From the Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8041
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ABSTRACT |
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Glucocorticoid in excess suppresses bone
formation in vivo and disrupts bone matrix protein
synthesis by osteoblasts in vitro. In contrast,
transforming growth factor (TGF-
) potently enhances bone matrix
apposition. The rat TGF-
type I receptor gene promoter contains
cis-acting elements for transcription factor CBFa1, which increases in
parallel with osteoblast differentiation. Here we present molecular
data linking these events. We show that previously unexplained effects
of glucocorticoid on bone loss may be mediated in part by suppression
of CBFa1, with a resultant decrease in the expression and activity of
the TGF-
type I receptor on matrix-producing bone cells.
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INTRODUCTION |
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Glucocorticoid-dependent bone loss by disregulated hormone expression or pharmacologic excess causes clinically significant osteoporosis in approximately 50% of affected individuals. Although changes in calcium absorption and effects on nonskeletal tissues contribute to the disease, striking effects occur directly on osteoblasts and at sites of active skeletal matrix deposition and remodeling (1, 2). A chronic reduction in osteoblast activity without corresponding changes in resorption would uncouple normal bone remodeling and decrease skeletal durability. Important genes targeted by glucocorticoid and molecular mediators for these events remain uncertain.
Transforming growth factor (TGF-
)1 enhances bone
matrix synthesis and repair, and bone contains perhaps the largest
store of TGF-
in the body (reviewed in Ref. 3). Bone cells exhibit conventional type II and type III TGF-
receptors (TRII and TRIII) that influence TGF-
binding to type I receptor (TRI) or its
activation, both essential for TGF-
-dependent events
(4-6). There are few systems where regulation of TRI expression has
been examined in detail and where functional changes correlate with
these variations. We found TRI levels specifically maintained on
differentiated bone cells in vitro, despite decreases in
TRII and TRIII in response to bone morphogenetic protein 2 (5). In
contrast, high levels of glucocorticoid rapidly reduce the proportion
of TGF-
binding to TRI on bone cells and correspondingly decrease
TGF-
activity (6).
To understand these events further, we cloned the rat TRI promoter and
observed higher promoter activity in osteoblast-like cells compared
with undifferentiated bone cells or dermal fibroblasts (7). The TRI
promoter includes a CpG island, several transcription factor Sp1
binding sites consistent with constitutive expression by many cells,
and six cis-acting elements for transcription factors, termed CBFa
(7-8).2 Whereas CBFa2 and
CBFa3 are important gene regulators in lymphoid cells (9), CBFa1
expression increases in parallel with osteoblast differentiation
in vitro (10).2 Moreover, targeted disruption of
the CBFa1 gene eliminates osteogenesis in mice, and insertion,
deletion, or missense mutations in CBFa1 occur in humans with the
skeletal disorder cleidocranial dysplasia (11). Genes directly affected
by CBFa1, especially those important for skeletal development, are
difficult to resolve when the factor is absent or dysfunctional and
when osteoblasts are absent or hard to detect.
Hormone-dependent decreases in CBFa1 could also challenge
skeletal integrity and indicate important downstream targets. Because
glucocorticoid suppresses TGF- activity and its binding to TRI on
osteoblasts and because binding sequences for CBFa1 occur in the TRI
promoter, we postulated molecular links among these events.
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EXPERIMENTAL PROCEDURES |
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Cells-- Primary osteoblast-enriched cultures were prepared by timed, sequential collagenase digestion of fetal rat parietal bone using procedures approved by the Yale Animal Care and Use Committee. Cells released during the third through the fifth 20-min collagenase digestion interval exhibit many physical and biochemical characteristics associated with bone-forming cells and are readily distinguished from less differentiated periosteal bone cells. Cells were plated at 3 × 103/cm2 and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as described (5-8).
Transfections--
Cultures at 50% confluence were transfected
with luciferase reporter plasmids pEN1.0 (7, 8) containing the fully
active TRI promoter, pSXN1C,2 containing two copies of a
CBFa response element inserted in vector pGL3-Promoter, p1233 (12),
containing 1.2 kbp of the TRII promoter, or pMMTV-Luc (13), containing
a well characterized glucocorticoid response element (GRE) from the
mouse mammary tumor virus. No GREs occur in pEN1.0 (7, 8). To assess
effects on TRI-dependent TGF- activity, cultures were
transfected with the TGF-
-responsive promoter/reporter construct
3TPLux (4). After 24 h, cultures were treated as indicated in each
experiment and rinsed, and extracts were used to measure luciferase
expression. There are only minimal variations in co-transfected
-galactosidase expression (± 6%) within experiments, and
correcting TRI promoter activity for
-galactosidase expression fails
to alter overall results (7, 8). However, TRI reporter constructs are
highly expressed by osteoblasts but exhibit significantly reduced
activity when co-expressed with secondary reporter constructs,
presumably from competition between plasmids for basal expression
elements. This study examines inhibitory effectors (glucocorticoids).
Therefore, promoter activities were compared in parallel cultures
rather than in the same cells in order not to add a confounder or to compromise TRI promoter/reporter expression further. Studies were repeated a minimum of three times to ensure reproducibility.
RNA Analyses--
Total RNA was extracted with acid guanidine
monothiocyanate, precipitated with isopropyl alcohol, and dissolved for
assay. Transcripts were assessed by RNase protection assay with
antisense RNA probes for rat TRI or 18 S rRNA. Probes were combined
with 5 µg of RNA, digested with 0.3 unit of RNase A and 14 units of RNase T1, and extracted with proteinase K and 0.5% SDS, and protected RNAs (TRI, 275 nucleotides; 18 S rRNA, 80 nucleotides) were collected in isopropyl alcohol, resolved on a 5% denaturing polyacrylamide gel,
and visualized by autoradiography (14). Alternately, total RNA was
fractionated on a 1.5% agarose/2.2 M formaldehyde gel, blotted onto charged nylon, and hybridized with a
[32P]-labeled cDNA restriction fragment of plasmid
bg7 encoding rat TRIII. Bound material was visualized by
autoradiography, and rRNA was assessed by ethidium staining of a
parallel sample (5).
TGF- Binding--
TGF-
1 was radioiodinated with chloramine
T. Cells were incubated in serum-free medium with 4 mg/ml bovine serum
albumin and 150 pM 125I-TGF-
1 for 3 h
at 4 °C. Rinsed cultures were chemically cross-linked and extracted
in reducing buffer, and samples were fractionated by electrophoresis on
5-15% polyacrylamide gels and examined by autoradiography (5,
14).
Cell and Nuclear Extracts-- As described previously (8), cells were collected by scraping and lysed in hypotonic buffer with phosphatase inhibitors, protease inhibitors, and 1% Triton X-100. Nuclei were collected by centrifugation, and supernatants were assessed for TRI protein. Nuclei were resuspended in hypertonic buffer with glycerol, phosphatase, and protease inhibitors and extracted for 30 min on ice. Insoluble material was cleared by centrifugation, and soluble protein was assessed for CBFa1 and Sp1.
Immunoblots-- For TRI, cell lysates (100 µg of protein), and for CBFa1, high salt nuclear extracts (40 µg of protein) were fractionated by polyacrylamide gel electrophoresis and electroblotted onto Immobilon P membranes (Millipore). Membranes were washed and blocked in 5% defatted milk, incubated with a 1:2,000 dilution of primary antibody (15), washed, incubated with a 1:3,000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase, developed with ECL (Amersham Pharmacia Biotech) reagents, and visualized by chemiluminescence (8).
Electrophoretic Gel Mobility Shift Assay--
Double strand
oligonucleotide probes were annealed, labeled with
[-32P]dCTP and Klenow fragment of Escherichia
coli DNA polymerase I, and gel-purified. Nuclear extracts (5 µg
of protein) were incubated with 32P probe. To assess
transcription factor immunologically, nonimmune (Santa Cruz) or rabbit
polyclonal anti-CBFa1 (15) was preincubated with nuclear extract
before adding 32P probe. Protein-DNA complexes were
resolved on 5% nondenaturing polyacrylamide gels and analyzed by
autoradiography (8).
Collagen and Non-collagen Protein Synthesis-- Cultures were pulsed with [3H]proline for the last 2 h of incubation. Cell layers were lysed by freeze-thawing and extracted in 0.5% Triton X-100. Samples were precipitated with trichloroacetic acid and chilled, and insoluble material was collected by centrifugation. [3H]Proline incorporation into collagen and non-collagen protein was measured by differential sensitivity to bacterial collagenase free of nonspecific protease activity (5, 6, 14).
Densitometry-- Relative differences on autoradiograms were assessed with a ScanMan densitometer and SigmaGel® (Jandel, San Rafael, CA).
Statistical Analysis-- Data were analyzed after multiple determinations for reproducibility. Biochemical data were expressed as means ± S.E. Statistical differences were assessed by analysis of variance with commercial software (SigmaStat®). After this, analysis was by the Student-Newman-Keuls method and considered significant with P values <0.05.
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RESULTS |
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Glucocorticoid Decreases TRI Promoter Activity-- Transfection construct pEN1.0, containing the 3' 1-kbp region of the rat TRI promoter, directs maximal reporter gene expression in primary and continuous osteoblast-enriched cultures (7, 8). Treatment for 24-48 h with the natural glucocorticoid hydroxycortisone or with the pharmacological glucocorticoid dexamethasone reduced TRI promoter activity by approximately 50% (Fig. 1, A-C). Treatment for less than 24 h did not cause significant reductions in reporter gene expression (Fig. 1A and other data not shown). This assay depends on competition between genomic and episomal elements for trans-acting factors, on reporter gene half-life, and on a balance between promoter plasmid expression and variations in specific trans-acting factors. Therefore, it may not reflect the timing of TRI mRNA expression with precision. Nevertheless, the effect was not nonspecific since neither steroid reduced reporter gene expression by the TRII promoter construct p1233 (12), and both agents enhanced expression by promoter construct pMMTV-Luc, containing a potent GRE (13) (Fig. 1D).
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Glucocorticoid Suppresses TRI mRNA and Protein
Levels--
Glucocorticoid also reduced steady state TRI mRNA
levels. Dexamethasone at 100 nM decreased TRI mRNA by
20% within 6 h, by 40% at 16 h, and by 65% after 48 h. As shown in Fig. 2A,
dexamethasone caused a dose-dependent inhibitory effect
after 48 h of treatment. This was not from general inhibition of
transcription or differential RNA recovery, since glucocorticoid
enhanced TRIII mRNA without proportional changes in rRNA
transcripts (Fig. 2B). Consistent with lower TRI promoter
activity (Fig. 1, A-C), less TRI mRNA (Fig.
2A), and a corresponding decrease in
125I-TGF- binding to TRI (Fig. 2C),
glucocorticoid decreased TRI protein levels by 50-60% within 24-48 h
and by greater than 90% after 72 h (Fig. 2).
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Glucocorticoid Decreases Nuclear Factor CBFa1 Levels-- We previously identified several inactive regions, 6 CBFa binding sequences, and 16 possible Sp1 binding sequences in the TRI promoter (7, 8).2 Therefore, we examined the effect of glucocorticoid on CBFa1 and Sp1 binding to 32P-labeled consensus oligonucleotide probes (Fig. 3A). Binding by osteoblast-derived nuclear factor to CBFa binding sequences in probes PC1 (5'-AACCACA-3') defined with lymphocyte and osteoblast extracts (9-10)2 and PS2 (5'-AACCGCG-3') from the rat TRI promoter2 decreased by 40-70% within 24 h of exposure to glucocorticoid and by 70-90% after 48 h. In contrast, nuclear factor binding to probe SP1 with Sp1 binding sequence (5'-GGGCGGGG-3') (8) decreased by 10% or less after 48 h of glucocorticoid treatment and by only 30% after 72 h (Fig. 3B). Studies with isoform-specific antibodies (15) reveal that CBFa1 forms the principal high molecular mass complex between nuclear protein from osteoblast-enriched cultures and TRI oligonucleotide PS2 (Fig. 3C) or other consensus DNA binding sequences for CBFas (9-10).2 By Western blot analysis (Fig. 3D), glucocorticoid rapidly and potently reduced full-length 55-kDa CBFa1, increased the appearance of a 46-kDa immunoreactive protein, and produced less variability in a 32-kDa immunoreactive band. The latter two proteins may be processed CBFa1 fragments or cross-reactive gene products. A small increase in complex formation appears to recur between 48 and 72 h of glucocorticoid treatment, but it is not yet known if it derives from the 46-kDa immunoreactive protein that accumulates during this treatment interval.
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Glucocorticoid Reduces TGF- Effects--
Up to 48 h of
treatment with glucocorticoid did not reduce basal protein synthesis
(Fig. 5A) or stimulatory
effects induced by platelet-derived growth factor (6), indicating that
decreases in TRI and CBFa1 were not from nonspecific inhibitory
effects. Nonetheless, glucocorticoid caused a small decrease in basal
collagen synthesis. Type I collagen, a major product of bone-forming
osteoblasts, comprises about 90% of the organic bone matrix. It is the
basic element of a network where other skeletal components assemble and
must be restored to areas resorbed during bone remodeling (1, 2) and is
greatly enhanced by TGF-
(3, 5, 6, 14). Consistent with a reduction
in TRI expression, stimulation by TGF-
was significantly suppressed
by glucocorticoid (Fig. 5, B and C). Because bone
matrix type I collagen expression may be regulated directly by changes
in CBFa1 (16-19) and, therefore, independently of variations in TRI,
we also examined the effect of glucocorticoid on reporter gene
expression induced by the TGF-
-responsive promoter/reporter
transfection construct 3TPLux (4). Glucocorticoid had no effect on
basal 3TPLux expression but significantly reduced the marked
stimulatory effect of TGF-
treatment (Fig. 5D).
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DISCUSSION |
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CBFa1 levels progressively increase with expression of the
osteoblast phenotype (10)2 in parallel with the increases
that we reported earlier on TRI promoter activity, TRI mRNA, and
cell surface TRI protein (5, 7). We now show that glucocorticoid
rapidly suppresses functional CBFa1 in nuclear extracts from osteoblast
cultures. Importantly, loss of CBFa1 by glucocorticoid treatment
corresponds with decreases in TRI promoter activity, TRI mRNA and
protein levels, and TGF--dependent biological effects.
Western blots with anti-CBFa1 specific antibody show a decrease in
full-length CBFa1 and an increase in protein of reduced molecular mass
in glucocorticoid-treated bone cells. In contrast, Northern blots do
not show coinciding reductions in CBFa1
mRNA,4 suggesting that
some of this effect may be post-transcriptional and perhaps produced by
changes in CBFa1 protein stability.
The decrease in cell surface TRI in glucocorticoid-treated cells is
consistent with our previous evidence for a short half-life of TRI
mRNA of approximately 6-7 h (14). A lower level of TRI expression,
driven principally by constitutive regulatory elements in the TRI
promoter (8), should not by itself fully eliminate osteogenic cell
activity. Clearly, genes other than TRI are important for osteoblast
function. Like osteocalcin, a protein expressed predominantly or
exclusively by osteoblasts, some may be directly regulated by CBFa1
(10, 11, 16-20). For example, basal type I collagen expression is
increased in cells transfected to express high levels of CBFa1 (16) and
is decreased in osteoblasts after 48 h of glucocorticoid treatment
(6, 21) when CBFa1 levels are near maximally
suppressed. Even so, the large increase in collagen
synthesis induced by TGF- treatment is even more strikingly reduced.
Accordingly, the stimulatory effect of TGF-
on collagen synthesis is
significantly lower in bone-derived cells that endogenously express
fewer osteoblast-associated features (5, 14), have lower osteogenic
potential in vitro (22), exhibit less TRI by relation to
TRII and TRIII (5, 14), and express less CBFa1.2 Loss of
TRI levels also limits the stimulatory effects of TGF-
on general
protein synthesis and on expression of 3TPLux (a reporter gene
sensitive to fluctuations in TGF-
receptors; Ref. 4), neither of
which are directly reduced by glucocorticoid treatment. Therefore,
variations in osteogenic events normally regulated by TGF-
, which
may be rapidly limited by the short functional half-life of TRI on
osteoblasts (14), may be sufficient to disrupt at least in part the
balanced bone remodeling cycle.
Whereas TGF- enhances replication and the synthesis of
type I collagen and other matrix components by bone
cells that are not yet fully differentiated, it inhibits biochemical
activities associated with later aspects of osteoblast differentiation
and mineralization in vitro (3, 5). Therefore, loss of TRI
in response to glucocorticoid would effectively suppress stimulatory effects by local TGF-
on osteoblast number and production of organic
bone matrix components required for later mineral deposition. However,
on well differentiated bone cells, lower levels of TRI would also
reduce the sensitivity of these cells to TGF-
and enable
mineralization in regions of bone where the organic matrix is already
sufficiently formed. In this regard, targeted overexpression of
TGF-
2 by late stage osteoblasts under control of the osteocalcin promoter correlates with abnormal bone formation (23), and
paradoxically, mineralized nodule formation is enhanced in
vitro by low dose or transient exposure to glucocorticoid in some
culture models (24). In the primary cell cultures used in our studies,
dexamethasone dose-dependently suppresses nodule formation
in vitro.3 This result is consistent with
studies in rat bone cell cultures where nodule formation was reduced by
the presence of antisense oligonucleotide to a domain common to all
CBFa subunits (10). Therefore, despite appropriate stimulatory or
permissive effects that occur with normally controlled expression or
release of TGF-
and glucocorticoid, inappropriate expression of
either agent or their receptors could disrupt the normal osteogenic
process.
CBFa1 knockout animals exhibit complete loss in mineralized skeletal
components and few if any differentiated osteoblasts (17, 18).
Consequently, it is not possible to assess directly the importance of
CBFa1 on specific genes expressed by bone-forming cells or their
precursors with those animals. Forced overexpression of CBFa1 has been
correlated with a number of osteoblast-related genes (16), but it is
not yet clear if all of these effects are direct. More subtle events
are more likely to regulate the gain or loss of CBFa1 expression during
normal bone growth or metabolic bone disease. Our results provide the
first evidence for glucocorticoid-dependent variations in
CBFa1 expression in bone and suggest that its loss rapidly
down-regulates TRI expression by bone cells. To date, little is still
known about the mechanisms that control TRI expression in bone or in
any tissue. Thus, our studies also provide new evidence for molecular
mechanisms that can account for changes in TRI expression in
combination with a functional consequence in a physiologically relevant
target tissue. Persistent decreases in matrix accumulation,
endogenously or in response to locally released TGF-, may contribute
substantially to bone fragility in glucocorticoid-dependent
osteoporosis. Identifying and understanding these control mechanisms
may assist the development of new ways to circumvent bone loss
associated with this and other aberrations in CBFa1 expression or its
activity.
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ACKNOWLEDGEMENTS |
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We are grateful to Patricia K. Donahoe
(Massachusetts General Hospital) for cDNA to assess rat
TRI transcripts, to Joan Massague (Memorial Sloan-Kettering Cancer
Center) for the 3TPLux transfection construct to assess TGF-
activity and cDNA to assess rat TRIII transcripts, to Seong-Jin Kim
(NCI, National Institutes of Health) and Ronald M. Evans (The Salk
Institute) for transfection constructs to assess TRII and
GRE-dependent promoter activity, to Scott W. Hiebert
(Vanderbilt University) for isoform-specific antibodies to CBFas, and
to Archibald Perkins (Yale University), Mary Goldring (Harvard
University), and Scott W. Hiebert for critical reading of our
manuscript.
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FOOTNOTES |
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* These studies were supported by National Institutes of Health awards AR39201 (to M. C.) and DK47421 (to T. L. M.) and Howard Hughes Medical Institute student fellowships (to D. J. C., and K. K. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Surgery
(Plastic Surgery), Yale University School of Medicine, 333 Cedar Street, P. O. Box 208041, New Haven, CT 06520-8041. Tel.:
203-785-4927; Fax: 203-785-5714; E-mail: michael.centrella{at}yale.edu.
1
The abbreviations used are: TGF-,
transforming growth factor
; TRI, TRII, TRIII, TGF-
receptor
types I, II, and III, respectively; GRE, glucocorticoid response
element; kbp, kilobase pair(s).
2 Ji, C., Casinghino, S., Chang, D. J., Chen, Y., Javed, A., Ito, Y., Hiebert, S. W., Lian, J. B., Stein, G. S., McCarthy, T. L., and Centrella, M. (1998) J. Cell. Biochem. (in press).
3 M. Centrella, unpublished observation.
4 T. L. McCarthy, D. J. Chang, and M. Centrella, unpublished observation.
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REFERENCES |
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