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INTRODUCTION |
Nucleotides exist in the extracellular microenvironment of bone
due to release from damaged cells at sites of injury, or via more
controlled nonlytic release (1), where they differentially activate
cell surface P2 receptors. Activation of Gq-coupled P2Y receptors leads to
[Ca2+]i1
mobilization which has ultimately been found to induce processes including proliferation (2) and differentiation (3) and to modulate
osteoblast-induced bone formation (4). The bone matrix synthesizing
osteoblasts are thought to express multiple P2Y receptor subtypes (5),
with expression profile changing as osteoblasts differentiate (6). The
mechanisms that determine focal responsiveness to systemic factors in
bone to allow a local tissue turnover, characteristic of the remodeling
process, remain unclear. However, our previous observations that
coapplication of nucleotides and parathyroid hormone (PTH) can result
in synergistic responses in osteoblasts (7) led us to hypothesize that
locally released extracellular nucleotides could sensitize surrounding cells to the action of circulating hormones.
Parathyroid hormone is one of the most important systemic regulators of
bone and mineral homeostasis. The action of PTH on skeletal cells is
complex and can result in the stimulation of both resorption and new
bone formation (8, 9). This ability to stimulate coupled, but opposing
processes, has been attributed to the nature of the receptor that
transduces signals arising from PTH stimulation. The PTH1 receptor
belongs to a subgroup of seven transmembrane receptors that include
those responsive to calcitonin, secretin, and VIP (10). These receptors
are distinct in their ability to couple to both Gs and
Gq and hence activate dual signal transduction pathways
leading to both cyclic AMP formation and release of Ca2+
from intracellular stores ([Ca2+]i).
The coupling of the PTH receptor to Gs in osteoblasts is
well characterized. Activation of the cAMP signaling pathway is
responsible for a number of PTH-induced downstream responses, including
expression of the c-fos proto-oncogene (11). In addition,
the use of truncated PTH fragments in vivo has confirmed
that complete N-terminal sequence (vital for Gs activation)
is essential to drive the anabolic skeletal effects of PTH (12) and
cAMP accumulation is often used as a measure of PTH receptor activation
(13). However, the nature of PTH receptor/Gq-coupling and
subsequent downstream responses in osteoblasts following activation
remains controversial. Both inositol
1,4,5-trisphosphate-dependent and independent mechanisms for PTH-induced [Ca2+]i release have been
reported (14, 15). To further complicate the issue of PTH-induced
[Ca2+]i release, recent studies in cell types
other than osteoblasts have demonstrated that while PTH alone is unable
to induce [Ca2+]i release, it can strongly
potentiate the [Ca2+]i elevations induced by
agonists acting at other Gq-coupled G-protein-coupled
receptor. The mechanism behind this potentiation remains unclear
but has been attributed to G protein subunit interaction or shuttling
of calcium between intracellular stores (16).
In a study by Kaplan et al. (17) using rat UMR-106 cells,
PTH alone was shown to produce small [Ca2+]i
elevations and also potentiated nucleotide-induced [Ca2+]i elevations. However, in contrast to these
findings, we previously demonstrated that PTH alone did not elevate
[Ca2+]i and was ineffective at potentiating
nucleotide-induced responses in the human osteosarcoma cell line SaOS-2
(7). In the same study, however, PTH and nucleotide costimulation of
SaOS-2 cells did result in synergistic induction of gene expression.
Considering the observations described above, the current study has
addressed: (i) the controversy concerning the ability of PTH to effect
[Ca2+]i release in the rat osteosarcoma cell line
UMR-106, (ii) the mechanisms by which PTH can potentiate
[Ca2+]i release induced by locally released
nucleotides, and (iii) whether this potentiated
[Ca2+]i response can ultimately drive downstream
cellular responses in osteoblasts.
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EXPERIMENTAL PROCEDURES |
Reagents--
Dulbecco's modified Eagle's medium (DMEM),
-modified Eagle's medium (
-MEM), Ham's F-12, and fetal
calf/bovine serum were purchased from Life Technologies (United Kingdom
or France). Human parathyroid hormones (PTH) 1-34 and 1-31, and
bovine PTH-(3-34) were purchased from Peninsula Laboratories (UK).
Nucleotides, bovine serum albumin, fluo-3 AM, potato apyrase, H-89,
IBMX, dibutyryl-cGMP, SKF96365, nitrocellulose membranes, and
peroxidase-coupled goat anti-rabbit antibodies were obtained from Sigma
(UK). Phospho-CREB and CREB specific antisera were obtained from New
England Biolabs (UK). Nitrocellulose membranes and enhanced
chemiluminescence reagents were acquired from Amersham Pharmacia
Biotech (UK) or PerkinElmer Life Sciences (Belgium). Zetabind
hybridization membrane was purchased from Cuno (Meriden, CT).
Luciferase lysis reagent and luciferase reagent were purchased from
Promega (UK). UMR-106 cells were kindly provided by T. J. Martin,
Melbourne, Australia.
Cell Culture--
UMR-106 rat osteoblast-like cells were
cultured in
-MEM supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM
L-glutamine. The cells were maintained at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. Confluent cells were either induced for subsequent preparation of whole cell
extracts or RNA isolation, or harvested upon confluence for Ca2+ release measurements in the FLIPR system (18). The
UMR-106 multi CRE c-fos reporter cell line was cultured in
Ham's F-12 and DMEM (1:1) under similar conditions.
[Ca2+]i Measurements--
Cells were
grown in 225-cm2 vented cap flasks, harvested upon
confluence, and resuspended in basal salt solution (BSS) composition (mM): NaCl (125), KCl (5), MgCl2 (1),
CaCl2 (1.5), HEPES (25), glucose (5), and 1 mg/ml bovine
serum albumin, pH 7.3. Cells were loaded with 17 µM
fluo-3-AM for 1 h at 37 °C with agitation and in the presence
of apyrase (2 units/ml), after which cells were washed 3 times in BSS
and aliquoted into 96-well black wall, clear base plates at a density
of 2.5 × 105 cells/well. These were then centrifuged
to obtain a confluent layer of cells on the base of the plate and cells
were subsequently washed 4 times in BSS. Ca2+ flux was
measured in all 96 wells simultaneously and in real time using a
Fluorescent Imaging Plate Reader (FLIPR). When effects of PTH and
nucleotides together were studied the cells were incubated for 60 s with PTH prior to exposure to nucleotide. The effects of IBMX and
forskolin were assessed by their introduction to the cells 6 min prior
to nucleotide addition, H-89 was introduced 10 min before the addition
of nucleotides and dibutyryl-cGMP 30 min before nucleotide addition. In
all cases [Ca2+]i release was measured for 1 min
after nucleotide addition, a measurement of fluorescence being taken
every second.
Whole Cell Extract Preparation--
Whole cell extracts were
prepared as previously described (19). At the appropriate time point,
plates were placed on ice and the cell layer quickly washed twice with
ice-cold phosphate-buffered saline (140 mM NaCl, 10 mM NaPO4, pH 7.3) containing 10 mM
NaF and 100 µM Na3VO4. Cells were
solubilized in 10 mM Tris-HCl, pH 7.05, 50 mM
NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM ZnCl2, 1% (v/v) Triton X-100, 100 µM Na3VO4, 20 mM
-glycerophosphate, 10 mM 4-nitrophenyl phosphate, 1 mM dithiothreitol, 0.5 mM benzamidine, 2.5 µg/ml aprotinin, leupeptin, pepstatin, 0.2 mM
phenylmethylsulfonyl fluoride, and 200 nM okadaic acid. The
cell layer was collected by scraping and lysis completed by vortexing
for 60 s. The lysate was clarified by centrifugation at
14,000 × g at 4 °C for 15 min and the protein
concentration determined with Bradford's reagent using bovine serum
albumin as standard. An aliquot for Western analysis was denatured
immediately in 2% SDS, 2% glycerol, 50 mM Tris-HCl, pH
6.8, 1% 2-mercaptoethanol and the remaining supernatant stored at
70 °C.
Western Analysis--
15 µg of whole cell extracts were
separated on an 8.5% SDS-polyacrylamide electrophoresis minigel and
transferred electrophoretically to a nitrocellulose membrane. The
filter was blocked for 1 h at room temperature in 5% (w/v) low
fat milk powder-TBST (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20), followed by incubation overnight at 4 °C with the specific antiserum indicated below in blocking buffer. After washing in TBST, the blots were incubated for 1 h at
room temperature with peroxidase-coupled goat anti-rabbit antibody,
diluted 1/1500 in 5% (w/v) low fat milk powder-TBST. After washing,
the immune complexes were visualized using enhanced chemiluminescence.
The antisera used were: anti-CREB and anti-phospho Ser133
CREB, diluted 1:1000.
RNA Isolation--
Total RNA was extracted from control and
stimulated cells according to the protocol of Chomczynski and Sacchi
(20). Briefly, cells were lysed in 4 M guanidine
thiocyanate, 0.5% Sarkosyl, 0.1 M mercaptoethanol, 25 mM sodium citrate, pH 7.0, followed by acid
phenol/chloroform extraction. RNA was stored as a precipitate at
70 °C.
Northern Analysis--
10 µg of total RNA was denatured and
electrophoresed through a 0.8% (w/v) agarose gel containing 3.7%
formaldehyde (v:v), followed by transfer to Zetabind hybridization
membrane. Blots were prehybridized at 65 °C in 50% formamide,
5 × SSC, 5 × Denhardt's reagent, 1% SDS, 50 mM sodium phosphate buffer, pH 6.8, 5 mg/ml total calf
liver RNA, 200 µg of tRNA, and probed with a c-fos riboprobe spanning exons 3 and 4 of the human c-fos gene
mixed with a riboprobe derived from the rat gapdh cDNA
as previously described (19). Membranes were washed for 30 min at
65 °C with 0.2 × SSC, 1% SDS solution and mRNAs
visualized using phosphorstorage technology and autoradiography with
Kodak X-AR film and intensifying screens at
70 °C. RNA loading and
integrity were measured by UV shadowing of the filter prior to hybridization.
Luciferase Reporter Gene Assays--
Reporter cells were seeded
into white 96-well plates (Dynex) at a density of 96,000 cells/well in
50% DMEM, 50% Ham's F-12 containing 10% fetal calf serum. Cells
were incubated for 24 h, after which time medium was replaced by
50% DMEM, 50% Ham's F-12 medium containing 0.1% bovine serum
albumin and incubated for a further 24 h. Cells were induced for
4 h with ×10 concentration of agonist stock solution prepared in
serum-free medium to the final concentrations indicated in Fig. 9.
After induction, cells were washed twice in cold phosphate-buffered
saline and lysed in luciferase cell culture lysis reagent for 15 min at
room temperature (10 µl/well). A microtiter plate luminometer (ML
3000, Dynex Technologies) was set up in the enhanced flash mode
(maximum sensitivity, delay time = 2 s, integration time = 10 s). Luciferase reagent (100 µl) was automatically added and
light emission was measured every 10 ms during a period of 10 s.
Data was recorded as the peak value of relative light units. Treatments
were performed in triplicate and the mean fold increase in luciferase
activity was calculated relative to mock-induced cells, which was taken
as 1-fold.
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RESULTS |
UMR-106 Cells Predominantly Express P2Y1
Receptor--
Since osteoblasts have been described to express
numerous subtypes of the P2Y family, and previous studies to receptor
profile UMR-106 cells have been incomplete, we used the FLIPR system to fully investigate P2Y receptor expression by these cells. The effects
of a series of known P2 agonists on [Ca2+]i
elevation were measured in fluo-3-loaded cells. The agonist potency
order (p[A]50) was as follows: 2-MeSADP (5.27 ± 0.09) > 2-MeSATP (4.89 ± 0.15) > ADP (4.60 ± 0.15) > ATP (4.57 ± 0.11), suggesting that P2Y1
is the predominant P2 receptor expressed by UMR-106 cells (Fig.
1). While AMP, 
MeATP, and UDP were
inactive at concentrations between 0.1 and 100 µM (data
not shown), UTP evoked a small [Ca2+]i elevation
(Fig. 1). This profile differs from that suggested by Kaplan et
al. (17) in an earlier study, where predominant functional effects
were elicited by P2Y2/Y4 receptor agonists. We
have previously reported heterogeneity of P2 receptor expression in
populations of primary and clonal osteoblasts (7), a process that
appears to be differentiation-dependent (6). Diverse
experimental culture methods or passage number may therefore account
for the apparent differences in receptor profile between cells in this study and those used in the study of Kaplan et al. (17).

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Fig. 1.
UMR-106 cells predominantly express
P2Y1 receptors. Fluo-3-loaded UMR-106 cells were
treated with various nucleotide agonists over a concentration range of
0.1-100 µM (as indicated on the x axis
scale). Ca2+ release was measured according to fluorescence
every second for 1 min after nucleotide addition by a FLIPR.
Fluorescence is expressed as a percentage of the response to 100 µM 2-MeSADP (n = 4). Errors represent
S.D. from the mean.
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PTH Potentiates P2Y1 Agonist-induced
[Ca2+]i Release--
In a previous study
using UMR-106 cells PTH potentiated [Ca2+]i
elevations induced by certain nucleotide agonists (17). In one of our
earlier studies, however, using the human osteosarcoma cell line
SaOS-2, we observed no potentiation of nucleotide-induced [Ca2+]i release following costimulation with PTH
(7). To clarify these differences, the effects of human PTH-(1-34)
(100 ng/ml), in combination with known nucleotide agonists, on
[Ca2+]i were investigated in UMR-106 cells using
the FLIPR. In contrast to the earlier study of Kaplan et al.
(17), we repeatedly saw no elevation of [Ca2+]i
in response to PTH (0.1-500 ng/ml, data not shown). We did, however,
observe a striking potentiation to the [Ca2+]i
response with all nucleotides tested (Fig.
2 shows just ADP for clarity). A
potentiated calcium response was observed when cells were stimulated
with UTP and PTH, therefore confirming that P2Y2 receptors
are expressed by these cells, albeit at low levels.

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Fig. 2.
Costimulation of UMR-106 cells with
nucleotides and PTH results in potentiated Ca2+
elevation. PTH-(1-34) (100 ng/ml) was introduced to fluo-3-loaded
UMR-106 cells 1 min before nucleotide addition (nucleotide
concentration range, 0.3 µM to 1 mM).
Ca2+ release was measured according to fluorescence every
second for 1 min after PTH addition, then for a further 1 min after
nucleotide addition. Fluorescence is expressed as a percentage of the
response to 1 mM ADP + PTH 100 ng/ml (n = 3). Errors represent S.D. from the mean.
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PTH-induced Potentiated Ca2+ Elevations Are Dependent
upon Intracellular Store Release--
To begin to investigate the
mechanism behind this observed potentiation we wondered whether calcium
influx contributed to this response. Again using the FLIPR we found
that in the absence of extracellular Ca2+, ADP and PTH
costimulation induced a [Ca2+]i response
approximately six times greater than that induced by ADP alone (Fig.
3A). However, this response
was depressed in comparison to that resulting from the same experiment
performed in the presence of extracellular Ca2+ (Fig.
3A). Interestingly, introduction of the receptor-activated calcium channel blocker SKF96365 resulted in an increase in the observed potentiation between ADP and PTH (Fig. 3B). Thus
calcium influx is not the major component in the potentiation we
observe.

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Fig. 3.
Potentiated Ca2+ elevations are
dependent upon intracellular store release. A, Fluo-3
loaded UMR-106 cells were divided into 2 groups, half were washed and
resuspended in Ca2+-free BSS, the remaining cells were
resuspended in normal BSS. PTH-(1-34) (100 ng/ml) was introduced 1 min
before ADP addition (0.3 µM to 1 mM, as shown
on the x axis scale) to all cells.
[Ca2+]i release was recorded as a fluorescence
reading by the FLIPR every second for 1 min after PTH addition, and
every second for 1 min after ADP addition (n = 3).
B, again fluo-3-loaded UMR-106 cells were divided, half were
resuspended in BSS containing SFK96365 (30 µM), remaining
cells were resuspended in normal BSS. Measurements were taken as for
A (n = 3).
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Truncated PTH Peptides Act Differentially to Potentiate ADP-induced
[Ca2+]i Release--
To further elucidate
the mechanisms behind the observed potentiated
[Ca2+]i release we used truncated forms of the
PTH peptide, which have previously been reported to have selective
stimulatory effects upon the G-proteins that couple second messenger
signaling pathways to the PTH receptor. It has been reported that
stimulation of the PTH receptor by PTH-(1-34) results in activation of
both Gq and Gs and therefore utilizes both PKC
and adenylyl cyclase to initiate signaling cascades (10). We report
here that PTH-(1-31) (100 ng/ml), which stimulates adenylyl cyclase
but not PKC (21), is able to potentiate ADP-induced Ca2+
responses as effectively as the full-length peptide (PTH-(1-34)) (Fig.
4A). In contrast, PTH-(3-34)
(100 ng/ml) which does not activate adenylyl cyclase (22) was unable to
potentiate the [Ca2+]i release induced by ADP
(Fig. 4A). To confirm that this peptide was functionally
interacting with the PTH receptor it was introduced in combination with
ADP and the active peptides, PTH-(1-34) or -(1-31). PTH-(3-34) was
shown to act as a functional antagonist at the PTH receptor as it
depressed the [Ca2+]i release induced by ADP in
combination with either PTH-(1-34) or PTH-(1-31) (Fig.
4B).

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Fig. 4.
Truncated PTH peptides act differentially to
potentiate ADP-induced [Ca2+]i release.
A, PTH-(1-31) (100 ng/ml), PTH-(3-34) (100 ng/ml), and
PTH-(1-34) (100 ng/ml) were assessed for their ability to potentiate
ADP-induced (0.3 µM TO 1 mM)
[Ca2+]i release in UMR-106 cells. All PTH
peptides were introduced to fluo-3-loaded cells in the FLIPR 1 min
before nucleotide addition. Ca2+ release was measured
according to fluorescence every second for 1 min after PTH addition,
then for a further 1 min after nucleotide addition. Fluorescence is
expressed as a percentage of the response to 1 mM ADP + PTH-(1-34) 100 ng/ml (n = 4). B,
PTH-(3-34) (100 ng/ml) was introduced in combination with both
PTH-(1-31) (100 ng/ml) and PTH-(1-34) (100 ng/ml) to UMR-106 cells.
ADP was added 1 min after PTH addition and fluorescence was measured as
for A (n = 3).
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H-89 Does Not Inhibit PTH/Nucleotide-potentiated
[Ca2+]i Release--
As a main downstream
target for activated adenylyl cyclase pathways, we next investigated
the potential role of PKA in driving potentiated
[Ca2+]i release in UMR-106 cells. Preincubation
of UMR-106 cells with the PKA inhibitor H-89 (1-100 µM)
did not affect potentiated [Ca2+]i release
induced by ADP and PTH, indicating that PKA activity is not necessary
to initiate the potentiation (Table I).
H-89 was inhibitory in these cells, since it blocked PTH-induced phosphorylation of CREB on Ser133, the residue
phosphorylated by active PKA (Fig.
5).
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Table I
H-89 does not depress ADP/PTH-induced potentiated Ca2+
elevations
H-89 (1-100 µM) was introduced to fluo-3-loaded UMR-106
cells 10 min before ADP (100 µM) and PTH (100 ng/ml)
addition. Ca2+ release was measured according to fluorescence
every 30 s for 10 min after H-89 addition, then every second for a
further 1 min after ADP/PTH addition. Fluorescence is expressed as a
percentage of the response to 100 µM ADP + 100 ng/ml
PTH. H-89 had no effect on the potentiation at any concentration
tested, figures show effects of 10 µM H-89
(n = 3).
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Fig. 5.
H-89 inhibits PTH-induced CREB
phosphorylation in UMR-106 cells. To confirm the bioactivity of
H-89 quiescent cells were treated with PTH (100 ng/ml) alone or in
combination with this compound (10 µM). H-89 was
introduced either 15 or 30 min before lysing cells, as indicated
above each lane, PTH was introduced 10 min before lysing in
all lanes. Whole cell extracts were prepared, and 15 µg of protein
per sample ran on an 8.5% SDS-polyacrylamide electrophoresis minigel.
After transfer to a nitrocellulose membrane the blot was immunodetected
with anti-phospho-Ser133 CREB antiserum, followed by
peroxidase-coupled anti-rabbit antiserum and detection using enhanced
chemiluminescence. The blot was stripped and re-probed with anti-CREB
antiserum to confirm the presence of similar levels of protein in each
lane. The absence of a signal for phospho-CREB in lanes treated with
H-89 confirms that this compound does inhibit PKA in this cell
type.
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Forskolin and IBMX Have Differential Effects on ADP-induced
[Ca2+]i Release--
The results above
showed that potentiation occurs with a PTH peptide that activates a
signaling pathway involving adenylyl cyclase but not PKA. To assess
whether elevated cAMP could drive potentiated
[Ca2+]i release, cells were costimulated with ADP
and the cell permeable adenylyl cyclase activator, forskolin (50 µM), in the absence of extracellular calcium. Forskolin
was unable to potentiate ADP-induced calcium responses (Table
II). Forskolin alone was shown to elevate
intracellular cAMP levels in these cells and to induce CREB
phoshorylation via PKA phosphorylation (data not shown). We also tested
the ability of the nonspecific phosphodiesterase inhibitor, IBMX (1 mM) to potentiate ADP-induced [Ca2+]i
release. In contrast to forskolin, IBMX effectively potentiated
ADP-induced [Ca2+]i release (Table II).
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Table II
IBMX, but not forskolin can potentiate nucleotide-induced
[Ca2+]i release
Forskolin (50 µM), IBMX (1 mM), and PTH (100 ng/ml) were introduced to fluo-3-loaded UMR-106 cells 6 min before ADP
addition (ADP concentration range, 0.3 µM to 1 mM). Ca2+ release was measured according to
fluorescence every second for 1 min after ADP addition. Fluorescence is
expressed as a percentage of the response to 100 µM
ADP + 100 ng/ml PTH (n = 3).
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cGMP Does Not Potentiate ADP-induced
[Ca2+]i Release--
Since the results
obtained with forskolin and IBMX suggested that accumulation of cGMP,
downstream of the activated PTH receptor, may be causing the
potentiation of ADP-induced [Ca2+]i levels, we
costimulated cells with ADP and the cell permeable cGMP analogue,
dibutyryl-cGMP (300 µM), in the absence of extracellular
calcium. Dibutyryl-cGMP was unable to potentiate ADP-induced
[Ca2+]i elevations (Fig.
6), eliminating accumulation of this cyclic monophosphate as a possible mechanism of potentiation. Dibutyryl-cGMP alone produced no [Ca2+]i
elevation (data not shown).

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Fig. 6.
cGMP accumulation is not involved in the
mechanism resulting in potentiated [Ca2+]i
elevation. The cGMP analogue dibutyryl-cGMP (300 µM)
was introduced to fluo-3-loaded cells 30 min before nucleotide addition
(nucleotide concentration range, 0.3 µM to 1 mM). Ca2+ release was measured according to
fluorescence every second for 1 min after nucleotide addition.
Fluorescence is expressed as a percentage of the response to 1 mM ADP + PTH 100 ng/ml (n = 3). Errors
represent S.D. from the mean. FCS, fetal calf serum.
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PTH and P2Y1 Receptor Agonists Induce Phosphorylation
of the Transcription Factor CREB--
Phosphorylation of CREB on
Ser133 is strongly linked to signaling-induced
transcriptional activation. Ser133 is a substrate for PKA
but also for kinases activated by elevated intracellular calcium. We
investigated whether ADP and PTH alone or in combination induced CREB
phosphorylation in UMR-106 cells. We performed Western analysis of
whole cell extracts using antisera directed against CREB phosphorylated
on Ser133. The P2Y1 agonist ADP (100 µM) induced low CREB activation following 15 min
stimulation, while PTH-(1-34) (100 ng/ml) gave rise to a robust
transcription factor phosphorylation. In combination, however, agonists
induced levels of CREB activation greater than those seen with either
agonist alone. Quantitative values for CREB and phospho-CREB were
obtained by densitometric analysis (Fig.
7).

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Fig. 7.
ADP and PTH stimulate CREB phosphorylation in
UMR-106 cells. Quiescent UMR-106 cells were stimulated with 100 µM ADP and 100 ng/ml PTH alone or in combination as
indicated above each lane. After 15 min cells were lysed and
Western analysis for CREB phosphorylation was performed as described in
the legend to Fig. 5. Following densitometry, phospho-CREB levels were
expressed as a ratio of CREB and then expressed as a percentage
increase over vehicle as indicated below each lane. FCS, fetal calf
serum.
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PTH and P2Y1 Receptor Agonists Synergistically Induce
the Endogenous c-fos Gene--
To determine the impact of the observed
potentiated calcium response on gene expression we measured the effects
of P2Y1 agonists and PTH on the levels of endogenous
c-fos transcription in UMR-106 cells. This proto-oncogene is
of particular relevance when studying osteoblasts as it has been
implicated in many of the processes that govern skeletal tissue
remodeling (23). Stimulation of quiescent UMR-106 cells with the
P2Y1 agonists ADP and 2-MeSATP (10 µM) for 45 min had a very weak effect on c-fos mRNA expression, as
assessed by Northern analysis (Fig. 8).
PTH alone led to a more robust c-fos induction that was
significantly enhanced by costimulation with PTH and P2Y1
agonists (Fig. 8).

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Fig. 8.
P2Y1 receptor agonists and PTH
induce synergistic endogenous c-fos gene expression in
UMR-106 cells. UMR-106 cells were serum-starved and then
stimulated for 45 min with 10 µM ADP, 2-MeSATP, and/or
PTH 100 ng/ml as indicated above each lane. 10 µg of total
RNA for each sample was analyzed by Northern blotting and hybridization
with c-fos, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) riboprobes mixed 15:1. The c-fos signal
in each lane was standardized to the GAPDH internal control, and the
level of induction relative to control expressed numerically below each
lane.
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The Ca/CRE Located at Position
60 in the c-fos Promoter Is
Sufficient to Drive Synergistic Gene Expression--
The Ca/CRE
element in the c-fos promoter responds to both cAMP and
certain types of calcium signals. Following the observation that
nucleotides and PTH could potentiate [Ca2+]i
release in UMR-106 cells, we wondered whether activation of the Ca/CRE
alone may be sufficient to generate the observed synergy on
c-fos gene expression. To assess this we utilized UMR-106 cells stably transfected with luciferase reporters driven by a truncated Ca/CRE promoter element. Costimulation of stable pools of
transfected UMR-106 cells with PTH and P2Y1 nucleotide
agonists resulted in synergistic luciferase activity (Fig.
9). These data show that this calcium
responsive region of the fos promoter is sufficient to drive
synergistic gene expression, and that the strong calcium signal induced
by ADP and PTH costimulation converges on this promoter element.

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Fig. 9.
PTH and nucleotides induce synergistic
c-fos gene expression via the Ca/CRE promoter
alone. UMR-106 cells stably transfected with the fos
Ca/CRE promoter-luciferase reporter were serum starved and then induced
for 4 h with 100 µM ADP and/or PTH 10 8
M. Luciferase activity was measured by a microtiter plate
luminometer in the enhanced flash mode (n = 3).
Error bars indicate the S.D.
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DISCUSSION |
The main findings of this study are: 1) the rat osteosarcoma cell
line UMR-106 primarily expresses P2Y1 receptors coupled to
[Ca2+]i mobilization, 2) PTH does not couple to
Gq in UMR-106 cells, but strongly potentiates
P2Y1 agonist-induced [Ca2+]i release
in these cells, 3) this potentiation is primarily dependent upon
calcium release from intracellular stores and relies upon the activated
PTH receptor coupling to Gs, 4) potentiated [Ca2+]i release occurs independently of cAMP/PKA
pathways or cGMP accumulation, 5) PTH and nucleotide costimulation
potentiates phosphorylation of the transcription factor CREB and leads
to a synergistic induction of the endogenous c-fos gene, and
6) the fos Ca/CRE alone is sufficient to obtain a similar
synergistic induction in the context of reporter genes stably
transfected into UMR-106 cells.
An important observation from these studies was that PTH-(1-34), over
a wide dose range, was unable to elevate [Ca2+]i
in UMR-106 cells, demonstrating that Gq is not activated in
response to PTH in this cell type (data not shown). This is in
contradiction to Kaplan et al. (17) who reported that PTH did induce a small [Ca2+]i elevation. Earlier
studies also confirm that the effect of PTH on
[Ca2+]i mobilization in osteoblasts is
controversial, one group finding no effect (24), but others reporting
various increases (25, 26). A possible explanation for these very
different reports may be the presence of ATP in the culture medium due
to cell lysis. We have also demonstrated that nucleotides can be constitutively released nonlytically from osteoblasts, and that this
may be positively regulated by fluid flow (1). Therefore one
consequence of agitating cell suspensions will be to increase release
of ATP, with the ability to modulate PTH-induced responses. Systems
fitting this criteria have been used in studies that report Gq coupling in UMR-106 cells in response to PTH. To avoid
this problem, we routinely treated the cells with apyrase to eliminate extracellular nucleotides released in this manner. It is also worth
noting that other studies have observed a Ca2+ response to
PTH in a subset of cells within a population, suggesting that this
response in osteoblasts is limited by the state of differentiation of
the cells (27, 28).
Our studies have shown huge potentiations of
[Ca2+]i in response to nucleotide and PTH
receptor costimulation which is consistent with previous findings (17).
This observed potentiation was primarily dependent on intracellular
store release, although the potentiation was slightly depressed in the
absence of extracellular Ca2+ (Fig. 3A).
Interestingly, an increased potentiation was observed in the presence
of the receptor-gated Ca2+ channel blocker SFK96365, (Fig.
3B), again supporting the theory that intracellular
Ca2+ store release, rather than Ca2+ influx was
primarily responsible for potentiation. These observations also
discount the possibility that potentiated cytosolic calcium levels
reflect the ability of PTH/cAMP-mediated stimulation of membrane
channels, since this process is dependent upon extracellular Ca2+ influx (29).
Although we found no evidence for Gq coupling to the PTH
receptor in UMR-106 cells, we utilized truncated PTH peptides to confirm that Gs activation was vital in driving the
potentiation mechanism. As expected, PTH-(1-31), which activates
G
s independently of Gq, was able to
potentiate ADP-induced [Ca2+]i release (Fig.
4A). Conversely PTH-(3-34), which reportedly activates
Gq (22), was ineffective in this respect, despite its
capacity to functionally interact with the PTH receptor, shown by its
ability to act as an antagonist to both PTH-(1-31) and PTH-(1-34)
(Fig. 4B). These data were suggestive of the involvement of
cAMP/PKA driven pathways in potentiating [Ca2+]i
release. Intracellular cross-talk may occur between cAMP and
[Ca2+]i driven pathways, cAMP can elicit
[Ca2+]i release via PKA phosphorylation of
inositol 1,4,5-trisphosphate receptor on serines 1755 and 1589. Kinetically, it seemed unlikely that this mechanism drove PTH
potentiation of ADP-induced [Ca2+]i release in
our system, however, we investigated this possibility using the
metabolic PKA inhibitor H-89. While H-89 effectively inhibited CREB
phosphorylation following PTH stimulation (Fig. 5), similar
concentrations were unable to depress [Ca2+]i
release in response to PTH and ADP (Table I).
Although activated PKA appeared not to be essential for a potentiated
[Ca2+]i release the possibility still remained
that elevated cAMP, through a PKA-independent mechanism, could play a
role. This was investigated by costimulating UMR-106 cells with ADP and
the adenylyl cyclase activator forskolin. In the absence of extracellular calcium the inability of forskolin to mimic PTH-induced responses strongly suggested that potentiated
[Ca2+]i release occurs independently of cAMP
accumulation. Unexpectedly, the nonspecific phosphodiesterase inhibitor
IBMX potentiated ADP-induced [Ca2+]i release in a
manner similar to PTH (Table II), suggesting that other cyclic
monophosphates may be involved in the potentiation mechanism in
osteoblasts. In contrast, Short and Taylor (16), using PTH1
receptor-transfected human kidney cells, were unable to potentiate
carbachol-induced calcium responses with IBMX, and in fact, inhibited
PTH-potentiated carbachol responses with this phosphodiesterase
inhibitor (16). To clarify this apparent contradiction, and to further
elucidate the potentiation mechanism, UMR-106 cells were costimulated
with ADP and the cGMP analogue, dibutyryl-cGMP. cGMP was unable to
potentiate ADP-induced Ca2+ elevations in these cells (Fig.
6). The discrepancies between the results obtained by Short and Taylor
(16) and our own, may perhaps be explained by the fact that in some
cell types IBMX has been found to elevate intracellular calcium (30,
31), whereas in other cell types, cGMP can dampen inositol
1,4,5-trisphosphate receptor-driven responses (32).
Our findings showing that neither elevated cAMP nor cGMP are able to
potentiate nucleotide-induced [Ca2+]i elevations
and the inability of IBMX to potentiate carbachol responses in kidney
cells demonstrate that an as yet unidentified mechanism is responsible
for [Ca2+]i potentiation observed upon dual
activation of two separate G-protein-coupled receptors. Other
investigators have suggested that PTH may regulate Ca2+
mobilization by facilitating translocation of Ca2+ between
discrete intracellular stores, thereby regulating the Ca2+
pool available to receptors linked to inositol 1,4,5-trisphosphate formation (16). Alternatively, it has been suggested that a direct
G-protein interaction may account for potentiated
[Ca2+]i release (33).
The significance of this calcium potentiation can be seen in its
effects on transcription factor activation and gene expression. The
transcription factor CREB binds the cyclic AMP response element (CRE)
and initiates transcription in response to a number of extracellular signals, including elevated [Ca2+]i. Phospho-CREB
plays a key role in signaling-driven activation of the proto-oncogene
c-fos which in turn has been strongly implicated in driving
many osteoblast functions, including proliferation and differentiation
(34, 35). PTH and ADP costimulation of UMR-106 cells resulted in
increased levels of phospho-CREB (Fig. 7), and potentiation of
endogenous c-fos mRNA expression (Fig. 8). Furthermore,
using UMR-106 reporter cells, stably transfected with a truncated
c-fos promoter consisting only of the Ca/CRE, we have
demonstrated that this promoter element alone is sufficient to drive
synergistic c-fos expression (Fig. 9). In contrast, in a
previous study using the human osteosarcoma cell line SaOS-2, we found
that both the Ca/CRE and the serum response element of the
c-fos promoter were required to drive the same synergy (7). This was consistent with an inability of PTH to potentiate
nucleotide-induced [Ca2+]i release in SaOS-2
cells. It would therefore appear that multiple pathways, both calcium
dependent and independent, exist to couple P2Y1 and PTH
receptor coactivation to synergistic gene expression in osteoblasts.
It has never been clear how systemic PTH can initiate remodeling that
is confined to a small area of bone. These studies suggest a mechanism
whereby the localized release of nucleotides, via lytic and nonlytic
mechanisms, can sensitize cells to surrounding systemic factors such as
PTH. This costimulation appears to have profound effects on
osteoblastic cells, initiating calcium-activated pathways that
ultimately result in transcription factor activation and synergistic
induction of gene expression.