 18), to 48%
of control values, paralleling the changes in mRNA levels (Fig. 4). On
day 18, we examined the inhibition of OC synthesis by various
concentrations of vitamin D (Fig. 5B). At 10-8
M and 10-9 M hormone, significant
decreases in OC synthesis were observed with no change in synthesis
with 10-10 M
1,25-(OH)2D3.
|
Hormonal responsiveness of the rat OC promoter construct in the
MC3T3-E1 cells and of the same construct in stably transfected ROS
17/2.8 cells were compared (Fig. 6). Increased activity
of the rat OC promoter, after vitamin D treatment (10-8
M, 48 h), was observed in both the rat and murine cell
lines. Furthermore, the combination of
1,25-(OH)2D3 with Dex resulted in reduction of
the vitamin D stimulated rat OC promoter activity. However, these
effects were more pronounced in rat ROS 17/2.8 cells than in mouse
MC3T3-E1 cells. Notably, Dex alone did not affect rat OC promoter
activity in the mouse cells. Synthesis of endogenous mouse OC in the
MC3T3-E1 cells carrying the pSRO-CAT construct shows a significant 50%
decrease in response to 1,25-(OH)2D3 treatment
similar to the response observed in Figure 5A. These findings support
the concept that down-regulation of the mouse OC gene by vitamin D may
be related to the mouse OC gene regulatory sequences, rather than to an
altered or species-specific representation of receptor and/or
associated transcription factors.
|
). A major complex (asterisk) was formed at
the mouse OC VDRE with mobility identical to the complex formed with
the rat OC VDRE sequence (Fig. 7A). The mouse OC VDRE binding complex
included the VDR and the RXR (Fig. 7B), as demonstrated by
supershift of the complex by antisera to VDR or RXR.
|
) demonstrated that the minor complexes were not
related to AP-1 (Fig. 7A). We also examined competency
of the mouse OC VDRE for binding the transcription factor YY1 (Fig. 7C). One of the minor bands (large arrow) was competed by a
YY1 consensus oligonucleotide but not by a mutant YY1 oligonucleotide
used as competitor. Thus, the mouse OC VDRE, like that of the rat,
contains a YY1 binding site. We also detect a high molecular mass
complex (Fig. 7C, designated by the small arrow), which is
not recognized by the rat OC VDRE sequence. Evidence for
species-restricted interactions is by both direct binding (panel A) and
oligonucleotide competition (panel C).
To determine whether the mouse OC VDRE domain was sufficient to mediate
the observed suppression of OC promoter activity in response to vitamin
D, we assessed the activity of the rat and mouse OC VDREs in the
context of a heterologous promoter (Fig. 8). Constructs containing a
single copy of the mouse or rat VDRE fused to a minimal TATA containing
promoter were transfected to MC3T3-E1 cells. The rat OC VDRE conferred
approximately 2-fold stimulation of transcription over the control
basal promoter activity in response to vitamin D. In contrast, the mOC
VDRE repressed activity of the heterologous promoter to 50% of the
control level in response to vitamin D. These results suggest that the
minimal mouse VDRE sequence is sufficient to confer suppressor activity
in response to vitamin D.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
The phenotypic stage of osteoblast maturation, from which the MC3T3-E1
clone was selected, may influence hormone responses. It is well
documented that hormone responsiveness can be attributed to the stage
of osteoblast differentiation in rat- and chick-derived osteoblasts (4, 23, 24). For example, vitamin D upregulates chicken OC in immature
osteoblasts (isolated from day 12 chick embryos) but down-regulates OC
in osteoblasts isolated from mineralized mature bone of day 17 embryos
(23). MC3T3-E1 cell properties are characteristic of committed
osteoblastic cells (e.g. early expression of OC), which may
account for: 1) the down-regulation of collagen, AP, and OC; and 2) the
quantitative changes in gene expression pre and post mineralization,
rather than the qualitative changes observed when marrow stromal or
mesenchymal stem cells are treated with steroid hormones (3, 22, 23).
However, some differences are apparent between pre- and
postmineralization osteoblasts (Fig. 4). Other investigators have
reported notable stage-specific differences in MC3T3-E1 properties (41, 42). Hiura et al. (41) showed that conditioned media from
early stage MC3T3-E1 cells inhibited osteoclast differentiation,
whereas media from late stage cells stimulated osteoclast formation.
These findings have been related to production of PGE2 and a
GM-CSF-like growth factor. The studies of Matsumoto et al.
(42) show that 1,25-(OH)2D3 stimulates AP
activity in both early and late cultures but can enhance mineral
deposition only in late-stage cultures. Thus, cellular levels of
cytokines, growth factors, and transcription factors present as a
function of the stage of osteoblast maturation can positively or
negatively influence expression of a gene or protein.
Another consideration of the findings we report in MC3T3-E1 cells relates to species-specific properties of murine-derived cells. The inhibited progression of MC3T3-E1 osteoblast differentiation and mineralization in response to Dex may not be restricted to this clonal line. For example, rat and human marrow-derived osteoprogenitor cells require the presence of Dex for formation of osteoblast colonies in culture (3, 17). In contrast, mouse marrow-derived adherent cells produce osteoblast colonies readily in the absence of Dex and require only the presence of ascorbic acid and ß-glycerol phosphate for mineralization (43). Exposure of cultured adherent mouse marrow cells to Dex blocks their differentiation to osteoblast colonies (44).
In addition to the above considerations, the decrease in collagen and AP activity and the striking inhibition of OC expression may contribute directly to the observed inhibition of mineralization of the mouse MC3T3-E1 osteoblasts. Both AP and OC are reciprocally related to proliferation, which is stimulated by Dex in MC3T3-E1 cells (except when initiated on day 4). However, the increase in cell growth is modest compared with the inhibitory effect on the osteoblast parameters, which are requisite for formation of a competent ECM for deposition of mineral. An interesting observation from our studies is that the combination of both steroids led to a significant enhancement of VDR mRNA, whereas each steroid alone either had no effect or decreased VDR levels. Perhaps this synergistic effect on the VDR mRNA by Dex and 1,25-(OH)2D3, which may occur via mRNA stabilization mechanisms rather than transcriptional control, relates to beneficial consequences of using 1,25-(OH)2D3 to counteract the inhibitory effects of glucocorticoids on bone clinically (1, 5).
The unusual response of murine MC3T3-E1 cells to glucocorticoid led us
to examine responsiveness of the cell line to the steroid hormone
vitamin D. Here we observed an absence of
1,25-(OH)2D3 enhancement of mouse OC gene
expression, in contrast to the effects of the hormone in all other
species examined (25). Multiple levels of steroid hormone mediated
transcriptional and posttranscriptional control of gene expression are
known. Therefore, the molecular mechanisms contributing to this finding
are likely to be complex. Cellular representation of species-specific
regulatory factors seems not to be a primary mechanism. Notably, when
the rat or human OC promoters are assayed in MC3T3 cells, either in
transient transfection assays (45, 46, and our studies, data not shown)
or as a stably integrated reporter construct (Fig. 6), the expected
up-regulation in response to vitamin D occurs. Serum OC (26) and mRNA
(47) levels in an in vivo mouse study were increased only
after several days exposure to 1,25-(OH)2D3 and
associated with hypercalcemia. Thus, the possibility exists of both
positive and negative vitamin D regulation of the mouse OC gene
in vivo, dependent on the biological circumstance. Other
preliminary reports support unique regulation of the mouse OC promoter
in vivo by 1,25-(OH)2D3, compared
with the rat and human genes (48, 49).
The nucleotide sequence variation in the distal steroid half motif of
the mouse OC VDRE, compared with sequences in the rat OC VDRE (see
Table 1), seems to contribute significantly to the altered response to
vitamin D. Several mechanisms involving VDR protein-protein and
VDRE-protein interactions, which can contribute to suppressor effects
of 1,25-(OH)2D3 on OC gene expression, are
known. The transcription factor YY1 (50) competes for VDR binding at
the VDRE and also disrupts TFIIB-VDR protein-protein interactions that
facilitate vitamin D-mediated transcriptional enhancement (50, 51, 52). The
sequences of the mouse OP VDRE do not allow binding of YY1. These
observations may explain the consistent up-regulation of OP by
1,25-(OH)2D3 in proliferating and
differentiated mouse and rat osteoblasts (23). Although we demonstrate
suppressor activity mediated by the mOC VDRE sequence, other mechanisms
for down-regulating the mouse OC gene within the context of the mouse
promoter, in response to vitamin D, may be operative. For example,
vitamin D regulation of other transcription factors, such as the
homeodomain protein Msx-2 (53, 54) may contribute to decreased mouse OC
expression (54, 55). Thus, multiple options for understanding the
observed responses of murine cells to vitamin D regulation of OC must
be explored.
|
Note Added In Proof |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
to the murine VDRE may reflect sequence
specific interaction with the longer murine OC VDRE probe used in our
study.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Present address: Amgen, Inc., 1840 DeHavilland Drive, Thousand Oaks,
California 91320-1789.
Received October 8, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added In ProofReferences |
|---|
1 and
2 collagen mRNA and their use in studying the regulation
of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23:6210–6216[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  J. Y. Kim, Y. L. Son, and Y. C. Lee Involvement of SMRT Corepressor in Transcriptional Repression by the Vitamin D Receptor Mol. Endocrinol., February 1, 2009; 23(2): 251 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Kearns, S. Khosla, and P. J. Kostenuik Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease Endocr. Rev., April 1, 2008; 29(2): 155 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-C. Huang, Z. Xie, H. Konaka, J. Sodek, H. E. Zhau, and L. W.K. Chung Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway Cancer Res., March 15, 2005; 65(6): 2303 - 2313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Smith and B. Frenkel Glucocorticoids Inhibit the Transcriptional Activity of LEF/TCF in Differentiating Osteoblasts in a Glycogen Synthase Kinase-3{beta}-dependent and -independent Manner J. Biol. Chem., January 21, 2005; 280(3): 2388 - 2394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Jiang, R. T. Franceschi, H. Boules, and G. Xiao Parathyroid Hormone Induction of the Osteocalcin Gene: REQUIREMENT FOR AN OSTEOBLAST-SPECIFIC ELEMENT 1 SEQUENCE IN THE PROMOTER AND INVOLVEMENT OF MULTIPLE SIGNALING PATHWAYS J. Biol. Chem., February 13, 2004; 279(7): 5329 - 5337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Luppen, N. Leclerc, T. Noh, A. Barski, A. Khokhar, A. L. Boskey, E. Smith, and B. Frenkel Brief Bone Morphogenetic Protein 2 Treatment of Glucocorticoid-inhibited MC3T3-E1 Osteoblasts Rescues Commitment-associated Cell Cycle and Mineralization without Alteration of Runx2 J. Biol. Chem., November 7, 2003; 278(45): 44995 - 45003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-C. Hsieh, G. K. Whitfield, P. W. Jurutka, C. A. Haussler, M. L. Thatcher, P. D. Thompson, H. T. L. Dang, M. A. Galligan, A. K. Oza, and M. R. Haussler Two Basic Amino Acids C-Terminal of the Proximal Box Specify Functional Binding of the Vitamin D Receptor to Its Rat Osteocalcin Deoxyribonucleic Acid- Responsive Element Endocrinology, November 1, 2003; 144(11): 5065 - 5080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Diefenderfer, A. M. Osyczka, J. P. Garino, and P. S. Leboy Regulation of BMP-Induced Transcription in Cultured Human Bone Marrow Stromal Cells J. Bone Joint Surg. Am., August 1, 2003; 85(90003): 19 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. GARDINER, P. A. BALDOCK, G. P. THOMAS, N. A. SIMS, N. K. HENDERSON, B. HOLLIS, C. P. WHITE, K. L. SUNN, N. A. MORRISON, W. R. WALSH, et al. Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage FASEB J, October 1, 2000; 14(13): 1908 - 1916. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. Struijs, A. Smals, S. A. de Witte, W. H. L. Hackeng, and H. Mulder Acute effects of etidronate on glucocorticoid-induced bone degradation Rheumatology, May 1, 2000; 39(5): 523 - 529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Boguslawski, L. V. Hale, X.-P. Yu, R. R. Miles, J. E. Onyia, R. F. Santerre, and S. Chandrasekhar Activation of Osteocalcin Transcription Involves Interaction of Protein Kinase A- and Protein Kinase C-dependent Pathways J. Biol. Chem., January 14, 2000; 275(2): 999 - 1006. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Javed, S. Gutierrez, M. Montecino, A. J. van Wijnen, J. L. Stein, G. S. Stein, and J. B. Lian Multiple Cbfa/AML Sites in the Rat Osteocalcin Promoter Are Required for Basal and Vitamin D-Responsive Transcription and Contribute to Chromatin Organization Mol. Cell. Biol., November 1, 1999; 19(11): 7491 - 7500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Ecarot and M. Desbarats 1,25-(OH)2D3 Down-Regulates Expression of Phex, a Marker of the Mature Osteoblast Endocrinology, March 1, 1999; 140(3): 1192 - 1199. [Abstract] [Full Text]
|
|
|
|
|
|
F. Aslam, L. McCabe, B. Frenkel, A. J. van Wijnen, G. S. Stein, J. B. Lian, and J. L. Stein AP-1 and Vitamin D Receptor (VDR) Signaling Pathways Converge at the Rat Osteocalcin VDR Element: Requirement for the Internal Activating Protein-1 Site for Vitamin D-Mediated Trans-Activation Endocrinology, January 1, 1999; 140(1): 63 - 70. [Abstract] [Full Text]
|
|
|
|
 |
|
 R.T. Franceschi The Developmental Control of Osteoblast-Specific Gene Expression: Role of Specific Transcription Factors and the Extracellular Matrix Environment Critical Reviews in Oral Biology & Medicine, January 1, 1999; 10(1): 40 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Yanaka, H. Akatsuka, E. Kawai, and K. Omori 1,25-Dihydroxyvitamin D3 upregulates natriuretic peptide receptor-C expression in mouse osteoblasts Am J Physiol Endocrinol Metab, December 1, 1998; 275(6): E965 - E973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Luegmayr, F. Varga, H. Glantschnig, N. Fratzl–Zelman, M. Rumpler, A. Ellinger, and K. Klaushofer 1,25-Dihydroxy Vitamin D3 and Tri-iodothyronine Stimulate the Expression of a Protein Immunologically Related to Osteocalcin J. Histochem. Cytochem., April 1, 1998; 46(4): 477 - 486. [Abstract] [Full Text]
|
|
|
|
|
|
B.-Y. Liu, J. Guo, B. Lanske, P. Divieti, H. M. Kronenberg, and F. R. Bringhurst Conditionally Immortalized Murine Bone Marrow Stromal Cells Mediate Parathyroid Hormone-Dependent Osteoclastogenesis in Vitro Endocrinology, April 1, 1998; 139(4): 1952 - 1964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. O. Carpenter, K. C. Moltz, B. Ellis, M. Andreoli, T. L. McCarthy, M. Centrella, D. Bryan, and C. M. Gundberg Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice Endocrinology, January 1, 1998; 139(1): 35 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Sims, C. P. White, K. L. Sunn, G. P. Thomas, M. L. Drummond, N. A. Morrison, J. A. Eisman, and E. M. Gardiner Human and Murine Osteocalcin Gene Expression: Conserved Tissue Restricted Expression and Divergent Responses to 1,25-Dihydroxyvitamin D3 in Vivo Mol. Endocrinol., October 1, 1997; 11(11): 1695 - 1708. [Abstract] [Full Text]
|
|
|
|
|
|
E. Smith, R. A. Redman, C. R. Logg, G. A. Coetzee, N. Kasahara, and B. Frenkel Glucocorticoids Inhibit Developmental Stage-specific Osteoblast Cell Cycle. DISSOCIATION OF CYCLIN A-CYCLIN-DEPENDENT KINASE 2 FROM E2F4-p130 COMPLEXES J. Biol. Chem., June 23, 2000; 275(26): 19992 - 20001. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, P <
0.05.
