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Departments of Cell Biology and Orthopedics and Yale Cancer Center, Yale University School of Medicine (D.I., C.S., W.C.H., R.B.), New Haven, Connecticut 06510; the Department of Biology and Anatomy, National Defense Medical Center (C.S.), Taipei, Taiwan 100, Republic of China; and the Department of Medicine, Harvard Medical School, New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center, Harvard Institute of Medicine (D.L.G., S.R.G.), Boston, Massachusetts 02115-5716
Address all correspondence and requests for reprints to: Dr. Roland Baron, Department of Orthopedics, Yale University School of Medicine, P.O. Box 208044, New Haven, Connecticut 06520-8044. E-mail: roland.baron{at}yale.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, complementary DNAs (cDNAs) encoding the CT receptor (CTR) in pig (7), human (8), rat (9), mouse (10), rabbit (11), and guinea pig (12) have been cloned and sequenced. Those studies revealed that the CTR belongs to a subfamily of G protein-coupled receptors that includes receptors for PTH/PTH-related peptide, secretin, vasoactive intestinal peptide, and others (reviewed in Ref. 13). Although the originally identified CTR in porcine kidney is highly conserved among species, other CTR isoforms (9, 10, 11, 14, 15, 16) have been reported in different species. In murine tissues, at least two different CTR isoforms are expressed: C1a, which corresponds to the common isoform conserved among species, and C1b, containing a 37-amino acid insert in the first extracellular loop (9, 10). Although the C1b isoform was originally identified in rat brain and was shown to be relatively specific to brain (9), its expression in rat and mouse osteoclasts has also been demonstrated by nonquantitative RT-PCR analysis (17). However, the relative expression levels and contributions to the CT-induced signaling of these two isoforms in osteoclasts have not been defined.
Molecular cloning of the CTR has made it possible to investigate the regulatory mechanism of CTR expression at the messenger RNA (mRNA) level. Recent studies have revealed that treatment with CT causes a substantial decrease in the steady state level of CTR mRNA and thus significantly contributes to homologous desensitization in cells of the osteoclast lineage (18, 19, 20, 21, 22, 23). These studies, however, have not resolved some issues regarding the mechanism of CTR mRNA down-regulation by CT, including whether the regulation occurs at the transcriptional level.
In the present study, we investigated the mechanism of CT-induced CTR mRNA down-regulation in cells of the osteoclast lineage, using an in vitro coculture system of mouse bone marrow and osteoblastic cells. By ribonuclease (RNase) protection assay and competitive PCR analysis, we demonstrated that in vitro-generated mouse osteoclast-like cells (OCLs) predominantly express the C1a isoform of the CTR and that its expression is negatively regulated by CT at least in part at the transcriptional level, independently of de novo protein synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and cell cultures
Newborn and 8-week-old male CD-1 mice were purchased from
Charles River Laboratories, Inc. (Wilmington, MA). A
murine macrophage cell line, P388D1, obtained from American Type Culture Collection (Rockville, MD), and a human T cell leukemia
cell line, MT-2 (24), a gift from Dr. N. Ruddle (Yale University, New
Haven, CT), were maintained in RPMI 1640 supplemented with 10% FBS.
TXB-1, a mouse marrow stromal cell line we have established from
transgenic animals expressing HTLV-I tax (25), was grown in 
MEM
containing 10% FBS. Primary osteoblastic cells were isolated from
newborn CD1 mouse calvariae by digestion with 0.1% collagenase type IA
and 0.2% dispase as previously described (26). Marrow cells were
obtained from aseptically isolated tibiae and femurs of 8-week-old CD1
mice by flushing out the marrow cavity with MEM using 25-gauge
needles. To generate OCLs in vitro, 2 x
107 marrow cells were cocultured with 2 x
106 primary osteoblasts in a 10-cm culture dish in the
presence of 10 nM 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] (26, 27). In these cultures,
differentiation of OCLs is dependent on calcitriol, and CTR-positive
osteoclasts and their precursors appear after 5–6 days. Before
purification, the tartrate-resistant acid phosphatase-positive
cells comprised between 5–10% of the cells present in the culture.
For OCL purification, cocultures on day 6 were treated sequentially for
10 min at 37 C with 0.1% collagenase-0.2% dispase in MEM without
FBS and 5 mM EDTA in PBS to remove osteoblastic cells,
whereas OCLs remained strongly attached to the culture dish. The
purified OCLs were highly spread, as previously described (27), and
covered 80–90% of the surface of the plate. After purification, more
than 80% of the cells were tartrate-resistant acid
phosphate-positive, and about 75+ of these were multinucleated,
with 3–10 nuclei/cell. The EDTA-resistant fraction similarly obtained
from cocultures grown in the absence of calcitriol was used as a bone
marrow-derived macrophage-enriched population. To analyze the
regulation of CTR mRNA expression, whole cocultures on day 5 were
deprived of calcitriol for 6 h before the experiments, treated
with various reagents for a period of time specified in each
experiment, and harvested for total RNA extraction.
Northern blot analysis
Total RNA was prepared according to an established method (28),
and the concentrations were determined by spectrophotometry. For
Northern blot analysis, 10 µg total RNA were separated on a 1%
denaturing agarose gel, transferred to a Hybond N+ membrane
(Amersham, Arlington Heights, IL) and hybridized with
32P-labeled mouse CTR or human glyceraldehyde phosphate
dehydrogenase (GAPDH) cDNA probe (106 cpm/ml) prepared by
the random primer labeling method (Boehringer Mannheim). The membrane
was washed under moderately high stringency (0.1 x SSC and 0.1%
SDS at 42 C) and autoradiographed.
RNase protection assay
Labeled complementary RNA (cRNA) probes were prepared by
subcloning cDNA fragments of mouse CTR and GAPDH into pBluescript II
(Stratagene, La Jolla, CA) and transcribing in
vitro in the antisense direction by T7 or T3 RNA polymerase in the
presence of [-32P]UTP. Cold UTP was also added to the
reaction for GAPDH to reduce the specific activity of the probe to
approximately 1/15th that of the CTR probe. The CTR probe corresponds
to nucleotides 1150–1677 of the reported mouse CTR cDNA sequence (10),
and the expected size of protected RNA fragments is 527 and 411 bases
for the C1b and the C1a isoforms, respectively. RNase protection assays
were performed using an RPA II kit (Ambion, Inc.,
Woodward, TX) according to the protocol suggested by the supplier.
Briefly, total RNA samples were incubated with CTR (50,000
cpm/reaction) and GAPDH cRNA probes (20,000 cpm/reaction) in a single
tube at 42 C for 16 h. After digesting single strand RNA with a
mixture of RNase A (5 U/ml) and RNase T (200 U/ml) at 37 C for 30 min,
protected RNA fragments were precipitated, resolved on a 5% denaturing
polyacrylamide gel, and visualized by autoradiography.
RT-PCR analysis
To avoid contamination of genomic DNA, RNA samples were first
treated with deoxyribonuclease I (
20 U/30 µg total RNA; Boehringer
Mannheim), extracted with phenol/chloroform, and reprecipitated with
isopropanol. RT was performed using random primer (Promega Corp., Madison, WI) and Superscript II reverse transcriptase
(Life Technologies, Inc., Gaithersburg, MD) according to
the protocol recommended by the supplier. Three micrograms of each RNA
sample were reverse transcribed in a 20-µl reaction, and 1 µl,
which corresponds to 150 ng RNA, was used for a PCR reaction. All of
the PCR reactions were performed under standard conditions using
AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ)
with cycles of 94 C for 30 sec, 58 C for 30 sec, and 72 C for 40 sec.
The oligonucleotides used for the amplification of CTR mRNA (primers
3+/3- in the lower part of Fig. 1
) were
5'-GTCTTGCAACTACTTCTGGATGC-3' (nucleotides 1308–1330, in exon 9) and
5'-AAGAAGAAGTTGACCACCAGAGC-3' (nucleotides 1562–1540, in exon 10),
which amplify 255 bp.
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. For
nonquantitative detection of hnRNA, oligonucleotides
5'-TTGGCTCATCAATGCACTCTT-3' (primer 1+, sense) and
5'-GGGTCCCTAGCT-CCTCAGAAT-3' (primer 1-, antisense) were used.
These primers are from the two introns that flank the putative exon 9
in the CTR genome (Galson, D. L., et al., manuscript in
preparation) and are expected to amplify 255 bp including the whole
exon 9 and juxtaposed intronic sequences. For competitive PCR, the
primer sets used to amplify the target are 5'-GGCACATGTTGACAAATACTC-3'
(primer 2+, sense) and 5'-GGGTCCCTAGCTCCTCAGAAT-3' (primer 2-,
antisense), which will amplify a 328-bp fragment that also contains the
whole exon 9 and some intronic sequences adjacent to the exon 9. To
generate a competitor, a chimeric sense primer,
5'-GGCACATGTTGACAA-ATACTCCAACCACATCTTCAACAA-3' and
antisense primer 2- were used. The underlined 3'-half of
the primer binds to the sequences 75 bp downstream of the sense target
primer-binding site and therefore amplified a shorter, 271-bp
competitor fragment with the same target primer-binding sites at both
ends. RT-PCR reactions were performed in the same way as described
above for the nonquantitative assay, except that an increasing amount
of competitor was added together with the sample in the reaction mixes.
The authenticity of the PCR products in competitive PCR analysis were
verified by Southern blot analysis, using a 32P end-labeled
oligonucleotide, 5'-ATGGCTGTGTTTACCGACGAGCAACGC-3' (primer C+), which
corresponds to coding sequences 1366–1392 in the exon 9, as a
probe. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The CTR
message was undetectable in all of the other cell types tested,
including calvaria-derived osteoblasts cultured with and without
1,25-(OH)2D3, a T cell line (MT-2), a marrow
stromal cell line (TXB-1), a macrophage cell line (P-388D1), and the
EDTA-resistant fraction from cocultures grown in the absence of
1,25-(OH)2D3, which represents marrow-derived
macrophages. These results confirmed that the CTR mRNA is specifically
expressed in cells of the osteoclast lineage in a vitamin D-dependent
manner in the coculture system used in this study.
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.) The results indicated that although C1a mRNA was clearly detectable
in OCLs over a wide range of total RNA levels (0.625–40 µg; Fig. 2B
and data not shown), C1b mRNA was below the detection limits at all
doses tested. In contrast, both C1a and C1b were expressed at more or
less comparable levels in the brain (Fig. 2B). By densitometric
analysis, we estimated the ratio of C1b to C1a at approximately 1.6 in
the brain and less than 0.01 in OCLs.
Thus, in the mouse, cells of the osteoclast lineage express
predominantly the C1a isoform, and among bone cells the expression is
osteoclast specific. Based on this fact, we decided to examine the
effect of CT on C1a-CTR mRNA expression in whole cocultures rather than
purified OCLs for the following reasons: 1) only cells of the
osteoclast lineage are expected to respond to CT; 2) the target (CTR)
mRNA is only expressed in cells of the osteoclast lineage; and 3)
mature OCLs generated in cocultures have a relatively short life span
after removal of the osteoblastic cells, which would have made the
experiments much more difficult to perform and interpret. We first
investigated the effects of varying concentrations of salmon CT (sCT)
on CTR mRNA expression in this system. As shown in Fig. 3A, when cocultures were treated with
various concentrations of sCT for 14 h, the steady state level of
C1a-CTR mRNA declined in a dose-dependent manner. The half-maximal
effect was observed at approximately 10 pM, which is close
to the reported EC50 of sCT-induced cAMP production in
osteoclasts (29). We next examined the effect of sCT at shorter times
(4 and 8 h) as shown in Fig. 3B. Treatment with 1 nM
sCT caused a time-dependent decrease in the C1a mRNA level, and
down-regulation of the message was detectable as early as 4 h
after the addition of sCT. Thus, sCT caused a dose- and time-dependent
reduction in the steady state level of C1a-CTR mRNA in cocultures.
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, continuous treatment with 1
mM (Bu)2cAMP or 50 µM
forskolin caused a clear reduction in the C1a mRNA level, mimicking the
effect of 1 nM sCT. In contrast, neither a PKC stimulator,
SC-9 (25 µM), nor a calcium ionophore, A23187 (100
nM), affected the message level significantly. Moreover,
the effects of (Bu)2cAMP and sCT, when present
throughout the incubation, were almost indistinguishable at all times
examined (Figs. 3B and 4A). These results support the idea that the
cAMP/protein kinase A pathway mediates the effect of CT on CTR mRNA
expression in osteoclasts (33), although we cannot completely exclude
the possibility that the effects of (Bu)2cAMP and
forskolin may be in part indirect, due to the heterogeneity of the
culture system used for these experiments. To provide further insight
into the regulatory mechanism of CTR mRNA expression, we examined the
effects of shorter treatment with human and salmon CTs and with
forskolin and (Bu)2cAMP. In these experiments,
cells were first treated for 1.5 h, then washed twice with PBS and
cultured in medium that lacked the agents for another 14 h. The
results indicated that a short exposure to sCT was as effective at
decreasing CTR mRNA as a continuous treatment (compare Fig. 4, A and
B), consistent with the extremely slow off-rate of sCT (34) and with
earlier reports that CT causes persistent elevation of intracellular
cAMP levels in osteoclasts (29, 35). Similar results were obtained with
human CT (Fig. 4B), suggesting that this peptide may also persistently
bind to and stimulate the CTR despite its lower affinity (34). In
contrast to the persistent effect of CT, short treatment with
(Bu)2cAMP did not cause persistent suppression of
CTR mRNA, indicating that cAMP analogs need to be continuously present
in the medium to keep CTR mRNA expression suppressed. Short term
forskolin treatment, however, elicited a long term suppression of CTR
mRNA expression (Fig. 4B), suggesting that it may induce signaling
events in addition to adenylyl cyclase (AC) activation in cells of the
osteoclast lineage.
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, treatment of
cocultures with DRB caused a rapid decrease in the C1a mRNA level, with
an estimated half-life of approximately 4–6 h. The relatively short
half-life is compatible with the presence of AU-rich motifs in the
3'-untranslated region of the CTR mRNA (10, 22), which are known to
confer instability on mRNA (36). Treatment with sCT or
(Bu)2cAMP did not cause any detectable changes in
mRNA stability, suggesting that the CTR down-regulation by CT does not
involve an acceleration of mRNA degradation. It is also noteworthy that
treatments with DRB (Fig. 5A) and sCT (Fig. 3B) showed similar temporal
patterns, indicating that the effect of CT can be mimicked by a
transcriptional inhibitor. Taken together, these results suggest that
the down-regulation of CTR by CT involves an inhibition of
transcription.
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). As the mRNA
level is not expected to change appreciably within 1 h, the CTR
mRNA level serves as a control for the number of CTR-expressing cells
in the culture. As shown in Fig. 6A, a clear-cut decrease in CTR hnRNA
was detectable 1 h after treatment with sCT by nonquantitative PCR
analysis. This effect was independent of de novo protein
synthesis, as it was also apparent in the presence of CHX. These
results were quantitatively confirmed by competitive PCR analysis (Fig. 6B). The estimated amount of CTR hnRNA in 150 ng total RNA was
approximately 0.01–0.03 fg in the control and 0.001–0.003 fg in the
CT-treated sample, respectively. In these experiments, treatment with
CHX did not alter the level of CTR hnRNA expression in either the
presence or absence of CT. Thus, treatment with sCT reduced the level
of CTR hnRNA to approximately 1/10th of the control level in 1 h,
independently of new protein synthesis. Taken together with the lack of
sCT effect on the stability of CTR mRNA, these findings strongly
suggest that CTR down-regulation by sCT in cells of the osteoclast
lineage occurs at least in part at the transcriptional level, and that
this effect is mediated by cAMP-dependent posttranslational
modifications of preexisting factors.
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Discussion |
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C1a being the only detectable CTR isoform in our experimental system, we next attempted to clarify the CT-induced downstream events leading to suppression of C1a-CTR mRNA expression. The results of the present study confirmed those of others (33), suggesting that cAMP is the key downstream signal mediating the effects of CT on CTR mRNA. (However, as we treated the complete coculture containing both the OCL and the supporting osteoblast-enriched calvarial cells, we cannot completely exclude the possibility that the (Bu)2cAMP and forskolin could also be having an effect on the other cells in the culture that might, in turn, influence the CTR expression by the OCL.) The long term suppression of CTR mRNA expression after a short exposure of the cells to the peptide is consistent with the prolonged effects of sCT on CTR expression in osteoclasts reported by others (19, 21) and with the persistent activation of AC after brief stimulation with CT (29, 35, 42). As (Bu)2cAMP mimicked the CT effect when constantly present in the cultures for longer periods of time, but not after a brief exposure, it is likely that persistent activation of AC by CT is both sufficient and necessary for the prolonged effects of CT on CTR mRNA expression. The persistent AC activation seems to be a unique feature of CT stimulation, as it has not been observed after stimulation by other ligands such as PGE2, which transiently activates AC (29, 42). The molecular basis for the CT-induced persistent AC activation is unclear at present. It is likely to be related at least in part to the prolonged binding of the peptide to the receptor, although the complex array of signals elicited through CTR upon ligand binding and/or the specific AC isoforms present in these cells might also contribute to this effect (43, 44). In this context, the prolonged effect of forskolin observed in this study is intriguing. Forskolin is a plant-derived diterpene that directly binds to and potently activates nearly all the known mammalian AC isoforms (reviewed in Ref. 45). The increase in intracellular cAMP that is induced by forskolin is in most cases rapid and reversible in nature, although both the efficacy and the kinetics vary among different tissues or cell types (45). If both forskolin and CT/CTR cause similar persistent AC activation, it might be attributed to the intrinsic nature of the particular AC isoform(s) activated by CT/CTR rather than specific CT-induced signals that act on AC and stabilize its activated state. Alternatively, forskolin, like CT, might elicit signaling events in addition to the direct activation of AC (46) that could result in the stabilization of active AC or induce the suppression of CTR gene expression by other as yet unknown mechanisms. Further studies on the kinetics of activation of different AC isoforms by CT and forskolin in osteoclasts and other cell types will be necessary to fully elucidate the mechanisms of persistent suppression of CTR expression.
The main objective of our study was to determine whether the regulation of CTR mRNA expression by CT occurs at the transcriptional level. As a surrogate for the classical nuclear run-on assay, we examined the level of CTR hnRNA to measure the transcriptional rate. We demonstrated that treatment with CT caused a prompt and clear-cut reduction in the CTR hnRNA level, strongly suggesting the involvement of a transcriptional mechanism. A recent report by Wada et al. (22) concluded, however, that CT had no significant effect on the CTR transcriptional rate measured by nuclear run-on assay. Although these results seem contradictory, there may be an explanation for this discrepancy; CT may not affect initiation of transcription but, instead, block elongation of the CTR transcript and cause transcriptional arrest. Indeed, several genes, including c-myc (47), c-fos (48), and c-myb (49), are known to be subject to such a regulatory mechanism at the level of transcriptional elongation. In that case, CT effects may have been masked if the full-length CTR cDNA was used to detect the run-on transcripts synthesized in vitro (22). It is also possible that CTR mRNA expression is regulated by a combination of transcriptional and posttranscriptional mechanisms, the latter making a greater contribution to the late phase of the prolonged effect of CT. Further extensive studies will be necessary to elucidate the mechanism by which CT down-regulates the level of CTR hnRNA expression.
In summary, we have demonstrated that in cells of the osteoclast lineage, expression of the C1a-CTR isoform mRNA, which accounts for more than 99% of the total CTR RNA in these cells, is rapidly and persistently down-regulated by the receptor’s cognate ligand. The fact that CT treatment causes an approximately 10-fold reduction in the steady state level of CTR-hnRNA in 1 h suggests the involvement of transcriptional inhibition. This down-regulation is independent of de novo protein synthesis and is thus most likely mediated by posttranslational modifications of a preexisting factor(s) by a mechanism that involves the cAMP/protein kinase A pathway.
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Footnotes |
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2 Present address: First Department of Internal Medicine, University
of Tokushima School of Medicine, 3–18-15 Kuramoto-cho Tokushima-shi,
Tokushima 770-8503, Japan.
Received May 20, 1998.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, EDTA-resistant fraction obtained from cocultures
grown in the absence of calcitriol, which represents marrow-derived
macrophages; POB ± D, calvaria-derived primary osteoblasts
cultured in the presence or absence of calcitriol; MT-2, human T cell
leukemia cell line; P388D1, murine macrophage cell line; TXB-1, murine
marrow stromal cell line. B, Various amounts of mouse brain and OCL
total RNA were analyzed for the expression of the C1a and C1b CTR
isoforms by RNase protection assay. P, Free probe; C, yeast transfer
RNA control. The positions of the free probe and the protected
fragments for C1a and C1b are indicated by arrows. Each
panel is representative of at least three independent experiments.
, sCT; 
, (Bu)2cAMP.