From the Human Bone Cell Research Group,
¶ Department of Human Anatomy and Cell Biology, and
Department of Clinical Chemistry,
University of Liverpool, Liverpool L69 3GE, United Kingdom, ** Novartis
Pharma AG, CH-4002, Basel, Switzerland, and
§§ Institut de Genetique Moleculaire de
Montpellier, UMR 5535, CNRS, 1919 Route de Mende,
34293 Montpellier cedex 5, France
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ABSTRACT |
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Extracellular nucleotides acting through specific
P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue,
the human osteosarcoma cell line SaOS-2 and primary osteoblasts express
P2Y1 and P2Y2 receptors, respectively.
Their activation by nucleotide agonists (ADP and ATP for
P2Y1; ATP and UTP for P2Y2) elevates
[Ca2+]i and moderately induces expression of the
c-fos proto-oncogene. A synergistic effect on
c-fos induction is observed by combining ATP and
parathyroid hormone, a key bone cell regulator. Parathyroid hormone
elevates intracellular cAMP levels and correspondingly activates a
stably integrated reporter gene driven by the
Ca2+/cAMP-responsive element of the human c-fos
promoter. Nucleotides have little effect on either cAMP levels or this
reporter, instead activating luciferase controlled by the full
c-fos promoter. This induction is reproduced by a stably
integrated serum response element reporter independently of
mitogen-activated protein kinase activation and ternary complex factor
phosphorylation. This novel example of synergy between the
cAMP-dependent protein kinase/CaCRE signaling module and a
non-mitogen-activated protein kinase/ternary complex factor pathway
that targets the serum response element shows that extracellular ATP,
via P2Y receptors, can potentiate strong responses to ubiquitous growth
and differentiative factors.
Extracellular stimuli regulate gene expression via the activation
of intracellular signaling systems that transduce the signal from
membrane-bound receptors to transcription factors. This results in the
rapid, transient induction of genes at the transcriptional level whose
products will mediate the appropriate cellular response. One of the
best characterized of these immediate early genes is the
c-fos proto-oncogene, whose activation can be linked to
different signaling cascades targeting distinct promoter elements
through the phosphorylation of transcription factors (1, 2).
In the c-fos promoter, the calcium/cAMP-responsive element
(CaCRE)1 (1), located just
upstream of the TATA box, can mediate activation following elevation of
intracellular cAMP, as well as Ca2+ in certain instances
(3, 4). Proteins of the CREB/activating transcription factor family
recognize this element, and transcriptional induction arises via their
phosphorylation on Ser133 (5). The serum response element
(SRE), at position c-fos induction plays an important role in vitro
in driving immortalized fibroblasts to enter the cell cycle and plays
an important role in vivo in the skeletal system. Mice
lacking the c-fos gene fail to develop osteoclasts and thus
show an osteopetrotic phenotype in which the dynamic process of bone
remodeling has been shifted toward bone accumulation (19, 20).
Conversely, constitutive overexpression of c-fos in the bone
environment of transgenic mice leads to the development of
osteosarcomas (21, 22). Accordingly, a number of proteins
characteristic of differentiating bone cells have regulatory activator
protein 1 sites in their promoters, and a variety of extracellular
factors documented to stimulate bone cell growth and differentiation
activate c-fos transcription in cultured bone cells in
vitro (2, 22-25).
Parathyroid hormone (PTH) is essential for the modeling and remodeling
of the skeleton. In bone-derived cell lines as well as in primary
osteoblasts in culture, PTH strongly induces transcription of the
c-fos gene (23, 24). This occurs via the
cAMP/cAMP-dependent protein kinase/CREB pathway in the
osteosarcoma cell line SaOS-2 (25) but may involve protein kinase C and
the ERK pathway in other cells (2). Because PTH is a systemic factor
and the process of bone remodeling is essentially a focal phenomenon,
cellular responsiveness to PTH in vivo is likely to be
modulated by other factors. We wondered whether ATP might play such a
modulatory role, since ATP can be released from osteoblasts into the
local bone microenvironment via a nonlytic mechanism (26) and since ATP
synergizes with mitogens to enhance DNA synthesis in a variety of cells
(27-29).
Extracellular nucleotides, such as ATP, exert stimulatory effects on
cells at micromolar concentrations through the P2 family of
membrane-bound receptors (30-33). Two major classes of P2 receptors have been delineated: P2X receptors, which are ligand-gated ion channels, and P2Y receptors, which are coupled to G proteins (32). More
pertinent to our hypothesis, P2Y receptors are expressed in
osteoblastic cells of rat (34, 35) and human origin (36, 37). Two major
subtypes of P2Y receptor, P2Y1 and P2Y2, are coupled through Gq to phosphatidylinositol 4,5-bisphosphate
hydrolysis and hence Ca2+ mobilization from intracellular
stores (32).
Here we show that both the osteosarcoma cell line SaOS-2 and primary
cells in culture express P2Y receptors that functionally couple to
c-fos activation. This involves increased intracellular Ca2+ and a signaling pathway that can activate an
SRE-driven reporter gene independently of the predominant ERK/TCF
signaling module. Co-activation of this pathway and that induced by PTH
increases c-fos mRNA levels well above those induced by
either stimulus alone, thereby providing a novel example of synergy
between a cAMP-dependent protein kinase and a
Ca2+-triggered signaling system not involving MAPK. These
data demonstrate that extracellular nucleotides can strongly potentiate
the response of bone cells to systemic factors and suggest that this
may be a common mechanism to generate strong localized responses to
systemic growth and differentiation factors.
Reagents--
Dulbecco's modified Eagle's medium, Cell Culture--
Human bone-derived cells (HBDC) were isolated
and cultured from explants of human bone as described previously (38).
In brief, specimens of human bone in Dulbecco's modified Eagle's medium were finely minced with a scalpel and then washed free of marrow
cells with several volumes of medium. The minced bone was cultured in
9-cm Petri dishes containing Dulbecco's modified Eagle's medium
supplemented with 10% FCS, 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 for 3 weeks. Confluent cells were either seeded
onto glass coverslips or induced for subsequent preparation of whole
cell extracts or RNA. SaOS-2 cells were maintained in Construction of c-fos-luciferase Reporter Genes for Stable
Transfection--
The fragment containing the c-fos
promoter (spanning positions Isolation and Culture of Stable Cell Lines Incorporating the
c-fos-luciferase Reporter Gene--
SaOS-2 cells (ATCC HTB 85) were
maintained in RPMI 1640 medium supplemented with 10% (v/v) FCS. These
cells were transfected with pfoslucneo1 or CaCRE-luc using Lipofectin
reagent according to the protocols of the manufacturer, and the cells
were maintained in RPMI/FCS for 24 h. Stably transfected cell
pools were selected for resistance to G418 following standard protocols
(39). UMR-106 cells were cultured in Ham's F-12 and Dulbecco's
modified Eagle's medium (1:1) supplemented with 10% FCS at 37 °C
in a humidified atmosphere containing 95% air and 5% CO2.
Stable pools of UMR-106 cells containing the SRE-luciferase reporter
were generated as described above.
Luciferase Reporter Gene Assays--
For reporter gene assays,
the cells were seeded into 96-well plates at a density of 96,000 cells/well. At near confluence, the medium was replaced with one
containing 0.5% FCS for 16 h, followed by serum-free RPMI
containing 0.1% BSA for 24 h. Agonists were added to cells as a
10× stock solution prepared in the same medium to the final
concentrations indicated in the figures. After a 4-h incubation, cells
were washed twice in cold PBS and lysed in luciferase cell culture
lysis reagent for 15 min at room temperature (25 µl/96-well plate).
The plates were subsequently centrifuged at 3000 rpm for 5 min at room
temperature before transferring 20 µl of each sample to a 96-well
microtiter plate (Dynatech) for analysis. A microtiter plate
luminometer (ML 3000, Dynatech Laboratories) 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 were recorded as the peak value of relative
light units.
Measurement of [Ca2+]i--
SaOS-2
cells and HBDC were grown to confluence on 22-mm diameter glass
coverslips. [Ca2+]i was measured after 2 h
of serum deprivation. Cells were loaded with fura-2 by incubation with
fura-2 acetoxymethyl ester (5 mM) for 20 min at 37 °C in
HEPES buffer (10 mM HEPES, 121 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4,
1.2 mM MgSO4, 2 mM
CaCl2, 5 mM NaHCO3, 10 mM glucose, pH 7.2) containing 2% BSA. Cells were subsequently washed three times in the same buffer containing 0.2%
BSA. Measurements were performed with a photon-counting
spectrophotometer on a Nikon TM Diaphot microscope with a × 40 oil immersion objective. The cell-coated coverslip was attached with
silicone grease to form the base of a stage-mounted, thermostatically
regulated chamber maintained at 37 °C. Groups of 6-8 cells were
illuminated with excitation light (340 and 380 nm) at a rate of 32 times/s, and the emission measurements (at 510 nm) were integrated into
1-s averages and stored. Agonists, in HEPES buffer with 0.2% BSA, were
added for 60-120 s, followed by at least 10 min of recovery prior to
further stimulation. Rmin,
Rmax, and autofluorescence values were obtained
in situ using ionomycin, as described previously (40).
[Ca2+]i was calculated from the ratio of
fluorescence at the two excitation wavelengths after subtraction of
autofluorescence (41). The results were evaluated statistically using
the Student's t test, assuming a significance of
p < 0.05.
RNA Isolation and cDNA Synthesis--
Total RNA was
extracted from control and stimulated cells with 4 M
guanidine thiocyanate, 0.5% sarkosyl, 0.1 M
mercaptoethanol, 25 mM sodium citrate, pH 7.0, followed by
acid phenol/chloroform extraction (39). RNA was stored in ethanol at
Northern Analysis--
5-10 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 as described by Evans et al. (25). Blots were
prehybridized at 42 °C in 40% formamide, 5× SSC, 10× Denhardt's
reagent, 1% SDS, 200 µg/ml denatured salmon sperm DNA, 200 µg of
tRNA and probed with a 487-base pair fragment spanning exons 3 and 4 of
the human c-fos gene labeled with
[ Polymerase Chain Reaction--
50-µl PCRs contained 0.25 units
of Taq DNA polymerase, 1 µg of sense and antisense primers
(see below), 200 µM dNTPs, 1.5 mM
MgCl2, 10 mM mercaptoethanol, 10 mM
Tris-HCl, pH 8.3, and 2 µl of cDNA. P2Y1,
P2Y2, and GAPDH were amplified for 40 cycles (94 °C for
10 s; 58 °C for 30 s; 72 °C for 30 s). The primer
sequences were as follows: P2Y1 sense,
TGTGGTGTACCCCCTCAAGTCCC; P2Y1 antisense, ATCCGTAACAGCCCAGAATCAGCA; P2Y2 sense,
CCAGGCCCCCGTGCTCTACTTTG; P2Y2 antisense,
CATGTTGATGGCGTTGAGGGTGTG; GAPDH sense, GGTGAAGGTCGGAGTCAACGG; GAPDH
antisense, GGTCATGAGTCCTTCCACGAT.
cAMP Measurements--
SaOS-2 cells were serum-starved as above
and then pretreated with 0.1 mM 3-isobutyl-1-methylxanthine
for 30 min at 37 °C. Agonist was then added as described in the
legend to Fig. 7. After a 20-min incubation, the cells were covered
with 1 ml of ice-cold 65% ethanol and scraped off with a rubber
policeman. Cells were collected by centrifugation at 8000 × g for 3 min and stored at Whole Cell Extract Preparation--
Whole cell extracts were
prepared as described previously (42). At the appropriate time point,
plates were placed on ice, and the cell layer was quickly washed twice
with ice-cold PBS (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
Gel Retardation Assay--
Reactions (7.5 µl) contained the
following components: 2.5 µg of poly(dI-dC)(dI-dC), 250 ng of calf
thymus DNA, 5% (v/v) glycerol, 66 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.35% (v/v)
Triton X-100, 0.05% (w/v) low fat milk, 15 mM
dithiothreitol, 15,000 cpm of 32P-labeled probe (0.2 ng/4
fmol), and 10 µg of whole cell extract. After a 30-min incubation at
room temperature, the entire reaction was loaded on a 4.5%
polyacrylamide gel containing 45 mM Tris borate, 1.5 mM EDTA (pH 8.3) and run at 1 mA/cm for 3-4 h. Gels were
dried, and the complexes were visualized by autoradiography using
intensifying screens or phosphor storage technology. Core SRF90-244 was produced in HeLa cells using a recombinant vaccinia virus (8). The probe corresponded to the c-fos SRE (see above) subcloned in front of a G-free cassette plasmid (44). After
EcoRI and NarI digestion, the ends were labeled
by a Klenow fill-in reaction containing [ Western Analysis--
15 µg of whole cell extracts were
separated on an 8.5% SDS-polyacrylamide gel electrophoresis minigel
and transferred electrophoretically to polyvinylidene difluoride. The
filter was blocked for 1 h at room temperature in 5% (w/v) BSA in
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 or sheep anti-mouse antibody,
diluted 1:1000 in 5% (w/v) low fat milk powder-TBST. After washing,
the immune complexes were visualized using enhanced chemiluminescence.
The antisera used were anti-pan-ERK, diluted 1:5000;
anti-phospho-Thr202/Tyr204 ERK, anti-phospho
Thr183/Tyr185 SAPK, anti-phospho
Thr180/Tyr182 p38, anti-SAPK, and anti-p38,
diluted 1:1000.
Differential Expression of P2Y1 and P2Y2
Receptors by SaOS-2 and Primary Human Bone-derived Cells--
Since
osteoblasts have been described to express different purinergic
receptors of the P2Y family, we used RT-PCR to analyze which subtypes
are expressed in the human osteosarcoma cell line SaOS-2. In addition,
we tested two populations of primary osteoblastic cells derived from
explants of human bone in vitro culture, termed HBDC1 and
HBDC2. RT-PCR on cDNA from SaOS-2 cells gave rise to an intense
signal for P2Y1 receptor transcripts but only a weak signal
for P2Y2 receptor (Fig. 1).
In contrast, only P2Y2 receptor transcripts were visualized
in the HBDC1 cDNA, while amplification of HBDC2-derived cDNA
showed similar levels of amplification for both P2Y1 and
P2Y2. The signal with primers specific for GAPDH was
similar in all reactions (Fig. 1), confirming the integrity and amount
of cDNA in each sample.
Elevation of [Ca2+]i in SaOS-2 Cells and
Primary Human Osteoblasts following P2 Receptor Stimulation--
P2Y
receptors are coupled to heteromeric G proteins intracellularly, and
have been reported to activate, via Gq,
phosphatidylinositol 4,5-bisphosphate hydrolysis and Ca2+
mobilization from intracellular stores (32). Since SaOS-2 cells and
HBDC1 express predominantly the P2Y1 and P2Y2
receptors, respectively, we tested whether different P2Y1
and P2Y2 receptor agonists could mobilize intracellular
Ca2+, measured using fluorescence increases in groups of
6-8 fura-2-loaded cells. ATP consistently induced a rise in
[Ca2+]i in both cell types (Fig.
2A and Table
I). In SaOS-2 cells, ATP was effective at
concentrations ranging from 1 to 100 µM (Fig.
2A). In these cells, the P2Y2 agonist UTP evoked
only a minor increase in [Ca2+]i at 10 µM and a more significant increase at 100 µM (Fig. 2B) that nevertheless was smaller
than that induced by 1 µM ATP (Fig. 2, compare
A and B). This difference in responsiveness was
also observed upon the sequential addition of UTP and ATP, thus
indicating that it did not reflect decreased sensitivity of the cells.
More importantly, UTP induced the same increase as ATP in HBDC1 (Fig.
3 and Table I), clearly showing that the difference is due to the P2Y receptor subtype expressed by the cells.
The effects of other agonists confirmed this differential
responsiveness between the primary cells and the established cell line.
In SaOS-2 cells, the ATP analogues ATP Induction of c-fos mRNA in SaOS-2 Cells following P2 Receptor
Stimulation--
Since nucleotide addition elevated intracellular
calcium levels, we tested whether this second messenger pathway
activated nuclear signaling, as measured by the induction of the
proto-oncogene c-fos. This event is particularly relevant in
bone-derived cells, since c-fos has been implicated in many
of the processes that govern skeletal tissue remodeling (21, 22).
ATP Synergistic Induction of c-fos by the Combination of P2 Receptor
Agonists and PTH in SaOS-2 Cells--
While P2Y1 receptor
agonists induced c-fos, the level of expression was low
relative to serum (Fig. 4C). Since nucleotides enhance the
proliferative response to mitogens in other cell types (27, 28), we
tested whether they might enhance c-fos induction in SaOS-2
cells by PTH, a potent stimulator of bone growth in vivo.
Quiescent SaOS-2 cells were treated with different nucleotides alone or
in combination with PTH and mRNA levels analyzed by Northern blotting. Cotreatment with PTH and either ATP or ADP resulted in a
synergistic induction of c-fos mRNA relative to either
inducer alone (Fig. 5A). In
contrast, UTP did not augment PTH-induced c-fos activation
in SaOS-2 cells, which is again consistent with its inability to
stimulate the P2Y1 receptor.
To confirm that this synergy arose from direct transcriptional
activation and facilitate further characterization of this effect, we
transfected SaOS-2 cells with a reporter gene in which the full
c-fos promoter, spanning positions Synergistic Induction of c-fos by the Combination of P2 Receptor
Agonists and PTH in HBDC--
To determine whether extracellular
nucleotides might also potentiate c-fos activation by PTH in
primary osteoblasts, we treated HBDC with PTH and a range of P2Y
receptor agonists. As above, ATP cooperated with PTH to strongly
enhance c-fos mRNA levels above those induced by either
factor alone (Fig. 6). In these cells,
the P2Y2 receptor agonist also synergized with PTH, whereas the P2Y1 agonist ADP was inactive (Fig. 6). Thus, the
primary cell population shows a response consistent with their receptor subtype and that contrasts with the response of the
P2Y1/SaOS-2 combination to the same nucleotides.
PTH and ATP Utilize Distinct Second Messengers in Human
Osteoblasts--
In clonal rat osteoblastic cells, ATP increases
PTH-induced [Ca2+]i responses (32); therefore, we
tested if the synergy between ATP and PTH resulted from enhanced
Ca2+ mobilization. In neither SaOS-2 cells nor the primary
osteoblasts did PTH, at 20-500 ng/ml, generate a significant calcium
response, nor did we ever observe that PTH enhanced the effect of
either ATP or UTP on
[Ca2+]i.3
In SaOS-2 cells, PTH-(1-34) induces c-fos expression
through cAMP-dependent phosphorylation of CREB
independently of protein kinase C activation, TCF phosphorylation, or
signal transducer and activator of transcription induction (25).
Therefore, we tested if the synergy on c-fos expression
following nucleotide/PTH stimulation might reflect modulation of cAMP
levels. A radioimmunoassay was used to assess the effects of nucleotide
stimulation, alone and in combination with PTH, on cAMP accumulation in
SaOS-2 cells. Forskolin, a potent activator of adenylyl cyclase at 10 µg/ml, elevated intracellular cAMP to 170 pmol/ml above the basal
level. On the other hand, the addition of 10 µM ATP,
ATP The CaCRE in the c-fos Promoter Is Insufficient for a Synergistic
Response--
The CaCRE in the c-fos promoter, which is
situated immediately upstream of the TATA box, can mediate activation
of reporter genes by certain Ca2+ signaling pathways (see
Introduction). We used stably transfected SaOS-2 cell lines to test the
possibility that the ATP/Ca2+ and PTH/cAMP pathways
converge on this site to synergistically induce c-fos. A
reporter driven by a truncated promoter, containing only the CaCRE
linked to luciferase, was compared with the full-length reporter
construct. Co-stimulation of the stable pools containing the truncated
reporter with PTH and nucleotides resulted in luciferase expression
levels slightly above those observed with PTH alone (Table
II). Notably, this increase reflected an
additive effect of ATP and PTH rather than the multiplicative effect
seen on the endogenous gene and the full promoter-driven reporter. This
strongly suggested that synergistic activation did not arise by the
convergence of these two signaling systems on the CaCRE alone.
ATP Activates the c-fos SRE Independently of ERK and
TCF--
These data implied that the ATP/Ca2+ signaling
pathway targeted another site in the c-fos promoter. The SRE
seemed the most likely candidate, since it can also mediate activation
by various calcium-dependent pathways. We again chose the
stable transfection approach. Unfortunately, SaOS-2 cells have proven
refractory to stable transfection with SRE-driven reporter constructs,
so we resorted to another bone-derived cell line, UMR-106, that shows the same responses as SaOS-2 cells to ATP and
PTH.4 Pools of UMR-106 cells
were generated that contain a reporter construct in which the
luciferase gene is controlled by three c-fos SREs cloned in
front of the c-fos TATA box. These cells responded strongly
to EGF and slightly less so to serum, which increased luciferase
activity 12- and 6-fold, respectively (Table II). Treatment with PTH
did not stimulate luciferase at all, while ATP caused a 4.6-fold
increase in luciferase activity. Thus, the SRE can mediate the response
to ATP-driven signals and is likely to be the promoter element targeted
by the ATP/Ca2+ pathway in bone cells.
The SRE integrates signals from many pathways but is particularly
responsive to activated MAPK cascades via the phosphorylation of TCF, a
major nuclear target for the MAPKs ERK, SAPK/c-Jun N-terminal kinase,
and p38 MAPK (2). We wondered whether this synergy reflected the
activation of one of these cascades by nucleotides, especially since
other bone growth factors can induce ERK activity (45-47). To test for
MAPK activation, we used antisera directed against the activated
kinases, which are highly specific for the molecules phosphorylated on
Thr and Tyr in their Thr-Xaa-Tyr activation motif. Western blots of
SaOS-2 whole cell extracts, probed with anti-Thr(P)202-Tyr(P)204 ERK antisera,
revealed that ATP did not activate ERK on its own or together with PTH
(Fig. 8). In contrast, EGF gave rise to a robust activation of ERK, as described previously in osteoblasts (48),
which was partially diminished by coinduction with PTH (Fig. 8).
UMR-106 cells show similar behavior (49), which may represent another
example of antagonism between cAMP-dependent protein kinase
and the ERK cascade apparent in some cell contexts (49-52). Thus, the
synergy between ATP and PTH does not involve the ERK pathway.
We used the same strategy to test whether ATP activated the
stress-responsive MAPKs, namely SAPK/c-Jun N-terminal kinase and p38
MAPK, in these cells. Neither kinase was induced by ATP (Fig. 8),
whereas they were weakly activated by EGF (Fig. 8) and strongly activated by the cellular stresses arsenite and
anisomycin.5
It remained possible that ATP activated a non-MAPK pathway that could
nevertheless target TCF, as has been documented in a mouse macrophage
cell line (53). TCF can be easily visualized in band shift assays,
where it forms a ternary complex on a 32P-labeled SRE probe
together with recombinant SRF deletion mutant that spans the MADS box
and neighboring amino acids (core SRF90-244 in Fig.
9). Phosphorylation of TCF leads to the
slowed mobility of the ternary complexes, as can be readily seen in the
complexes formed by the EGF-treated whole cell extract (Fig. 9).
Antibody supershift experiments show that complex I1 contains the TCF
Elk-1 and that I2 is formed by
SAP-1a.6 Neither ATP, PTH,
nor the two together induced any change in TCF complexes compared with
untreated, control extracts (complex U; Fig. 9). Thus, ATP activates
the SRE through a MAPK- and TCF-independent signaling pathway.
The major findings of this study are as follows: 1) bone-derived
cells, both the osteosarcoma cell line SaOS-2 and primary cultures of
osteoblasts from human bone, express different subtypes of the
purinergic receptor class P2Y; 2) extracellular nucleotides elevate
[Ca2+]i but not [cAMP]i in both cell
types; 3) their response to different nucleotides reflects the receptor
subtype identified by RT-PCR; 4) these receptors functionally couple to intracellular signaling pathways that weakly activate both the endogenous c-fos gene and stably transfected
c-fos promoter-luciferase reporter constructs; 5) the P2Y
signaling pathway combines with one induced by PTH to synergistically
elevate c-fos mRNA levels and luciferase activity; 6)
the P2Y component in this synergy involves the c-fos SRE in
a MAPK- and TCF-independent manner.
We have investigated two different bone cell types in this study. The
immortalized cell line SaOS-2 is derived from a human osteosarcoma and
shows osteoblastic characteristics (54). To confirm that results
obtained from these cells were representative of osteoblasts, we also
used primary cultures of cells derived from human bone explants. These
contain almost exclusively osteoblasts at different states of
differentiation (38), which helps explain the slight differences in P2Y
receptor expression between different cultures and between individual
cells within a culture (55). This variability means that the responses
measured using these cells represent that of a population of cells
rather than a clone.
Purinergic receptors of the P2Y class are strongly implicated in
mediating the response of these cells to NTPs. P2Y receptor expression
has previously been demonstrated in rat osteoblastic cell lines (34,
35) and human bone-derived cells (36, 37). Here we report that, in
SaOS-2 cells, ADP, ATP, and 2-meSATP induced increases in
[Ca2+]i of similar magnitude, data consistent
with expression of the P2Y1 receptor subtype. Only marginal
levels of P2Y2 mRNA could be detected, and accordingly
the P2Y2 receptor agonist UTP was only weakly effective at
elevating [Ca2+]i. In contrast, UTP and ATP
strongly elevated [Ca2+]i in primary osteoblast
population HBDC1, which correlated with high levels of P2Y2
receptor mRNAs. Consistent with the very low levels of
P2Y1 mRNA, these cells showed no response to either ADP
or 2-meSATP, while intermediate levels of P2Y1 receptor
expression by HBDC2 were consistent with the ability of ADP/2-meSATP to
mobilize intracellular calcium in a subpopulation of these cells as
previously reported (55). As indicated above, we attribute this
heterogeneity of receptor expression to the well characterized
differentiation-dependent heterogeneity of osteoblast
phenotype within primary populations cultured from explants of bone.
Notably, nucleotide treatment did not lead to increased
[cAMP]i in either cell type, thus eliminating cAMP as an
intracellular second messenger for P2Y receptors in these osteoblasts.
To assess the biological activity of NTPs in osteoblasts, we have
analyzed the activation of the c-fos proto-oncogene, which is strongly implicated in controlling the proliferation and
differentiation of bone cells (see Introduction). We find that P2
receptor agonists induce c-fos expression in correspondence
with the elevation of [Ca2+]i via specific
receptor subtypes (discussed above). This correlation is upheld in the
primary population of HBDC1; UTP and ATP induced c-fos,
while ADP and 2-meSATP were ineffective. Similarly, in SaOS-2 cells ADP
and ATP also led to increased c-fos mRNA levels, while
UTP did not. However, while 2-meSATP effectively increased
[Ca2+]i in SaOS-2 cells, this agonist failed to
induce c-fos gene expression. This indicates that increased
[Ca2+]i alone is not sufficient for induction of
the c-fos gene in SaOS-2 cells. Many reports have
demonstrated that single G protein-coupled receptor species can be
linked to multiple effector systems (55, 56). Occupation of the
P2Y1 receptor by 2-meSATP could induce a receptor
conformation that activates G protein-dependent hydrolysis
of phosphatidylinositol 4,5-bisphosphate, but not the G protein(s) that
trigger other effector pathways leading to c-fos induction.
Consistent with this notion, others have documented agonist-dependent coupling of G protein-coupled receptors
to various intracellular effectors (57, 58).
The relatively modest increase in c-fos mRNA levels
induced by nucleotides contrasted with the robust increase generated by PTH treatment. Even more striking was the synergistic effect on c-fos by the combination of NTPs and PTH, which resulted
from the activation of two distinct signaling pathways. While NTPs lead
to increased [Ca2+]i without affecting cAMP, PTH
does the opposite, namely elevating the levels of cAMP,
cAMP-dependent protein kinase activity, and CREB
phosphorylation independently of increased Ca2+ (25, 59,
60). CREB phosphorylation alone might suffice to explain the
synergistic induction of c-fos, since its major binding site
in the fos promoter, the CaCRE, can integrate both cAMP and
Ca2+ signals in transfection assays (61). However, the
assays using stably transfected reporter genes suggest that the
NTP-Ca2+ and PTH-cAMP pathways do not converge on the same
promoter element. The CaCRE reporter was strongly induced by PTH. This
induction was not significantly increased by including promoter
sequences out to position The SRE is bound and activated in vitro by a ternary
complex, containing SRF and TCF, that reproduces the pattern observed in genomic footprints (6, 7, 11). Transactivation by TCF is strongly
potentiated through its phosphorylation by the MAPKs, and the MAPK ERK
can be activated by P2Y receptor agonists, as well as other
Ca2+ signals, in different cell types (29, 66-70).
Surprisingly, band shift and Western blotting show that ATP treatment
of SaOS-2 cells does not lead to activation of any MAPK pathway or to
detectable levels of TCF phosphorylation, thus ruling out a
contribution by this signaling module (Figs. 8 and 9). The SRF·SRE
complex can also mediate the calcium-driven activation of transiently introduced reporter genes, either alone (12, 13) or together with the
FAP element located immediately downstream of the SRE (64, 65). Taken
together, these observations suggest that synergy represents the
combined effect of the NTP-Ca2+ pathway targeting the SRE
via SRF independently of TCF and the PTH-cAMP pathway targeting CREB.
Notably, a similar synergism between CREB and SRF has been proposed to
mediate c-fos induction by neurotrophins in neuronal cells
(18). It will be interesting to determine the mechanism behind this
effect and whether the MAPK/TCF signaling module can also contribute to
it. Considering the strong effect on the endogenous c-fos
gene, it is also possible that other regulatory elements, located
intragenically, have a role in this synergy (65, 71).
The fact that NTPs and PTH cooperated in both osteoblast-like
osteosarcoma cell lines and primary osteoblasts suggests that this
phenomenon is physiologically relevant. PTH is a systemic hormone that
functions as a principal regulator of mineral homeostasis that affects
primarily the skeletal system and kidney. In bone, the PTH-responsive
cells are osteoblasts (72), which, following activation by this
hormone, produce factors that drive osteoclastic differentiation and
subsequent bone resorption (73). These data suggest that NTPs amplify
the responsiveness of bone cells to PTH, thus providing a means of
mounting a localized response to a systemic factor like PTH. This seems
reasonable, since ATP can exist transiently in the extracellular
environment following both cell damage and release via a regulatory
nonlytic mechanism and, furthermore, can act synergistically with
factors to enhance DNA synthesis and promote cell proliferation in
Swiss 3T3 and 3T6 fibroblasts, A431 cells (27), porcine aortic smooth
muscle cells (28), and thyroid cells (29). Thus, the localized release of nucleotides as a result of different physiological stresses, along
with the differential expression of P2 receptors, provides the means
for a highly targeted response in vivo to ubiquitous extracellular factors like PTH. This raises the possibility that this
type of signal integration represents a universal means for generating
a selective response in many in vitro and in vivo situations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
300 in the promoter, mediates induction by many
extracellular signals via a ternary complex composed of a dimer of
serum response factor (SRF) together with one molecule of the TCF
family of Ets proteins (Elk-1, SAP-1a, or ERP/NET/SAP-2 (6-10)). TCFs,
particularly Elk-1 and SAP-1a, are important nuclear targets of various
MAPK cascades (11), while SRF is apparently sufficient for activation
by certain Ca2+ signals and signals emanating from the
Rho/Rac/CDC42 family of small GTPases (12-14). Upstream of the SRE is
the v-sis-inducible element, the binding site for homo- and
heterodimers of signal transducer and activator of transcription 1 and
3 upon their cytoplasmic activation by cytokines and certain growth
factors (15, 16). While transient transfections have proven useful in
attributing a role to each element, the results from mice containing
c-fos transgenes (17) and more recent data in
vitro indicate that multiple elements are necessary for a strong
response (18),2 thereby
implying that they are targeted simultaneously by intracellular signals.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified
Eagle's medium, Ham's F-12, and RPMI 1640 were obtained from Flow
Laboratories (United Kingdom). Fetal calf serum (FCS) was purchased
from Life Technologies Ltd. and fura-2 acetoxymethyl ester was from
Molecular Probes, Inc. (Eugene, OR). dNTPs, oligo(dT), RNase inhibitor, and some restriction enzymes were from Roche Molecular Biochemicals, while Taq DNA polymerase and Superscript 2 reverse
transcriptase were from Life Technologies, Inc. NTPs and poly(dI-dC)
were obtained from Amersham Pharmacia Biotech, while EGF came from
Upstate Biotechnology, Inc. (Lake Placid, NY). Zetabind hybridization
membrane was purchased from Cuno (Meriden, CT). Human parathyroid
hormone (PTH)-(1-34) was purchased from Peninsula Laboratories.
Nucleotides, bovine serum albumin (BSA), EGF, and peroxidase-coupled
goat anti-rabbit antibodies were obtained from Sigma. Luciferase lysis
reagent (25 mM Tris-phosphate, pH 7.8, 2 mM
dithiothreitol, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100) and luciferase reagent were
purchased from Promega. Phospho-MAPK-specific antisera and NarI were obtained from New England Biolabs, and
anti-pan-ERK serum was purchased from Transduction Laboratories.
Peroxidase-coupled donkey anti-rabbit antibody and enhanced
chemiluminescence reagents were acquired from Amersham Pharmacia
Biotech. 32P nucleotides and Renaissance chemiluminescence
reagents were purchased from NEN Life Science Products. Polyvinylidene
difluoride membrane was purchased from Millipore Corp.
-modified
Eagle's medium supplemented as above, and seeded onto glass coverslips or harvested upon confluence.
721 to
1, accession number M16287)
linked to the luciferase gene was subcloned from pUC19fosluc1 (kindly
provided by Dr. L. Runkel) into pSV2neo, to create pfoslucneo1. A
multimerized SRE-luciferase reporter gene vector was obtained by
ligating the oligonucleotide CCGCAGGATGTCCATATTAGGACATCTGTGTGCCGTCCTACAGGTATAATCCTGTAGACACGCCGG that was used to replace the promoter upstream of the
c-fos TATA box in the same vector. Clones containing three
copies of the SRE were identified and verified by sequencing. The
vector containing a single copy of the c-fos CaCRE was
generated by deleting sequences upstream of position
88 in the
fos promoter in pfoslucneo1. Its identity was verified by sequencing.
20 °C. Prior to first strand cDNA synthesis, RNA was
DNase-treated with RNase-free DNase I (35 units/ml). 5 µg of
DNase-treated total RNA was used as template for first strand cDNA
synthesis in a 50-µl reaction containing 0.5 mM dNTPs,
1.25 µg of oligo(dT), 20 units of RNase inhibitor, 10 mM
dithiothreitol, 6 mM MgCl2, 40 mM
KCl, 50 mM Tris-HCl, pH 8.3, and 1000 units of Moloney
murine leukemia virus reverse transcriptase. After 1 h at
37 °C, the reaction was frozen at
20 °C.
-32P]dCTP (3000 Ci/mmol) by random priming. Membranes
were washed for 30 min at 65 °C with 0.2× SSC, 1% SDS solution,
and mRNAs were visualized using phosphor storage technology and
autoradiography with Kodak XAR film and intensifying screens at
70 °C. RNA loading and integrity were followed by ethidium bromide
staining or hybridization with a random primed fragment purified from a
cDNA clone containing rat GAPDH. The blot in Fig. 6 was hybridized
with c-fos and GAPDH riboprobes in the same mix as described
previously (42).
20 °C until cAMP levels were
determined using a radioimmunoassay (43). The statistical significance
was assessed by a one-way analysis of variance followed by the
Tukey-Kramer multiple comparisons post-test.
-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 was completed by
vortexing for 45 s. The lysate was clarified by centrifugation at
10,000 × g at 4 °C for 30 min, and the supernatant
was stored at
70 °C.
-32P]dATP and
cold dCTP, dGTP, and dTTP. Fragments were isolated from polyacrylamide
gels by electroelution.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Differential P2 receptor expression by SaOS-2
and primary human bone-derived cells. PCR amplification of
cDNAs with P2Y1, P2Y2, or GAPDH primer
pairs. cDNAs were synthesized from RNA templates prepared from
SaOS-2 cells, two different primary osteoblast populations (HBDC1 and
-2), and an osteoclastoma tumor. The tumor expresses both receptor
subtypes and thus serves as the positive control (37). As indicated on
the left, the bands of 259 and 362 base pairs
(bp) correspond to amplification products specific for
P2Y1 and P2Y2 receptor cDNAs, respectively,
and GAPDH yields a larger 519-base pair band. The control reaction
contained H2O instead of template cDNA, and the
unmarked lanes contain molecular size
markers.
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Fig. 2.
Nucleotide-induced increases in
[Ca2+]i in SaOS-2 cells. Groups of
fura-2-loaded SaOS-2 cells (6-8 per point) were sequentially
stimulated with increasing concentrations of ATP (A) and UTP
(B) for the times indicated on the x axis scale.
The curves plot intracellular calcium concentration in nM,
as indicated on the y axis. The values are representative of
the following numbers of measurements. A, 1 µM, n = 4; 10 µM,
n = 16; 100 µM, n = 3. B, n = 3.
[Ca2+]i elevation in SaOS-2 and human bone-derived
cells in response to P2 receptor agonists
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Fig. 3.
Nucleotide-induced increase in
[Ca2+]i in primary human bone-derived cells.
Primary cultures of human osteoblasts (HBDC1 cells, 6-8 per point)
were loaded with fura-2 and stimulated sequentially with the different
P2Y agonists (10 µM) indicated at the top.
Table I presents the statistics concerning these measurements.
S and 2-meSATP, as well as ADP
(all 10 µM), induced [Ca2+]i
increases comparable with ATP (Table I). The primary cells did not show
the same behavior, since these P2Y1 agonists did not
elevate [Ca2+]i in HBDC1 (Fig. 3). These
differences in the functional response to extracellular nucleotides are
consistent with and thereby confirm the receptor profiles obtained by
RT-PCR (Fig. 1).
S stimulation of quiescent SaOS-2 cells led to a
dose-dependent induction of c-fos mRNA,
measured by Northern blotting (Fig.
4A), which was maximal at 10 µM ATP
S. This represented a typical transient
activation in which fos mRNA levels peaked 45 min after
stimulation and then rapidly decayed (Fig. 4B). In addition,
we tested the same range of P2Y receptor agonists used to analyze
Ca2+ mobilization. 10 µM ATP
S, ATP, and
ADP stimulated c-fos expression, whereas UTP and 2-meSATP
did not (Fig. 4C). While this generally reflects the
activation of the P2Y1 receptor, this correlation is not
universal, as shown by the lack of induction by 2-meSATP and the
decrease between 10 and 100 µM ATP
S. This was not due to differing levels of RNA, since rehybridization of the blots with a
GAPDH probe and/or ethidium bromide staining confirmed equal loading of
mRNA in all lanes. This apparent discrepancy between increased
[Ca2+]i and c-fos induction will be
discussed in more detail below.
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Fig. 4.
Induction of c-fos mRNA
in SaOS-2 cells following nucleotide stimulation. A,
SaOS-2 cells were induced for 45 min with ATP as indicated. RNAs were
isolated and purified using the guanidine one-step procedure and
visualized by hybridizing Northern blots containing 10 µg of total
RNA/lane with a 32P c-fos probe
labeled by random priming (top). Equal RNA loading and
integrity were confirmed by ethidium bromide staining
(bottom). Numerical values for the c-fos
mRNA:28 S ribosomal RNA ratio are as follows: 10 7
M ATP
S, 0.195; 10
6 M ATP
S,
2.50; 10
5 M ATP
S, 11.59; 10
4
M ATP
S, 3.10. B, kinetics of c-fos
mRNA induction in response to 10 µM ATP
S. Northern
blots were hybridized as above, and the maximal induction level, which
occurred 45 min postinduction, was taken as 100%. Numerical values for
the c-fos mRNA:28 S ribosomal RNA ratio are as follows:
30 min, 0.23; 45 min, 0.41; 60 min, 0.23. C, SaOS-2 cells
were stimulated for 45 min with a range of nucleotide agonists at 10 µM concentration, and c-fos mRNA induction
was analyzed as described above (top). The membrane was
stripped and rehybridized with a GAPDH probe 32P-labeled by
random priming to confirm equal loading and mRNA integrity
(bottom). Numerical values for the c-fos:GAPDH
mRNA ratio are as follows: 10% FCS, 4.58; ADP, 0.19; ATP
S, 0.1;
ATP, 0.134. Medium, UTP, and 2-meSATP did not induce c-fos
expression.
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Fig. 5.
Nucleotides synergize with PTH to induce
c-fos expression in SaOS-2 cells. A,
SaOS-2 cells were stimulated with PTH (100 ng/ml) and nucleotide
agonists (10 µM) for 45 min. RNAs were analyzed by
Northern blotting (top) and ethidium staining
(bottom) as described in the legend to Fig. 4. The
c-fos signal in each lane was standardized to 28 S ribosomal RNA, and the level of induction relative to control was
expressed numerically. B, SaOS-2 cells stably transfected
with the fos ( 711/
1) promoter-luciferase reporter were
serum-starved and then induced for 4 h with PTH (100 ng/ml), ATP
(10 µM), UTP (10 µM), or the combination of
PTH and NTP. Luciferase activity was measured as described under
"Experimental Procedures." The data represent the average of three
measurements. Error bars indicate the S.D.
711 to
1, was linked to the firefly luciferase gene. To avoid the problems inherent in
assaying signaling by transient transfection of nonphysiological quantities of DNA, we selected pools of stably transfected cells using
the neomycin resistance gene present in the same construct. Both serum
and PTH induced a robust response of this reporter construct, elevating
luciferase activity 15-20-fold in the stable transfectants (Fig.
5B). ATP showed only a weak effect on its own, consistently
severalfold above the background level. However, the combination of ATP
and PTH cooperatively induced the c-fos reporter, which is
in direct contrast to the failure of UTP to significantly augment
luciferase activity in combination with PTH (Fig. 5B). Thus,
we are able to reproduce the synergy between ATP and PTH using the
c-fos promoter in stable transfection assays, which
indicates that this effect arises primarily from increased transcriptional activation and not from stabilization of the
fos mRNA. Furthermore, it suggests that ATP and PTH
activate intracellular signals that target regulatory elements in the
c-fos promoter.
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Fig. 6.
Synergy between nucleotides and PTH in
c-fos induction in primary human osteoblasts.
Populations of primary human bone-derived cells were serum-starved and
then stimulated for 45 min with 10 µM ATP, ADP, UTP,
and/or 100 ng/ml PTH. 5 µg of total RNA were analyzed by Northern
blotting and hybridization with c-fos, and 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 was expressed numerically.
S, UTP, or ADP had no significant effect on cAMP levels either
alone (Fig. 7A) or in
combination with PTH (Fig. 7B).
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Fig. 7.
Nucleotides do not elevate intracellular cAMP
levels in SaOS-2 cells. A, the effect of nucleotide
agonist stimulation on intracellular cAMP levels. SaOS-2 cells were
starved and then stimulated with 10 µg/ml forskolin or 10 µM nucleotides as indicated. After 20 min, cells were
treated, and cAMP levels were determined by radioimmunoassay.
B, nucleotides fail to inhibit PTH-induced cAMP
accumulation. SaOS-2 cells were starved and then stimulated with 20 ng/ml PTH-(1-34) alone or together with the nucleotide agonists
indicated (10 µM). All data are represented as mean ± S.E. (n = 6). An asterisk denotes
significance at p < 0.001.
ATP and PTH activation of truncated c-fos elements driving
luciferase expression
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Fig. 8.
ATP does not stimulate ERK, SAPK, or p38 MAPK
phosphorylation in SaOS-2 cells. SaOS-2 cells rendered quiescent
by serum deprivation were stimulated with 10 µM ATP S,
50 ng/ml EGF, and 100 ng/ml PTH alone or in combination as indicated
above each lane. After 10 min, whole cell
extracts were prepared, and 15 µg of protein was loaded per
lane on 8.5% SDS-polyacrylamide gel electrophoresis
minigels, run in triplicate. After transfer to polyvinylidene
difluoride, the blots were immunodetected with the following antisera:
anti-phospho-Thr202/Tyr204ERK1/2,
anti-phospho-Thr183/Tyr185SAPK, and
anti-phospho-Thr180/Tyr182p38, followed by
peroxidase-coupled anti-rabbit antiserum and detection using enhanced
chemiluminescence. The blots were stripped and reprobed with
anti-pan-ERK, anti-SAPK, and anti-p38 MAPK antiserum to confirm the
presence of similar levels of the kinases in each
lane.7
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Fig. 9.
TCF phosphorylation is not induced by ATP
stimulation in SaOS-2 cells. The SaOS-2 whole cell extracts
described in the legend of Fig. 8 cells were analyzed for TCF
phosphorylation by band shift assay. Extracts were prepared from
uninduced cells (vehicle) or cells induced with 10 µM
ATP S, 50 ng/ml EGF, 100 ng/ml PTH, or ATP
S and PTH. In addition
to 10 µg of whole cell extract, binding reactions also contained
32P-labeled c-fos SRE and a SRF deletion
mutation, core SRF90-244. Core SRF readily allows
visualization of induced related changes in TCF independently of other
cellular proteins. After a 30-min incubation, the complexes were
resolved by prolonged electrophoresis on 5% polyacrylamide gels. After
drying, the complexes were visualized by autoradiography.
Phosphorylation of TCF changes the mobility of its ternary complexes
with core SRF and the SRE from the uninduced position (U) to
the slowed, induced positions (I1 and I2). This
mobility change reflects hyperphosphorylation of TCF resulting from
activation of the ERK signaling cascade (42). Complex I1 contains
hyperphosphorylated Elk-1, while complex I2 contains predominantly
hyperphosphorylated SAP-1a (53).6
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
711, ruling out the contribution of other
cryptic cAMP response elements active in transient transfection
analyses (3, 4). On the other hand, the full promoter mediated the synergy between NTPs and PTH. NTPs alone consistently gave a
severalfold induction of the fos
711-linked reporter gene
and background levels with the CaCRE. There are several possible
explanations for this observation. One is that the NTP-Ca2+
pathway weakly targets the CREB/activating transcription factor complexes bound to cryptic cAMP response elements located elsewhere in
the c-fos promoter. This seemed unlikely, since NTPs
activated neither the CaCRE reporter gene nor one driven by an array of six cAMP response elements.7
The other is that synergism arises from the effects of the
NTP-Ca2+ pathway targeting another promoter element, either
the SRE or the v-sis-inducible element. Ca2+
signals can selectively target the SRE in neuronal (62, 63), mesangial
(64), and T cells (65). Accordingly, we observed that NTPs induced
luciferase expression from a SRE-driven reporter gene stably
transfected into bone cells.
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ACKNOWLEDGEMENT |
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We thank Professor P. Cobbold for access to equipment and helpful discussion of the manuscript.
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FOOTNOTES |
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* 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.
§ Supported by the Arthritis and Rheumatism Research Council. To whom correspondence should be addressed: Tel.: 44-151-794-5505; Fax: 44-151-794-5517; E-mail: wbb{at}liv.ac.uk.
Supported by the Wellcome Trust.
¶¶ A researcher of the CNRS. Supported by the Fondation pour la Recherche Medicale, Association pour la Recherche sur le Cancer, and Novartis Inc.
2 M. Bebien, C. Becamel, V. Richard, and R. A. Hipskind, manuscript in preparation; R. A. Hipskind, C. Halleux, S. Decker, M. Bebien, D. B. Evans, and G. Bilbe, manuscript in preparation.
3 C. J. Dixon and W. B. Bowler, unpublished observations.
4 C. Halleux and G. Bilbe, unpublished observations.
5 W. Bowler and R. A. Hipskind, unpublished observations.
6 R. A. Hipskind, unpublished observations.
7 W. Bowler, V. Richard, and R. A. Hipskind, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
CaCRE, calcium and
cAMP response element;
SRE, serum response element;
PCR, polymerase
chain reaction;
RT-PCR, reverse transcript PCR;
MAPK, mitogen-activated
protein kinase;
ERK, extracellular signal-regulated kinase;
SAPK, stress-activated protein kinase;
ATPS, adenosine
5'-O-(3-thiotriphosphate);
2-meSATP, 2-methylthioadenosine
5'-triphosphate;
PTH, parathyroid hormone;
BSA, bovine serum albumin;
FCS, fetal calf serum;
CREB, cAMP response element-binding protein;
SRF, serum response factor;
TCF, ternary complex factor;
HBDC, human
bone-derived cell(s);
SSC, saline-sodium citrate;
EGF, epidermal growth
factor;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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