(Received for publication, February 12, 1997, and in revised form, May 7, 1997)
From the ¶ Department of Medicine (Arthritis), Calcium deposition diseases caused by
calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate
(BCP) crystals are a significant source of morbidity in the elderly. We
have shown previously that both types of crystals can induce
mitogenesis, as well as metalloproteinase synthesis and secretion by
fibroblasts and chondrocytes. These responses may promote
degradation of articular tissues. We have also shown previously that
both CPPD and BCP crystals activate expression of the c-fos
and c-jun proto-oncogenes. Phosphocitrate (PC) can
specifically block mitogenesis and proto-oncogene expression induced by
either BCP or CPPD crystals in 3T3 cells and human fibroblasts,
suggesting that PC may be an effective therapy for calcium deposition
diseases. To understand how PC inhibits BCP and CPPD-mediated cellular
effects, we have investigated the mechanism by which BCP and CPPD
transduce signals to the nucleus. Here we demonstrate that BCP and CPPD
crystals activate a protein kinase signal transduction pathway
involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear
transcription factor, cyclic AMP response element-binding protein
(CREB), on serine 133, a residue essential for CREB's ability to
transactivate. Treatment of cells with PC at concentrations of
10 Crystalline calcium pyrophosphate dihydrate
(CPPD)1 and basic calcium phosphate (BCP; a
term including carbonate-substitute apatite, octacalcium phosphate, and
tricalcium phosphate) are the two commonest forms of pathologic
articular mineral. Each occurs frequently in degenerative joints, and
each may be phlogistic, causing acute attacks of pseudogout in the case
of CPPD crystals and acute calcific periarthritis in the case of BCP
crystals (1, 2).
Calcium-containing crystals such as BCP and CPPD in concentrations
found in pathologic human joint fluids, stimulate fibroblast, synovial
cell, and chondrocyte mitogenesis in vitro by a process similar to that of platelet-derived growth factor (PDGF) (3). Moreover,
BCP crystals and PDGF exert similar biologic effects on cultured cells,
such as stimulation of PGE2 production via the
phospholipase A2/cyclo-oxygenase pathway (4), activation of
phospholipase C and inositol phospholipid hydrolysis (5), induction of
collagenase and neutral protease synthesis (6-8), and induction of
proto-oncogenes (c-fos and c-myc) (9, 10).
Based on clinical and synovial fluid findings and in vitro
data (11), a hypothesis has been formulated concerning the pathogenesis of calcium-containing crystal deposition diseases. Synovial lining cells phagocytose crystals in the joint fluid. In the process and/or as
a result of endocytosis, synovial cells respond with: 1) protease
synthesis and secretion, which in turn releases additional crystals and
collagen from the surrounding tissue, 2) PGE2 production, 3) DNA synthesis as a result of protein kinase C activation and crystal
dissolution. Proteases and PGE2 cause the
"degeneration" of the periarticular tissue. DNA synthesis leads to
an increase in synovial cells that generate more proteases and
PGE2 (12).
Members of the mitogen-activated protein kinase (MAPK) family are key
regulators of a variety of signal transduction cascades that play a
central role of mediating cellular responses elicited by many different
environmental agents. Three distinct MAPK-dependent signaling cascades have been identified in mammalian cells. These can
be distinguished based on the particular MAPK members activated: p42/p44 MAPKs, p38 MAPK, or p46/p54 stress-activated protein
kinase/c-Jun N-terminal kinases. p38 MAPK and the stress-activated
protein kinase/c-Jun N-terminal kinases mediate signals in response to cytokines and environmental stress. The p42/p44 MAPK pathway was the
first identified and is the best understood ERK-based signaling pathway
(reviewed in Ref. 13). This pathway is required for cell proliferation
elicited by growth factors and Ras transformation of cells. The p42/p44
MAPKs are believed to regulate proliferation by a mechanism that
involves activation of genes associated with cell proliferation,
including primary response genes such as c-fos and
c-jun. These kinases can also phosphorylate other kinases, such as the RSK family, thereby regulating their action. In the case of
c-fos gene, p42/p44 MAPKs have been shown to activate expression by phosphorylating members of the Elk-1 family of
transcription factors (reviewed in Ref. 14). The Elk-1 factors bind to
the c-fos serum response element by virtue of their
association with serum response factor (SRF), which directly interacts
with the serum response element (14).
PC is a naturally occurring compound which has been identified in
mammalian mitochondria (15) and crab hepatopancreas (16). It has been
speculated that PC may have an important role in preventing calcium
phosphate precipitation in cells or cellular compartments maintaining
high concentration of calcium and phosphate (15). PC prevents soft
tissue calcification in vivo and does not produce any
significant toxic side effect in rats or mice when given in doses up to
150 µmol/kg/day (17, 18).
Our earlier studies have shown that PC specifically inhibits CPPD and
BCP crystal-induced proto-oncogene (c-fos and
c-jun) expression, metalloproteinase synthesis, and
mitogenesis in human fibroblasts in vitro, while PC has no
effect on similar biologic responses induced by EGF, PDGF, and serum
(19).
In the present study, we examine whether BCP and CPPD crystals activate
cells through the protein kinase signal transduction pathway involving
p42/p44 (ERK 2 and ERK 1) or p38 MAP kinases. We also investigated
whether BCP and CPPD crystals could activate the CREB transcription
factor. CREB is important for mediating gene activation in response to
calcium signals, and it has recently been demonstrated that it can be
activated by a p42/p44 MAPK-dependent pathway (27). The
effects of PC, n-sulfo-2-aminotricarballylate (SAT, a PC
analogue) and citrate (Fig. 1) on this signal
transduction pathway were also examined.
Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced
salt solution without calcium and magnesium (HBSS), fetal bovine serum
(FBS), Pen-Strep-Fungizone, and HEPES buffer were obtained from Life
Technologies, Inc. Human IL-1 PC was prepared as the sodium salt by phosphorylation of tribenyl
citrate followed by deprotection through hydrogenation. The monosodium
salt was crystallized from water (20). SAT was synthesized according to
the procedure of Brown et al. (18).
BCP crystals were prepared by alkaline hydrolysis of brushite using a
modification of the method of Bett et al. (21). These crystals have a calcium/phosphate ratio of 1.59 and contained partially
carbonate-substituted hydroxyapatite mixed with octacalcium phosphate
by FTIR spectroscopy. Crystals were crushed in an agate mortar and
sieved to yield 10-20-µm aggregates, which were sterilized and
rendered pyrogen-free by heating at 200 °C for 90 min. CPPD crystals
were made by methods published earlier (22) and sieved, sterilized, and
treated in a similar fashion as BCP crystals.
Human foreskin fibroblast cultures were established from explants and
transferred. They were grown and maintained in DMEM supplemented with
10% FBS containing penicillin and streptomycin. All cultures used were
third- or fourth-passage cells. Experiments were performed on confluent
cell monolayers that had been rendered quiescent by removing the
medium, washing with DMEM containing 0.5% FBS, and subsequently
incubating in this medium for 24 h. PC, SAT, or citrate was added
to the medium 30 min before stimulation at the desired concentration.
Cells were stimulated with BCP (50 µm/ml), IL-1 Cells were grown to confluence in culture dishes containing 24 wells,
each 16-mm in diameter (Multiwell, Life Technologies, Inc.) and
rendered quiescent by incubating in 0.5% FBS for 24 h. Quiescent
cells were then stimulated with 10% FBS, BCP crystals, or CPPD
crystals in the presence and absence of inhibitors (PC, SAT, and
citrate) in DMEM, 0.5% FBS. [3H]Thymidine (1 µCi/ml)
was added to the wells 23 h after stimulation and pulsed for
1 h. The cells were washed three times with phosphate-buffered saline, and macromolecules were precipitated with 5% trichloroacetic acid solution. The precipitate was washed again with phosphate-buffered saline and dissolved in 1 ml of 0.1 N NaOH, 0.1% sodium
dodecyl sulfate. Trichloroacetic acid-precipitable radioactivity was
determined in a liquid scintillation counter (Packard Instrument,
Downer's Grove, IL). In previous experiments, the optimal
concentrations to induce thymidine incorporation in human foreskin
fibroblasts were BCP crystals (100 µg/ml) and CPPD crystal (200 µg/ml) (12). Unless otherwise specified, these were the
concentrations of BCP and CPPD crystals used in all experiments.
To elucidate a pathway by which BCP elicits its
many biological effects, the effect of these crystals on p42/p44 MAPK
activity was examined. By measuring the levels of phosphorylated
p42/p44 MAPK, the degree of activation was determined. After 15-min
stimulation, serum, IL-1, and BCP induce phosphorylation of p42/p44
MAPK significantly over basal levels (Fig. 2a, lanes 2, 4,
and 6). These results show that BCP
stimulates the p42/p44 MAPK pathway as strongly as serum. IL-1 also
induced phosphorylation of p42/p44 MAPK (Fig. 2a, lane 4),
which suggests that this pathway is not always specific to mitogenic
signals. The presence of 10
To clarify the
time dependence of p42/p44 MAPK phosphorylation by BCP, a time course
study was carried out (Fig. 3). After an initial 5 min
delay, the levels of P-p42/p44 MAPK induced by BCP increase quickly and
by 15 min reach a maximum level. Surprisingly, IL-1
The p38 MAPK
pathway, associated with stress and cytokine responses (23), was
examined to further characterize cellular responses to BCP. The time
course of p38 MAPK phosphorylation was studied. BCP did not
significantly induce phosphorylation of p38 MAPK (Fig.
4). IL-1, on the other hand, causes a transient increase
in P-p38 MAPK levels that begins within 5 min of stimulation and ends
by 30 min (Fig. 4). Serum does not cause any increase in P-p38 MAPK
levels. The IL-1-induced response is much stronger than the responses
elicited by either serum or BCP.
CPPD was studied to see whether it also acted
through the p42/p44 MAPK pathway. A time course study of P-p42/p44 MAPK
levels shows that, like BCP, CPPD also induces phosphorylation of
p42/p44 MAPK (Fig. 5a, lanes 2-4). Unlike
BCP, there is no initial 5-min delay before phosphorylation of p42/p44
MAPK. By 5 min after stimulation, P-p42/p44 MAPK levels reach a maximal
level, which remains sustained throughout the time course. As with BCP,
incubation with 10
The p38 MAPK pathway is not activated by CPPD crystals (Fig.
5b). Even at 30 min after stimulation, P-p38 MAPK levels are not elevated over basal levels. The upper band in Fig. 3b is
a cross-reactive band which is not relevant to p38 MAPK activation. Overall, CPPD has a very similar effect to BCP on the p42/p44 and p38
MAPK pathways.
To
ascertain what concentration of PC is needed to block BCP-induced
p42/p44 MAPK phosphorylation, a dose-response study was carried out
(Fig. 6). Using PC concentrations ranging from
10
A dose-response study of SAT and citrate was also carried out (Fig.
7). While 10
Both BCP and CPPD have been shown to exert
their effects at least in part by a mechanism that involves an influx
of extracellular calcium.2 Since the CREB
transcription factor has been shown to be an important regulator of
gene expression in response to calcium signals, we investigated the
activation of CREB by treatment of cells with BCP or CPPD crystals
using a phosphoserine 133-specific antibody. Serum, CPPD, and BCP all
induced CREB serine 133 phosphorylation (Fig. 8).
However, serum and CPPD crystal induced maximal phosphorylation of CREB
serine 133 within 15 min, while there appeared to be a delay of 15 min
in the BCP crystal induction.
Effects of PC, SAT, and citrate on BCP crystal-induced and IL-1 induced
thymidine incorporation are summarized in Fig. 9, a and
b, respectively. As reported before, PC
(10
The observation that BCP and CPPD can activate p42/p44
MAPK suggested that crystal-induced biological effects may be mediated by a MAPK pathway. To begin to address this issue the effect of a
selective inhibitor of the MAP kinase cascade, PD98059, on
crystal-induced p42/p44 MAPK activities and [3H]thymidine
incorporation was studied. As can be seen in Fig. 10, a and
b, PD98059 blocked BCP and CPPD
crystal-induced p42/p44 MAPK activities and thymidine incorporation in
a dose-dependent fashion. These results indicate that the
BCP and CPPD crystal-induced cell activation is mediated at least in
part by a MAPK-dependent pathway.
In the present study, we have demonstrated that BCP and CPPD
crystals differentially activate members of the MAPK signal
transduction cascade. Calcium-containing crystals activate the MAPK
p42/p44 and not the p38 protein kinase cascade pathway, and induction of p42/p44 MAPKs by BCP crystals occurs with a 5-min delay relative to
serum and IL-1 The induction of c-fos and c-jun transcription
are among the earliest responses to serum and growth factors in culture
cells (28, 29). The Fos protein, in combination with the protein products of c-jun, can serve as a trans-acting activator of
the expression of other genes such as collagenase and stromelysin (28-31). McDonnell et al. (29) reported that EGF
stimulation of stromelysin required induction of c-fos,
c-jun, and activation of PKC. Since BCP crystal-induced
collagenase and stromelysin synthesis was preceded by a transient
increase in the level of intracellular Ca2+, PKC
activation, and c-fos, c-myc, c-jun
induction (12), it is likely that these same factors are required for
crystal-induced metalloproteinase synthesis and that BCP may be
mediating its tissue destructive effects as a result of induction of
the c-fos gene.
The RAS/p42/p44 MAPK pathway mediates activation of the
c-fos gene in response to a variety of mitogenic agents,
including serum and purified growth factors such as EGF.
Transcriptional activation of the gene is a synergistic process in
which multiple c-fos promoter elements are targeted to
effect maximal activation. In one case, activated p42/p44 MAPKs
directly phosphorylate members of the Elk-1 family of SRF-associated
transcription factors, thereby stimulating transcription in an
SRF-dependent manner (reviewed in Ref. 14). In addition,
p42/p44 MAPK can also activate RSK family members, which can
phosphorylate CREB Ser133 (27). This
RSK-dependent pathway has been shown to also contribute to
activation of the c-fos gene in response to treatment of
cells by purified growth factors (27). In the case of crystal-induced c-fos expression, we have shown that both SRF and CREB
binding sites in the c-fos promoter are necessary for a
maximal response.3 Whether Elk-1 related
factors contribute to the response remains to be determined.
In this study, we have also shown that PC (10 PC restricts transformations involved in the nucleation, growth, and
aggregation of many calcium salts, including phosphate, oxalate, and
carbonate (32, 33). The strong binding affinity that PC possesses for
growing crystals accounts for the superior inhibitory capacity compared
with citrate and SAT and is believed to result from both its
multinegative charges (Fig. 1) and natural stereochemistry (34, 35).
Recent insights into the mechanism of PC action have been gained by
exploring its interaction with the crystal faces of calcium oxalate
monohydrate crystal using both experimental evidence and computer
modeling (32). PC with both its PO4 and carboxylate groups
contributing has been shown to bind more favorably than citrate
(possessing carboxylates only] with calcium ions distributed on the
( Our working hypothesis of the mechanism of action of PC on crystal is
that the binding of PC to calcium-containing crystals change the Our earlier studies had shown that PC specifically inhibits CPPD and
BCP crystal-induced metalloproteinase synthesis and mitogenesis in
human fibroblasts in vitro, while PC has no effect on
similar biologic responses induced by EGF, PDGF, and serum (19). PC (10-1000 µM) blocked both the BCP and CPPD crystal
formation in a dose-dependent fashion in both articular
cartilage vesicle and articular cartilage models, while SAT and citrate
(1 mM) blocked only BCP crystal formation in similar models
(23, 36). PC prevents disease progression in murine progressive
ankylosis (an animal model of crystal deposition diseases), a condition
marked by extensive BCP deposition (24).2 Taken together
with the present data, PC may be considered a potential therapeutic
agent for both CPPD and BCP crystal deposition diseases. Our working
hypothesis is that PC will have dual beneficial effects of blocking the
degeneration-promoting effects of crystals, e.g.
metalloproteinase synthesis and mitogenesis (12), and of inhibiting
further BCP and CPPD crystal formation in articular tissue (23).
We acknowledge the expert technical
assistance of Elizebeth McDonough and Michele Brogley.
Geriatric Research,
Department of Biochemistry, Medical
College of Wisconsin, Milwaukee, Wisconsin 53226, and the
§ Department of Biochemistry, University of Tasmania,
Hobart, Tasmania 7001, Australia
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3 to 10
5 M blocked both the
activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation,
in a dose-dependent fashion. At 10
3
M, a PC analogue,
n-sulfo-2-aminotricarballylate and citrate also modulate
this signal transduction pathway. Inhibition by PC is specific for BCP-
and CPPD-mediated signaling, since all three compounds had no effect on
serum-induced p42/P44 or interleukin-1
induced p38 MAP kinase
activities. Treatment of cells with an inhibitor of MEK1, an upstream
activator of MAPKs, significantly inhibited crystal-induced cell
proliferation, suggesting that the MAPK pathway is a significant
mediator of crystal-induced signals.
Fig. 1.
The formulae of phosphocitrate, citrate and
SAT at physiological pH (7.4).
[View Larger Version of this Image (10K GIF file)]
was obtained from Sigma (I-4019).
Phospho-specific antibodies for p42/p44 MAPKs, p38 MAPK, and a
chemiluminescent Western detection system were purchased from New
England Biolabs (Beverly, MA). The MEK1 inhibitor PD98059 was also
purchased from New England Biolabs.
(10 ng/ml), or
20% serum for 15 min except in the time course studies. Cells were
lysed with SDS sample buffer, heated to 100 °C for 5 min, and
briefly centrifuged. Samples were then stored at
20 °C. To detect
activated forms of p44/42 MAPK, antibodies that specifically recognize
p44/42 MAPK phosphorylated on Tyr204, or antibodies that
specifically recognize Tyr182 on p38 MAPK, were purchased
from New England Biolabs and used in immunoblot analyses.
Phosphorylation on Tyr204 is diagnostic for activated
p44/42 MAPK, and phosphorylation on Tyr182 is diagnostic
for activated p38 MAPK. Detection of antibody binding was carried out
as described by the manufacturer using the Photope chemiluminescent
detection system (New England Biolabs).
BCP Activates the p42/p44 MAPK Pathway and Can be Specifically
Blocked by PC
3 M PC has no
effect on the serum- or IL-1-induced response, but completely blocks
the BCP-induced response (Fig. 2a, lanes 3, 5, and
7) implying relative specificity.
Fig. 2.
BCP activates the p42/p44 MAPK pathway and is
specifically blocked by PC. a, phosphorylated p42/p44 MAPK;
b, p42/p44 MAPK. Lane 1, unstimulated; lane
2, serum (10%); lane 3, serum + PC; lane 4,
IL-1 (10 ng/ml); lane 5, IL-1 + PC, lane 6, BCP (50 µg); lane 7, BCP + PC. All cells were stimulated for
15 min. Concentration of PC = 103
M.
[View Larger Version of this Image (28K GIF file)]
causes a
transient increase in P-p42/p44 MAPK levels that ends 30 min after
stimulation. Serum induces a rapid increase in P-p42/p44 MAPK levels
that remains sustained throughout the time course.
Fig. 3.
Time course of BCP, IL-1, and serum
activation of the p42/p44 MAPK pathway. a, phosphorylated
p42/p44 MAPK; b, p42/p44 MAPK. Lane 1,
unstimulated; lane 2, 5 min; lane 3, 10 min;
lane 4, 15 min; lane 5, 30 min.
[View Larger Version of this Image (46K GIF file)]
Fig. 4.
Effect of BCP, IL-1, and serum on the p38
MAPK pathway. Lane 1, unstimulated; lane 2, 5 min; lane 3, 10 min; lane 4, 15 min; lane
5, 30 min.
[View Larger Version of this Image (35K GIF file)]
3 M PC completely blocks
CPPD induction of p42/p44 MAPK phosphorylation. The PC analogues SAT
and citrate have a less appreciable effect on this activation.
Fig. 5.
Time course of CPPD activation of the p42/p44
MAPK and p38 pathways and effect of PC, SAT, and citrate. a,
phosphorylated p42/p44 MAPK; b, p38 MAPK. Lane 1,
unstimulated; lane 2, 5 min (CPPD); lane 3, 15 min (CPPD); lane 4, 30 min (CPPD); lane 5, CPPD + PC (15 min); lane 6, CPPD + SAT (15 min); lane 7,
CPPD + citrate (15 min); lane 8, BCP (15 min).
Concentrations of BCP (50 µg/ml), CPPD crystals (50 µg/ml), PC
(103 M), SAT (10
3
M), and citrate (10
3 M) were
used.
[View Larger Version of this Image (25K GIF file)]
3 to 10
6 M, P-p42/p44 MAPK
levels induced by BCP were assayed. The study showed that PC inhibited
BCP activation of the p42/p44 MAPK pathway in a
dose-dependent manner. While 10
6
M PC did have an appreciable effect on activation,
10
3 M PC was necessary to completely block
activation (Fig. 4, lanes 4-7).
Fig. 6.
Dose-response study of PC on BCP-induced
p42/p44 MAPK phosphorylation. a, phosphorylated p42/p44
MAPK; b, p42/p44 MAPK. Lane 1, unstimulated;
lane 2, serum; lane 3, BCP; lane 4,
BCP + 103 M PC; lane 5, BCP + 10
4 M PC; lane 6, BCP + 10
5 M PC; lane 7, BCP + 10
6 M PC. Cells were incubated with
inhibitors for 30 min followed by 15-min stimulation.
[View Larger Version of this Image (34K GIF file)]
3 M SAT and
10
3 M citrate reduced BCP induction of
p42/p44 MAPK, neither was as effective as PC. At lower concentrations,
SAT seems to block the response more effectively than citrate. PC, SAT,
and citrate, which only differ in the side group attached to their
tricarboxylic acid chain, clearly have very different biological
effects. PC is much more effective than either of its analogues, SAT
and citrate, at blocking BCP-induced p42/p44 MAPK phosphorylation.
Fig. 7.
Dose-response study of SAT and citrate on
BCP-induced p42/p44 MAPK phosphorylation. a, phosphorylated
p42/p44 MAPK; b, p42/p44 MAPK. Lane 1, BCP;
lane 2, BCP + 103 M SAT;
lane 3, BCP + 10
4 M SAT;
lane 4, BCP + 10
5 M SAT;
lane 5, BCP + 10
6 M SAT;
lane 6, BCP + 10
3 M citrate;
lane 7, BCP + 10
4 M citrate;
lane 8, BCP + 10
5 M citrate;
lane 9, BCP + 10
6 M citrate. Time
of stimulation was 15 min.
[View Larger Version of this Image (26K GIF file)]
Fig. 8.
CPPD- and BCP-mediated phosphorylation of
CREB Ser133 can be blocked by PC. Lane 1,
unstimulated; lane 2, CPPD crystal (15 min); lane
3, CPPD (30 min); lane 4, CPPD + PC (30 min);
lane 5, BCP (15 min); lane 6, BCP (30 min);
lane 7, BCP + PC (30 min); lane 8, 10% serum (15 min).
[View Larger Version of this Image (28K GIF file)]
3 to 10
5 M) blocked
crystal-induced mitogenesis while SAT or citrate had little effect
(Fig. 9a). Interleukin-1
did not induce
[3H]thymidine incorporation in human foreskin fibroblasts
above unstimulated control (Fig. 9b).
Fig. 9.
a, effects of PC, SAT and citrate on BCP
crystal-induced [3H]thymidine incorporation.
1, unstimulated control; 2, BCP crystal; 3, inhibitors (103 M);
4, inhibitor (10
4); 5,
10
5 M; 6, inhibitors
(10
6 M). b, effect of PC, SAT and
citrate on IL-1 induced [3H]thymidine incorporation.
1, unstimulated control; 2, 10% serum; 3, IL-1 (10 ng); 4, IL-1 + inhibitors
(10
3 M); 5, IL-1 + Inhibitors
(10
4 M).
[View Larger Version of this Image (27K GIF file)]
Fig. 10.
a, effect of PD98059, a selective
inhibitor of MAP kinase on BCP crystal-induced p42/p44 MAPK activities.
Lane 1, unstimulated control; lane 2, 10% FBS;
lane 3, 10% FBS + 10 µM inhibitor; lane 4, 10% FBS + 50 µM inhibitor; lane 5,
10% FBS + 100 µM inhibitor; lane 6, 50 µg/ml BCP; lane 7, 50 µg/ml BCP + 10 µM
inhibitor; lane 8, 50 µg/ml BCP + 50 µM
inhibitor; lane 9, 50 µg/ml BCP + 100 µM
inhibitor. b, effect of PD 98059 on BCP and CPPD
crystal-induced [3H]thymidine incorporation. Experiments
were done in the presence of 10 µM inhibitor (lane
1), 50 µM inhibitor (lane 2); and 100 µM inhibitor (lane 3).
[View Larger Version of this Image (55K GIF file)]
induction. We also have shown that treatment of cells
with BCP crystals leads to phosphorylation of CREB Ser133.
CREB is a key transcriptional regulator of the c-fos gene
that has been shown to be important for mediating c-fos
activation in response to elevated levels of intracellular calcium (25) and cAMP (26). Serine 133 is a critical residue whose phosphorylation is necessary for CREB-mediated transactivation. Recently, CREB has also
been shown to be important for mediating activation of the
c-fos gene by purified growth factors, through a
MAPK-dependent pathway (27). Since the p42/p44 MAPK pathway
is necessary for cell proliferation our results suggest that calcium
containing crystals may mediate their mitogenic effects by activating
this pathway. Consistent with this, inhibition of MEK1, an upstream activator of MAPKs, with the inhibitor PD98059, significantly inhibits
crystal-mediated cell proliferation. Since p42/p44 MAPKs are
differentially activated by BCP and CPPD, relative to p38 MAPK, this
observation suggests that p42/p44 MAPKs are important mediators of the
crystal response. Our results also suggest that c-fos
activation may occur by a mechanism that involves activation of the
CREB factor.
3 to
10
6 M) can specifically block BCP and CPPD
crystal activation of MAPK in a dose-dependent manner, at
concentrations that have previously been shown to block mitogenesis.
SAT and citrate in high concentration (10
3 M)
partially block MAPK and mitogenesis. Consistent with these observations, our earlier studies had shown that PC specifically inhibits CPPD and BCP crystal-induced proto-oncogene (c-fos
and c-jun) expression, metalloproteinase synthesis, and
mitogenesis in human fibroblasts in vitro, while PC has no
effect on similar biologic responses induced by EGF, PDGF, and serum
(19).
1 0 1) and (0 1 0) surfaces of calcium oxalate monohydrate crystal,
thus blocking growth. We speculate that the inhibitory action of PC on
the CPPD unit cell lattice, although different from calcium oxalate
monohydrate or BCP, would be achieved by a similar inhibitory
mechanism. SAT is not as efficient an inhibitor as PC for calcium salts
examined so far, which probably reflects its stereochemistry and the
fact that both a sulfate moiety (one less charge than PO4)
and the presence of a nitrogen atom reduce its capacity to position
itself correctly for crystal face interaction.
potential of the crystal surface, thus interfering with the
crystal-membrane interactions that lead to cellular responses. This
would explain why PC does not inhibit the monosodium urate crystal and
peptide growth factor induction of mitogenesis and metalloproteinase
synthesis (19).
*
This work was supported in part by United States Public
Health Service Grant AR-38421 and a grant from the Arthritis
Foundation, Florida Chapter (both to H. S. C.), an American Cancer
Society Institutional Seed Grant Award from the Medical College of
Wisconsin Cancer Center, a grant-in-aid from the American Heart
Association, Wisconsin Division, and Shannon Award R55GM/OD51856 (to R. P. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Arthritis Division
(D-26), Dept. of Medicine, University of Miami School of Medicine, 1201 N. W. 16th St., Miami, FL 33135. Tel.: 305-243-5735; Fax: 305-243-5655; E-mail: hcheung{at}mednet.med.miami.edu.
1
The abbreviations used are: CPPD, calcium
pyrophosphate dihydrate; MAPK, mitogen-activated protein kinase;
P-MAPK, phosphorylated MAPK; SAT,
n-sulfo-2-aminotricarballylate; BCP, basic calcium phosphate; PC, phosphocitrate; PDGF, platelet-derived growth factor; SRF, serum response factor; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium; IL, interleukin; FBS, fetal bovine serum; CREB, cyclic AMP response element-binding protein.
2
P. B. Halverson, A. Greene, and H. S. Cheung,
submitted for publication.
3
D. Nair and R. Misra, unpublished
observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.