Inflammatory cytokines (IL-1
, TNF-
) and LPS modulate the
Ca2+ signaling pathway in
osteoblasts
Vincent K.
Tam,
Sandra
Schotland, and
Jacob
Green
Laboratory of Membrane Biology, Research Institute, Division of
Nephrology and Department of Medicine, Cedars-Sinai Medical Center,
Los Angeles, California 90048
 |
ABSTRACT |
Locally derived growth factors and cytokines in bone play a
crucial role in the regulation of bone remodeling, i.e., bone formation
and bone resorption processes. We studied the effect of interleukin
(IL)-1
, tumor necrosis factor (TNF)-
, and
Escherichia coli lipopolysaccharide
(LPS) on the hormone-activated
Ca2+ message system in the
osteoblastic cell line UMR-106 and in osteoblastic cultures derived
from neonatal rat calvariae. In both cell preparations, IL-1
,
TNF-
, and LPS did not alter basal intracellular
Ca2+ concentration
([Ca2+]i)
but attenuated Ca2+ transients
evoked by parathyroid hormone (PTH) and
PGE2 in a dose (1-100 ng/ml)-
and time (8-24 h)-dependent fashion. The cytokines modulated
hormonally induced Ca2+ influx
(estimated by using Mn2+ as a
surrogate for Ca2+) as well as
Ca2+ mobilization from
intracellular stores. The latter was linked to suppressed production of
hormonally induced inositol 1,4,5-trisphosphate. The effect of
cytokines on
[Ca2+]i
was abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml).
The cytokine's effect was, however, independent of nitric oxide (NO)
production, since NO donors (sodium nitroprusside) as well as permeable
cGMP analogs augment, rather than attenuate, hormonally induced
Ca2+ transients in osteoblasts.
Given the stimulatory role of cytokines on NO production in
osteoblasts, the disparate effects of cytokines and NO on the
Ca2+ signaling pathway may serve
an autocrine/paracrine mechanism for modulating the effect of
calciotropic hormones on bone metabolism.
interleukin-1
; tumor necrosis factor-
; lipopolysaccharide; nitric oxide; intracellular calcium
 |
INTRODUCTION |
NORMAL BONE REMODELING is achieved by a balance of bone
formation and bone resorption. These processes are closely regulated and are under the control of both systemic hormones as well as locally
derived growth factors and cytokines (11). The inflammatory cytokines
interleukin (IL)-1 and tumor necrosis factor (TNF), as well as
bacterial endotoxin [lipopolysaccharide (LPS)], have been
shown to exert complex effects on bone remodeling (18). IL-1
and
IL-1
, which have similar biological potency, as well as TNF-
,
stimulate bone cell replication (9), stimulate osteoclastic bone
resorption (6, 10, 37, 49), and inhibit bone formation (e.g.,
production of type I collagen and expression of alkaline phosphatase
activity) (9, 48). Both cytokines also stimulate prostaglandin
synthesis by osteoblasts (3, 60) and thus may affect bone remodeling
via this pathway as well. As the bone cell responsible for bone
formation, the osteoblast appears to be the bone cell targeted by IL-1
and TNF, in mediating their inhibitory effect on bone formation.
However, it is now clearly established that the process of osteoclastic
bone resorption mediated by both cytokines is also dependent on the
coexistence of osteoblasts, suggesting that IL-1 and TNF stimulate the
release of soluble factors from osteoblasts, which, in turn, stimulate
osteoclasts to resorb bone (49, 50).
In addition to IL-1 and TNF, LPS has been shown to exert direct effects
on bone remodeling, as well as to stimulate the production of other
cytokines (e.g., IL-1, TNF, and IL-6) by osteoblasts (18). Recent
evidence indicates that TNF-
and LPS induce nitric oxide (NO)
production in osteoblastic cells. This effect is enhanced by IL-1
(13). The production of NO by osteoblasts, or by adjoining endothelial
cells in bone marrow, leads to a reduction of osteoclastic bone
resorption (13, 27). It is therefore postulated that NO production by
cytokines represents a protective mechanism against unopposed bone
resorption by the very same cytokines, such as, for example, during
inflammation.
This article focuses on the interaction between inflammatory cytokines
and systemic hormones at the cellular level. In vivo, there may be
synergistic interaction of local mediators such as TNF-
, IL-1
,
and bacterial endotoxin with circulating calciotropic hormones in
stimulating bone resorption (2, 35, 39). Inasmuch as the osteoblast
serves as a target cell for calciotropic hormones in mediating changes
in both bone formation and bone resorption (31, 32), it is conceivable
that hormones exert their effect after the generation of second
messengers inside the osteoblastic cytosol. The two major signaling
pathways activated by calciotropic hormones include the adenylate
cyclase/cAMP system and intracellular Ca2+ concentration
([Ca2+]i)
(57, 58). Various studies have shown that TNF-
and IL-1 inhibit
parathyroid hormone (PTH)-responsive adenylate cyclase in osteoblastic
cells (7, 21, 23, 44, 46). In view of the paucity of data regarding the
effect of cytokines on the [Ca2+]i
message system, and given the cardinal role played by
[Ca2+]i in bone
remodeling (22, 40), we were prompted to study the effects of IL-1
,
TNF-
, and LPS on the
[Ca2+]i transduction
pathway stimulated by calciotropic hormones (PTH, PGE2) in osteoblasts.
For the studies presented herein, we used the clonal osteoblastic cell
line UMR-106. These cells share many phenotypic features with normal
osteoblasts, including the responsiveness to calciotropic hormones and
cytokines (36). Because these are transformed osteoblastic cells, we
verified the reproducibility of our results by repeating several key
experiments in cultured cells derived from neonatal rat calvariae.
 |
METHODS |
Cell culture conditions.
The UMR-106 cell line was a generous gift of Dr. T. J. Martin
(University of Melbourne, Melbourne, Australia) to Dr. T. J. Hahn
(Veterans Affairs Medical Center, West Los Angeles, CA), who in turn
generously supplied us with these cells. Cells were used between
passages 10 and 12 and subpassages 3 and 14. Cells were seeded at a density of 2.5 × 104
cells/cm2 in tissue culture flasks
or multiwell plates and grown at 37°C in a humidified 95% air-5%
CO2 atmosphere in Ham's F-12-DMEM
(1:1) supplemented with 14.3 mM
NaHCO3, 1.2 mM
L-glutamine, 7% fetal bovine
serum, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. The cells
reached confluence within 6-7 days in culture and were used on
days 6-8 of growth.
Osteoblastic cell cultures were also prepared from calvariae of 1- to
2-day-old Sprague-Dawley rats by using a successive enzymatic digestion
method (16). Briefly, calvariae were removed and cleaned from the
adherent tissue under a dissecting microscope. The bone tissue was cut
into small pieces and digested with a mixture of trypsin/collagenase
and bacterial collagenase (2.0 mg/ml) for 20 min at 37°C. The bone
pieces were minced with divalent ion-free PBS, then resuspended in
trypsin/collagenase solution for 40 min and washed with PBS. After a
second 40-min digestion, the pieces were vortexed and filtered through
a metal mesh (Millipore, Bedford, MA) and then through 10-µm
nylon membrane. The filtered solution was mixed with an equal volume of
PBS and diluted to 30 ml with
-MEM. Cells were pelleted by
centrifugation at 800 g for 5 min. The
yielded cells were plated in
-MEM, supplemented with 10% FBS,
and grown to confluence. Experiments were performed in cells from
subpassages 2-5.
Determination of
[Ca2+]i.
Changes in
[Ca2+]i
were monitored fluorometrically by use of the AM of the
Ca2+-sensitive probe fura 2. Cells
grown to confluence on 25-mm-diameter glass coverslips were washed and
suspended in a balanced salt solution (BSS) containing (in mM) 140 NaCl, 1 MgCl2, 4 KCl, 10 HEPES-Tris, 1.5 CaCl2, 5 glucose,
and 5 sodium pyruvate, pH 7.4 (adjusted with 1 M NaOH). The cells were
then loaded with 5 µM fura 2 (in DMSO) for 15-20 min at
37°C. Extracellular dye was removed by three washes with BSS. The
coverslip with attached cells was then mounted in a perfusion chamber
and continuously perfused at a rate of 10-12 ml/min. The perfusate
was delivered through an eight-way valve to a heat exchanger and then
to the chamber and maintained at 37°C. The recording system
included a Diaphot inverted microscope (Nikon, Melville, NJ) equipped
with a high numerical aperture Neofluor ×100/1.3 numerical
aperture (Carl Zeiss, Thornwood, NY) oil-immersion objective. The
microscope was attached to a Photon Technology International Deltascan
spectrofluorometer (PTI, S. Brunswick, NJ), which provided a
dual-wavelength excitation light. To initiate an experiment, cells were
bathed in the Ca2+-replete assay
buffer or (where required) in a
Ca2+-free buffer that was devoid
of Ca2+ and contained 0.1 mM EGTA.
The final osmolarity of all solutions was 300 mosM. To evoke a change
in
[Ca2+]i,
test compounds were added to the assay buffer (where indicated) in a
1:1,000 dilution from a stock solution. Photon emission was monitored
at 510 nm with excitation wavelength alternating between 340 (F340) and 380 nm
(F380). At the end of each
experiment, the minimum (Rmin)
and maximum (Rmax) ratio of
F340 and
F380
(R340/380) was determined in BSS
containing no added Ca2+, 4 mM
EGTA, and 1 µM ionomycin and in BSS containing 10 mM
CaCl2 and 1 µM ionomycin,
respectively. Each coverslip was individually corrected for
autofluorescence by Mn2+
quenching, and
[Ca2+]i
was calculated according to the formula (20)
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|
where
Kd represents the
dissociation constant of fura 2 for
Ca2+ (224 nM), R represents the
ratio of
F340/F380,
Sf2 is the
F380 intensity obtained from
Rmin (free fura 2), and
Sb2 is the
F380 when the dye is fully
saturated with calcium collected during the
Rmax determination.
Determination of water-soluble inositol phosphates.
UMR-106 cells in 12-well plates were labeled with 3 µCi/ml
myo-[3H]inositol
(Amersham, Arlington Heights, IL) in Trowell's
T8 (inositol-free) medium
supplemented with 15 mM HEPES, 1.2 mM
L-glutamine, 2% FCS, 100 U/ml
penicillin, and 0.1 mg/ml streptomycin for 24 h. On the day of the
experiment, the unincorporated
myo-[3H]inositol
was washed away with serum-free Trowell's
T8 medium with the above additives
and kept in that same medium for 1 h in the presence of 10 mM LiCl.
Cells were stimulated with various agonists for the designated times.
The reaction was terminated by the aspiration of the medium, followed
by two 10% trichloroacetic acid washes. The washes were pooled, and
trichloroacetic acid was extracted with water-saturated diethyl ether.
The ether extracts were loaded onto glass wool-plugged Pasteur pipette
columns containing ~2 g of anion exchange resin (AG 1-X8 formate,
Bio-Rad Laboratories, Richmond, CA).
Inositol 4-monophosphate (IP1),
inositol 1,4-bisphosphate (IP2),
and inositol 1,4,5-trisphosphate
(IP3) were sequentially eluted
from the columns by increasing concentrations of ammonium formate,
according to the method of Berridge et al. (5). Elution fractions of 2 ml each were diluted into liquid scintillation fluid and counted in a
beta counter. Data are expressed as total counts per minute (cpm) of
6-ml eluates for each inositol phosphate metabolite per
106 cells.
Determination of cellular cAMP levels.
Determination of cellular cAMP levels was done in culture plates
containing 24 multiwells per plate. Cells preincubated with cytokines
or vehicle (PBS + 0.1% albumin) were acutely (from 5 min to 2 h)
stimulated with agonists dissolved in 1 ml BSS solution at 37°C in
the presence of 0.2 mM IBMX. The reaction was terminated by aspirating
the medium containing the stimulant and then adding 0.5 ml
L-propanol to extract the cAMP
from the cell layer. The cell layers were then kept at 4°C for 1 h.
The propanol extract was removed to glass tubes. The propanol was
evaporated under a stream of nitrogen gas, and the dried extract was
kept at
70°C until assay. Before assay, the extract was
reconstituted with sodium acetate buffer, pH 6.2. Assay of cAMP was
carried out by RIA with minor modification.
125I-succinyltyrosine ester of
cAMP (ICN, Irvine, CA) (10,000 cpm/100 µl) was utilized.
Antigen-antibody precipitation was done by 100% ethanol. Results are
expressed as picomoles of cAMP per
106 cells.
PTH-related protein binding studies.
Human
[36Tyr]parathyroid
hormone-related protein
(PTHrP)-(1
36)-NH2 was
radioiodinated and purified by HPLC to a final estimated specific
activity of 2,200 Ci/mmol. Binding studies were carried out in
24-multiwell dishes. Binding of the radioligand was initiated by
removing the medium and incubating cells at 15°C in binding buffer
containing (in mM) 50 Tris · HCl, pH 7.5, 100 NaCl, 2 CaCl2, 5 KCl, as well as 0.5%
heat-inactivated BSA and 20% fetal bovine serum containing 250,000 cpm
of tracer in the presence or absence of different concentrations of
unlabeled PTHrP-(1
34)-NH2.
Reactions were terminated by aspirating the buffer and washing the
monolayers three times with ice-cold 0.9% NaCl. Cells were then
treated with 200 µl of 1.0 N NaOH and transferred quantitatively to
test tubes, and cell-associated radioactivity was determined by gamma
counting.
Determination of cell growth.
Cell mitogenic activity was assessed by the incorporation of
[3H]thymidine.
Briefly, cells were grown in 24-well plates. Twenty-two hours before
experimentation, the medium was changed to serum-free Ham's F-12-DMEM.
Three hours before the harvest, cells were pulsed with 0.2 µCi/ml
[3H]thymidine (6.7 Ci/mmol). Cells were harvested by three PBS washes to remove
unincorporated label followed by two washes with 10% trichloroacetic
acid.
The cell layers were solubilized in 1 N NaOH, and aliquots of the
solubilized cells were diluted into liquid scintillation fluid after
neutralization with HCl and counted in a beta counter. Data are
expressed as cpm per well. Cell counts were performed by releasing
cells with trypsin-EDTA and counting with a hemocytometer.
Statistics.
Results are expressed as means ± SD. Nonlinear square curve fitting
was used to assess dose-response curves to estimate mean half-maximally
effective and maximally effective concentrations of agonist with 67%
confidence limits, assuming highly correlated asymmetric variance
spaces. One- and two-way ANOVAs to test for differences among treatment
means were performed as indicated where appropriate. Each experiment
was performed at least four times with separate batches of cells to
confirm reproducibility of the results.
Reagents and hormones.
Recombinant murine IL-1
was purchased from Genzyme (Cambridge, MA).
TNF-
and LPS were from R&D Systems (Minneapolis, MN). The cytokines
were reconstituted in PBS containing 0.1% BSA to yield a working stock
solution of 1 µg/ml. PGE2 was
purchased from Upjohn (Kalamazoo, MI), and bovine PTH-(1
34) was from
Peninsula Laboratories (San Carlos, CA). Fura 2-AM was obtained from
Molecular Probes (Eugene, OR). Herbimycin A was from GIBCO (Grand
Island, NY). A stock solution of the drug was made in 100% DMSO and
diluted with the culture medium before addition to cells. The culture media containing the equivalent concentrations of DMSO served as
vehicle controls. Cell culture media and other chemicals were purchased
from Sigma (St. Louis, MO), with the exception of those specifically
described.
 |
RESULTS |
Effect of cytokines on agonist-induced
Ca2+ transients.
Calcemic hormones (e.g., PTH,
PGE2) have been shown to induce
Ca2+ transients
([Ca2+]i)
in osteoblasts.
[Ca2+]i
can be elevated either by opening of a plasma membrane
Ca2+ channel (54, 55) or through
release of Ca2+ from intracellular
stores after hormonal activation of phospholipase C and
phosphatidylinositol breakdown (57). Figure
1 describes the effect of TNF-
, LPS, and
IL-1
on hormonally stimulated
Ca2+ transients. Under control
conditions (vehicle-treated cells, Fig.
1A), acute exposure of UMR-106
cells to 10
8 M PTH-(1
34)
in the presence of 1.5 mM Ca2+ in
the extracellular buffer elicited a
[Ca2+]i
rise from a baseline value of 114 ± 12 nM to a peak of 224 ± 14 nM. When cells were preincubated for 24 h with TNF-
(100 ng/ml), LPS
(10 ng/ml), or IL-1
(10 ng/ml), resting
[Ca2+]i
was not altered. However, the PTH-induced
Ca2+ transient was significantly
attenuated by each of the cytokines (Fig. 1,
B-D;
P < 0.01 vs. control). The
combination of TNF, LPS, and IL-1
at the same concentration as
before had a greater suppressive effect on
[Ca2+]i
responses, compared with the effect observed when each cytokine was
given alone (Fig. 1E).

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Fig. 1.
Effect of cytokines and lipopolysaccharide (LPS) on hormonally induced
Ca2+ transients. UMR-106 cells
were preincubated for 24 h in serum-free DMEM containing either vehicle
alone (A and
a) or different cytokines as
indicated. On day of experiment, cells were loaded with
Ca2+ indicator fura 2 and mounted
onto stage of a Nikon Diaphot microscope attached to a Deltascan
spectrofluorometer as described in
METHODS. Cells were perfused with
balanced salt solution (containing 1.5 mM
CaCl2), pH 7.4 at 37°C, and
basal cytosolic Ca2+ concentration
([Ca2+]i)
was recorded. Parathyroid hormone (PTH;
10 8 M) or
PGE2
(10 6 M) was acutely added
to cells (arrows), and fluorescence was recorded.
[Ca2+]i
was calculated after calibration of fluorescent signal. This experiment
represents 1 of 7 experiments with similar results. TNF, tumor necrosis
factor; IL-1, interleukin-1.
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Similar results to those obtained with PTH were also obtained when the
effect of cytokines on
PGE2-induced
Ca2+ transients was studied. Thus,
under control conditions, PGE2 (10
6 M) stimulated
[Ca2+]i
rise from a baseline of 114 ± 9 nM to a peak value of 185 ± 11 nM (Fig. 1A). The
[Ca2+]i
response was significantly attenuated
(P < 0.01 vs. control) after 24-h
incubation of the cells with TNF-
, LPS, IL-1
, or all three
cytokines combined (Fig. 1,
B-E,
respectively).
After 24-h incubation with any cytokine, cell viability was not
affected and was higher than 95% as judged by trypan blue exclusion.
To evaluate the contribution of extracellular
Ca2+ to the effect of cytokines on
hormonally stimulated Ca2+
transients, we repeated the experiment described in Fig. 1 by bathing
the cells in Ca2+-free medium. Any
residual Ca2+ in the buffer was
removed by the addition of EGTA. Under these conditions, resting
[Ca2+]i
falls significantly, because, in the absence of extracellular Ca2+,
[Ca2+]i
is pumped out from the cytosol by the plasma membrane
Ca2+-ATPase at a faster rate than
when there is Ca2+ in the media.
Thus, after 4-5 min of incubation in
Ca2+-free conditions,
[Ca2+]i
stabilized at a value of 52 ± 6 nM (Fig.
2, A and
a). This value was not altered after
preincubation of the cells for 24 h with cytokines. However, TNF-
,
LPS, and a combination of the two cytokines together brought about an
attenuation of hormonally induced
Ca2+ transients. Acute exposure of
the cells to 10
8 M PTH
elicited an immediate
[Ca2+]i
rise from 52 ± 6 to 145 ± 12 nM (Fig.
2A). In the absence of Ca2+ in the extracellular medium,
this Ca2+ transient reflects
Ca2+ release from intracellular
stores. This component of
[Ca2+]i
rise was significantly blunted by preincubation of the cells for 24 h
with TNF-
(100 ng/ml)
([Ca2+]i = 97 ± 3 nM, P < 0.01 vs. control) or LPS (10 ng/ml)
([Ca2+]i = 85 ± 6 nM, P < 0.01 vs.
control). When the two cytokines were combined, there was an additive
effect compared with the effect of each cytokine given alone (Fig.
2D). Similar qualitative results to
those obtained with PTH were also obtained when the effect of cytokines
on PGE2-induced
[Ca2+]i
rise was tested (Fig. 2,
a-d).
Twenty-four-hour incubation of UMR-106 cells with the cytokines brought
about a 70-80% suppression of the peak
[Ca2+]i
response to PGE2
(P < 0.01 vs. control).

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Fig. 2.
Effect of cytokines and LPS on hormonally induced
Ca2+ transients under
Ca2+-free conditions. UMR-106
cells were preincubated for 24 h in serum-free DMEM containing either
vehicle alone (A and
a) or different cytokines as
indicated. On day of experiment, cells were loaded with
Ca2+ indicator fura 2 and perfused
with Ca2+-free balanced salt
solution containing 0.1 mM EGTA. Basal
[Ca2+]i
was recorded and calculated as described in
METHODS. After stabilization of
signal, PTH (10 8 M) or
PGE2
(10 6 M) was acutely added
to cells (arrows). Fluorescence was recorded, and
Ca2+ transients were calculated
after calibration of signal as described in
METHODS. This experiment represents 1 of 6 experiments with similar results.
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To further distinguish between the effect of cytokines on
hormone-dependent Ca2+ entry
across the plasma membrane vs. an effect on
Ca2+ mobilization from
intracellular stores, we used Mn2+
as a surrogate for Ca2+.
Mn2+ uses the same influx channels
as Ca2+ and has a very high
affinity for fura 2. The binding of
Mn2+ to the fura 2 molecule
quenches its fluorescence (47). Thus changes in fura 2 fluorescence
induced by Mn2+ can be used to
estimate Ca2+ influx pathways. In
the experiment described in Fig. 3, UMR-106 cells were exposed to Ca2+-free
media (containing EGTA), with (a and
b) or without
(A and B) 10 µM
Mn2+. The addition of
10
6 M
PGE2 to the cells caused an acute
rise of
[Ca2+]i
from 55 ± 4 to 122 ± 9 nM (Fig. 3,
A and
a). Under
Ca2+-free conditions, this
response signifies Ca2+ release
from intracellular stores. Preincubation of the cells for 24 h with
TNF-
(100 ng/ml) resulted in a significant attenuation of the
[Ca2+]i
increment (Fig. 3, B and
b)
(P < 0.01 vs. control). In cells bathed in a medium containing
MnCl2, the acute initial rise in [Ca2+]i
was followed by fluorescence quenching, signifying
Mn2+ entry across the plasma
membrane (Fig. 3, a and
b). As shown in this experiment,
pretreatment of the cells with TNF-
caused a decline in the rate of
fluorescence quenching (Fig. 3, b vs. a), suggesting an inhibitory effect
on hormonally induced Ca2+ entry
brought about by the cytokine. LPS and IL-1
showed a similar effect
to that of TNF-
(data not shown). Also, the
Mn2+ experiments, as performed
with PGE2, were also carried out
with PTH. Similar qualitative results were obtained.

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Fig. 3.
Cytokines inhibit Ca2+ entry
pathways in osteoblasts. UMR-106 cells were preincubated for 24 h in
serum-free DMEM containing either vehicle alone
(A and
a) or 100 ng/ml TNF-
(B and
b). On day of experiment, cells were
loaded with fura 2 as described in
METHODS and then perfused with
Ca2+-free balanced salt solution
in absence (A and
B) or presence
(a and
b) of 10 mM
MnCl2.
PGE2
(10 6 M) was acutely added
to cells (arrow), and fluorescence was recorded. This experiment
represents 1 of 5 similar experiments.
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Both PTH and PGE2 have been shown
to hydrolyze phosphatidylinositol phosphate and to activate protein
kinase C (PKC), after the generation of diacylglycerol (12, 57). Also,
PKC has been shown to activate
Ca2+ channels in osteoblastic
cells, including the UMR-106 cells (59). It is assumed, therefore, that
activation of PKC by calciotropic hormones is partly responsible for
Ca2+ influx mediated by these
agonists. We therefore tested the effect of cytokines on PKC-dependent
Ca2+ channel. Figure
4 demonstrates that under control
conditions, phorbol 12-myristate 13-acetate (1 µM), a phorbol ester
which activates PKC, acutely stimulates a
[Ca2+]i
rise from a baseline value of 122 ± 8 to 417 ± 15 nM (Fig. 4A). Preincubation of the cells for
24 h with TNF-
(100 ng/ml) (Fig.
4B), LPS (10 ng/ml) (Fig.
4C), or a combination of both cytokines at the same concentrations (Fig.
4D) brought about an attenuation of
the PKC-dependent
[Ca2+]i response (peak
[Ca2+]i response = 278 ± 4, 246 ± 8, and 163 ± 10 nM, respectively, P < 0.01 for each cytokine vs. control). The suppressive effect on
PKC-activated Ca2+ channel was
also shared by IL-1
(data not shown).

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Fig. 4.
Cytokines attenuate protein kinase C (PKC)-activated
Ca2+ channel. UMR-106 cells were
preincubated for 24 h in serum-free DMEM containing either vehicle
alone (A) or TNF- , LPS, or LPS + TNF- as indicated
(B-D).
On day of experiment, cells were loaded with fura 2 as described in
METHODS and perfused with BSS
containing 1.5 mM CaCl2. After
stabilization of fluorescent signal, active phorbol 12-myristate
13-acetate (PMA; 1 µM) was acutely added to cells (arrows), and
fluorescence was recorded. This experiment represents 1 of 6 experiments with similar results.
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The experiments described in Figs. 1-4 indicate that IL-1
,
TNF-
, and LPS modulate Ca2+
transients evoked by PTH and PGE2.
The cytokines exert their effect by attenuating both
Ca2+ entry pathways and
Ca2+ mobilization from
intracellular stores.
PTH/PTHrP receptor binding.
To explore the possibility of altered hormonal binding brought about by
cytokines, we tested the effect of LPS on PTH receptor binding. Figure
5 shows that UMR-106 cells possess
high-affinity binding of
125I-PTHrP to the PTH/PTHrP
receptor. There was, however, no effect of LPS (10 ng/ml) on the
binding properties of PTH. The
Kd values estimated by the concentration required to displace 50% of the radioligand in the cells (Fig. 5A)
as well as by a Scatchard plot (Fig.
5B) were 8.4 and 8.7 nM in cells
preincubated with vehicle and cells preexposed to LPS, respectively.

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Fig. 5.
LPS does not affect PTH receptor binding.
A: UMR-106 cells were preincubated for
24 h in serum-free DMEM containing either vehicle alone ( ) or 10 ng/ml LPS ( ). On day of experiment, cells were incubated with
250,000 cpm of tracer in presence of increasing concentrations of
unlabeled PTH-related protein
(PTHrP)-(1 34)-NH2. Binding assay
was then performed as described in
METHODS.
B: Scatchard plot for specific binding
of tracer. Data are means ± SD
(n = 4) from 3 independent
experiments. B/F= bound/free.
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Role of tyrosine kinase activation in mediating cytokines effect on
[Ca2+]i.
Tyrosine kinase (TK) activation is a central signal transduction event
in IL-1, TNF, and LPS biological responses (1, 43). We therefore tested
the possibility that the suppressive effect of cytokines on hormonally
stimulated
[Ca2+]i
responses is mediated through the activation of intracellular TK.
Toward that end, we studied the effect of cytokines on
Ca2+ transients in vehicle
(DMSO)-treated UMR-106 cells as compared with cells pretreated for 24 h
with 50 ng/ml herbimycin A. This antibiotic drug has been shown to
inhibit a number of intracellular TKs, including the
src TK, while having no effect on
serine/threonine kinase, protein kinase A, and PKC (51). Figure
6 shows that in DMSO-treated cells, LPS (10 ng/ml) causes inhibition of Ca2+
transients induced by either PTH
(10
8 M) or
PGE2
(10
6 M), as seen before.
However, in cells pretreated with herbimycin A, while the drug itself
did not have an effect on hormonally induced
Ca2+ rise, it completely abrogated
the inhibitory effect of LPS on Ca2+ signals generated by PTH and
PGE2. Qualitative results similar to those seen with LPS were also observed with TNF-
(100 ng/ml), namely, abrogation of the TNF effect on PTH-induced
[Ca2+]i
signals in cells preexposed to herbimycin A (data not shown).

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Fig. 6.
Herbimycin A abrogates inhibitory effect of LPS on hormonally induced
Ca2+ transients. UMR-106 cells
were preincubated for 24 h in serum-free DMEM containing the following:
DMSO (1:1,000 dilution), control (A
and a), DMSO + 10 ng/ml LPS
(B and
b), herbimycin A (50 ng/ml)
(C and
c), and herbimycin A (50 ng/ml) + LPS (D and
d). On day of experiment, cells were
loaded with fura 2 and perfused with BSS containing 1.5 mM
CaCl2. PTH
(10 8 M) or
PGE2
(10 6 M) was added acutely
as indicated by arrows, and fluorescence was recorded. This experiment
represents 1 of 5 experiments with similar results.
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Effect of cytokines on
Ca2+ transients
in calvarial osteoblasts.
Because UMR-106 is a transformed cell line, we verified the
reproducibility of our results by repeating some of the experiments in
cell cultures derived from neonatal rat calvariae. As shown in Table
1, TNF-
, LPS, and IL-1
had a marked
suppressive effect on hormonally induced
[Ca2+]i
responses in a pattern similar to that observed in UMR-106 cells. The
effect of the cytokines was observed in both the presence and absence
of Ca2+ in the extracellular
media.
Because the UMR-106 cells are osteoblast-like cells of clonal origin
and respond to cytokines in a manner similar to nontransformed osteoblastic cells, they were used in all further studies unless otherwise indicated.
Effect of cytokines in cells treated with indomethacin.
In view of the fact that IL-1 and TNF can stimulate the production of
prostaglandins in osteoblasts (3, 60), we studied the effect of these
two cytokines on hormonally stimulated
[Ca2+]i
rise in UMR-106 cells preincubated for 24 h with 1 µM indomethacin. The suppressive effect of 24 h of pretreatment with IL-1
(10 ng/ml)
and TNF-
(100 ng/ml) on the
[Ca2+]i
signal in response to PGE2
(10
6 M) and PTH
(10
8 M) was not altered by
indomethacin (data not shown). It appears, therefore, that the
modulatory effect of cytokines on
Ca2+ transients was independent of
prostaglandin production.
Dose dependency of the effect of cytokines on
[Ca2+]i
signals.
Figure 7 shows that the suppressive effect
of TNF-
and LPS on PTH
(10
8 M)-induced
Ca2+ transients was dose
dependent. The effect of TNF-
was maximal at 100 ng/ml (~70%
suppression of
[Ca2+]i
response after 24 h of preincubation). The effect of LPS was maximal at
a dose of 100 ng/ml (~90% suppression). A dose-response curve was
also established for IL-1
(data not shown). The maximal effect of
IL-1
was achieved at 50 ng/ml (~80% suppression of Ca2+ transients).

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Fig. 7.
Dose dependency for effect of cytokines and LPS on hormonally evoked
[Ca2+]i
response. UMR-106 cells were preincubated for 24 h in serum-free DMEM
containing either vehicle alone (control) or indicated concentration of
TNF- or LPS. On day of experiment, cells were loaded with fura 2, and
[Ca2+]i
was measured and calculated after acute stimulation (arrow) with PTH
(10 8 M), as described in
METHODS. This experiment represents 1 of 5 experiments with similar results.
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A qualitatively similar dose response was obtained when the effect of
cytokines on PGE2-evoked
[Ca2+]i
signals was studied.
Time course for the effect of cytokines on
[Ca2+]i
responses.
Figure 8 demonstrates that the first
significant inhibition of a
PGE2-induced
Ca2+ transient was observed after
8 h of preincubation with 10 ng/ml LPS (26 ± 4% inhibition,
P < 0.05 vs. control). The maximal
inhibitory effect of LPS was observed after 24 h of preincubation (72 ± 8% inhibition, P < 0.01 vs.
control) with no additional effect observed after 48 h. TNF-
and
IL-1
showed a similar time course pattern (data not shown).

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Fig. 8.
Time course for effect of LPS on hormonally induced
Ca2+ transients. UMR-106 cells
were preincubated for indicated time periods in serum-free DMEM
containing either vehicle alone or 10 ng/ml LPS. At each time point,
cells were loaded with fura 2, and fluorescence was recorded at
baseline and after acute stimulation with
10 6 M
PGE2, as described in
METHODS. Percent inhibition of
hormonally evoked
[Ca2+]i
rise above baseline in LPS-treated cells vs. vehicle-treated cells was
calculated and plotted. Results are means ± SD from 6 independent
experiments. * P < 0.05, LPS-treated cells vs. control.
** P < 0.01, LPS-treated cells
vs. control.
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Effect of cytokines on hormonally induced phosphoinositol
production.
Because the release of Ca2+ from
intracellular stores is closely linked to the inositol phosphate
turnover, we next measured the effect of IL-1
, TNF-
, and LPS on
phosphatidylinositol metabolism. Figure 9
describes the effect of LPS and IL-1
on the production of
IP1,
IP2, and
IP3 mediated by
PGE2 in UMR-106 cells. We used PGE2 as an agonist in this study,
since we have shown that in UMR-106 cells,
PGE2 is a potent stimulator of
IP3 generation (57). In
preliminary experiments, we found a significant increase in IP3 after 10 s of stimulation with
PGE2. After 30 s of stimulation, IP1 was elevated whereas
IP3 was undetectable. This finding
is probably related to the fact that 30 s is a long enough time during which IP3 is already degraded to
IP2 and
IP1. Based on these findings, we
measured phosphoinositol production after 10-s stimulation with
PGE2. As shown in Fig. 9,
pretreatment of the cells with LPS (10 ng/ml) or IL-1
(100 ng/ml)
for 24 h significantly inhibited the generation of inositol phosphates
by PGE2.
IP3 was 1,720 ± 33, 750 ± 17, and 615 ± 22 cpm/106 cells
in control (vehicle-treated cells), LPS-treated, and IL-1
-treated cells, respectively (P < 0.01 for
cytokines + PGE2 vs.
PGE2 alone). IP2 was 630 ± 15, 380 ± 12, and 500 ± 15 cpm/106 cells
in control, LPS-treated, and IL-1
-treated cells, respectively (P < 0.05, cytokines + PGE2 vs.
PGE2 alone).
IP1 was 1,924 ± 24, 1,180 ± 18, and 1,112 ± 15 cpm/106 cells in the three groups
of cells, respectively (P < 0.01, cytokines + PGE2 vs.
PGE2 alone). TNF-
showed
~30% suppression of IP1 and IP3 generation by
PGE2 (data not shown).

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Fig. 9.
Cytokines attenuate phosphoinositol production by
PGE2. UMR-106 cells in 12-well
plates were prelabeled with 3 µCi/ml
myo-[3H]inositol
as described in METHODS. On day of
experiment, cells were stimulated with
10 6 M
PGE2 for 10 s. Reaction was
terminated with TCA, and inositol 4-monophosphate
(IP1), inositol 4,5-bisphosphate
(IP2), and inositol
1,4,5-trisphosphate (IP3) were
separated on AG-1X8 columns as described in
METHODS. Experiment was performed
under the following conditions: 1)
control (no PGE2 added);
2) cells preincubated for 24 h with
vehicle (PBS) followed by acute (10 s) stimulation with
PGE2;
3) cells preincubated for 24 h with
LPS (10 ng/ml) followed by acute (10 s) exposure to
PGE2;
4) cells preincubated for 24 h with
interleukin (IL)-1 (100 ng/ml) followed by 10-s stimulation with
PGE2. Values are means ± SD
(n = 4) from 3 independent
experiments. * P < 0.05, PGE2 with vehicle vs.
PGE2 with cytokines.
** P < 0.01, PGE2 with vehicle vs.
PGE2 with cytokines.
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The initial inhibitory effect of the cytokines on phosphoinositol
breakdown was observed after 8 h of incubation, and the overall time
course corresponded to the time course of the cytokine effect on
hormonally induced Ca2+
transients. Although our experiment did not separate the different IP3 isomers, these results clearly
demonstrate that these cytokines blunt the hydrolysis of
phosphatidylinositols triggered by
PGE2. None of the cytokines, when
tested alone, had any effect on the basal levels of phosphoinositols.
Effect of cytokines on cell growth.
We have recently shown that various osteotropic factors (e.g., PTH,
PGE2) mediate their effects on
osteoblast function through the
Ca2+ and cAMP signaling systems
(56, 57). Among the pleiotropic effects mediated by these messenger
systems, cAMP is antimitotic in osteoblasts, whereas the
Ca2+ signaling system, while not
having an effect on cell mitogenic activity by itself, antagonizes the
antimitotic effect of cAMP (56, 57). We, therefore, reasoned that
because the cytokines attenuate
[Ca2+]i
rise by calciotropic hormones, they may impinge on the effects of
hormones on cell growth. Figure 10
demonstrates that in UMR-106 cells, PTH dose dependently inhibits
[3H]thymidine uptake.
Pretreatment for 24 h with LPS (10 ng/ml), by itself, did not have an
effect on cell mitogenesis. However, when LPS was combined with PTH,
there was a marked potentiation of the antimitotic effect of PTH.

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Fig. 10.
LPS potentiates antimitotic effect of PTH. UMR-106 cells grown in
24-well plates were preincubated for 24 h in serum-free DMEM containing
different concentrations of bovine PTH (bPTH) as indicated, PTH at
different concentrations together with 10 ng/ml LPS, or 10 ng/ml LPS.
LPS was added 15 min before addition of PTH and was then kept for
entire 24-h incubation period with PTH. Three hours before harvest,
cells were pulsed with
[3H]thymidine and, at
end of incubation period,
[3H]thymidine uptake
was done as described in METHODS.
Results are expressed as percent uptake relative to 100% control
([3H]thymidine uptake
where no agonist was added). PBS (vehicle for cytokines) did not have
any effect on uptake. Data are means ± SD
(n = 4) from 4 independent
experiments.
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The synergistic antimitotic effect induced by LPS could also be
demonstrated when it was combined with
PGE2. Thus
PGE2 at doses of 1 and 10 µM
inhibited
[3H]thymidine uptake
by 52 and 76%, respectively (control 100%). Pretreatment of the cells
for 24 h with 10 ng/ml LPS had no effect on cell growth, but, when
combined with PGE2, LPS enhanced
the antimitotic effect of PGE2
(reduction by 75 and 92% from control at 1 and 10 µM
PGE2, respectively,
P < 0.01, PGE2 alone vs.
PGE2 + LPS).
The inhibitory effect of LPS on cell growth also could be demonstrated
in cultured calvarial osteoblasts. The results were qualitatively
comparable to those obtained in UMR-106 cells (Table 2). Once again, the potentiating
antimitotic effect of LPS was evident when combined with either PTH or
PGE2 (Table 2).
In contrast to the effect of LPS, IL-1
did not modulate the
hormonally (PGE2, PTH) induced
antimitotic effect. TNF-
(100 ng/ml), while having no effect on cell
growth by itself, amplified the antimitotic effect of
PGE2 (~40% increase in the
antimitotic effect of 10
6 M
PGE2) but did not influence PTH
effect on all growth. We reasoned that this may have been related to
the fact that TNF-
and IL-1
affect hormonally induced cAMP
generation in addition to their effect on
[Ca2+]i.
Table 3 describes the effect of LPS,
TNF-
, and IL-1
on agonist-mediated cAMP production. We measured
cAMP production in response to PTH,
PGE2, forskolin, and cholera
toxin. Forskolin and cholera toxin were used respectively to activate
the catalytic and stimulatory
(Gs) subunit of adenylyl cyclase
independent of hormonal binding to a receptor. The data presented in
Table 3 demonstrate that LPS did not affect cAMP production by either receptor (PTH, PGE2) or
nonreceptor (forskolin and cholera toxin) mechanisms. IL-1
downregulated cAMP production by both receptor (PTH,
PGE2) and postreceptor
mechanisms. TNF-
inhibited PTH-mediated cAMP generation while having
no effect on cAMP production mediated by
PGE2, cholera toxin, or forskolin.
Role of NO in mediating the effects of cytokines on
[Ca2+]i.
IL-1, TNF-
, and LPS have been shown to induce NO production in
primary rat osteoblast-like cells, and the UMR-106 cells (13) as well
as in the
MC3T3-E1
mouse clonal osteoblastic cells (14). We therefore asked whether the
effects of these cytokines on hormonally induced
[Ca2+]i
responses could be mediated through NO production. To address this
question, we used two approaches: 1)
studying the effect of NO on hormonally induced
Ca2+ transients and
2) blocking NO production in cells
that were or were not exposed to cytokines.
Figure 11 shows that the NO donor sodium
nitroprusside (SNP) dose dependently (10 and 100 µM) augmented
[Ca2+]i
rise evoked by acute stimulation with
10
8 M PTH (Fig. 11,
B and
C vs.
A). Peak
[Ca2+]i
signal was 239 ± 10, 292 ± 8, and 348 ± 9 nM in control
cells (PTH alone) and cells that were pretreated with 10 and 100 µM SNP, respectively (P < 0.01 SNP vs.
control). The effect of SNP was abrogated by LY-83583
(6-anilino-5,8-quinolinedione; Research Biochemical), an inhibitor of
soluble guanylate cyclase (34) (Fig.
11D). This compound was employed
because, in most tissues studied, NO exerts its action by activating
soluble guanylate cyclase followed by the generation of the second
messenger cGMP. Indeed, in cells pretreated with the cGMP analog
8-bromo-cGMP (8-BrcGMP), the
PGE2-induced
Ca2+ transients were enhanced
(Fig. 11,
a-c).
Peak
[Ca2+]i
signal was 176 ± 5, 226 ± 7, and 284 ± 11 nM in control
cells (PGE2 alone) and in cells
preexposed (10 min) to 8-BrcGMP at 10 and 100 µM, respectively
(P < 0.01, 8-BrcGMP vs.
control). The effect of 8-BrcGMP was abrogated by
Rp-8-BrcGMP (BIOLOG Life Science Institute, La Jolla, CA), a
selective inhibitor of cGMP-dependent protein kinase (8) (Fig.
11d). Neither LY-83583 nor
Rp-8-BrcGMP, when used alone, had any effect on PTH- and
PGE2-induced
Ca2+ transients, respectively
(Fig. 11, E and
e).

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Fig. 11.
Nitric oxide (NO) and cGMP augment hormonally induced
Ca2+ transients in osteoblasts.
UMR-106 cells were preincubated for 24 h in serum-free DMEM. On day of
experiment, cells were loaded with fura 2, and
[Ca2+]i
was measured as described in METHODS
after acute stimulation with
10 8 M PTH
(A-D)
or 10 6 M
PGE2
(a-d).
Ten minutes before addition of PTH, cells were exposed to the following
agents: vehicle only (A); 10 µM of
the NO donor sodium nitroprusside (SNP)
(B); 100 µM SNP
(C); SNP (100 µM) together with
LY-83583 (1 µM), an inhibitor of soluble guanylate cyclase
(D); and LY-83583 (1 µM) alone
(E). Ten minutes before acute
stimulation with PGE2, cells were
exposed to the following agents: vehicle only
(a); 10 µM 8-bromo-cGMP (8-BrcGMP)
(b); 100 µM 8-BrcGMP
(c); 100 µM cGMP together with 100 mM Rp-8-BrcGMP, an inhibitor of cGMP-dependent protein
kinase (d); and
Rp-8-BrcGMP (100 µM) alone
(e). This experiment represents 1 of
7 experiments with similar results.
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The experiments described in Fig. 12 show
the effect of NO blockade that was achieved by using two techniques:
1) culturing the cells in
L-arginine-free media and
2) employing the competitive inhibitor of NO production
NG-monomethyl-L-arginine
(L-NMMA). In control cells
(i.e., cells that were not exposed to LPS),
L-arginine-free conditions
attenuated Ca2+ transients evoked
by PGE2 (Fig. 12,
A and
B) from a peak value of 192 ± 6 to 178 ± 9 nM. Exposure of the cells to
L-NMMA (in the presence of
sufficient L-arginine) brought
about attenuation of PTH-induced
Ca2+ transients (Fig. 12,
a and
b) from a peak value of 232 ± 6 nM to a value of 184 ± 7 nM (P < 0.05).

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Fig. 12.
Blocking NO synthesis results in attenuation of hormonally induced
[Ca2+]i
rise. This inhibitory effect is additive to effect exerted by LPS.
UMR-106 cells were preincubated for 24 h in serum-free DMEM containing
either vehicle alone (A,
B, a,
and b) or 10 ng/ml LPS
(C,
D, c,
and d). In cells preincubated with
vehicle alone (control), media either contained a sufficient amount of
L-arginine (0.4 mM)
(A and
a) or was deprived of
L-arginine
(B). Some of the control cells
incubated in sufficient
L-arginine conditions were
preexposed for 24 h to 1 mM of the NO synthase inhibitor
NG-monomethyl-L-arginine
(L-NMMA)
(b). Cells preincubated with LPS had
either normal L-arginine
concentration in media (C and
c) or were deprived of
L-arginine
(D). Some of the cells
(d) were preexposed for 24 h to 1 mM
L-NMMA (in presence of
sufficient L-arginine). On day
of experiment, cells were loaded with fura 2, and
[Ca2+]i
was measured as described in METHODS
after acute stimulation with either
10 6 M
PGE2
(A-D)
or 10 8 M PTH
(a-d).
This experiment is 1 of 6 experiments with similar results.
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As shown before, LPS (10 ng/ml) blunted hormonally induced
[Ca2+]i
rise to both PGE2
(10
6 M) (192 ± 7 and
142 ± 5 nM in control and LPS-treated cells, respectively,
P < 0.01) (Fig.
12C) and to PTH
(10
8 M) (232 ± 8 and
148 ± 5 nM in control and LPS-treated cells, respectively,
P < 0.01) (Fig.
12c). When LPS was combined with either L-arginine-free
conditions (Fig. 12D) or with
L-NMMA (Fig. 12d), there was an even greater
suppression of hormonally induced Ca2+ transients
(P < 0.001 vs. hormone alone with
normal L-arginine in media,
P < 0.01 vs. LPS in the presence of
sufficient L-arginine). The
experiments described here were repeated with IL-1
and TNF-
. Similar results were obtained (data not shown).
The data presented in Figs. 11 and 12 suggest that in osteoblasts, NO
has a stimulatory effect on hormonally induced
[Ca2+]i
responses. Inhibition of NO production results in blunting of
hormonally stimulated
[Ca2+]i
signals and is additive to the effect of cytokines in this regard. It
appears, therefore, that the suppressive effect of cytokines on
hormonally evoked Ca2+ transients
is not mediated by the
L-arginine/NO pathway.
 |
DISCUSSION |
Our studies show that the proinflammatory cytokines IL-1 and TNF, as
well as bacterial endotoxin, influence the
Ca2+ signal transduction pathway
activated by calciotropic hormones in osteoblasts. IL-1
, TNF-
,
and LPS, while having no effect of their own on resting
[Ca2+]i,
attenuated
[Ca2+]i
responses generated by PTH and
PGE2 both in the UMR-106
osteoblastic cell line and in nontransformed osteoblasts from neonatal
rat calvariae. The effect of the cytokines on the
Ca2+ messenger system could be
initially observed only after 8 h of incubation and was attributable
both to the inhibition of Ca2+
release from intracellular stores and to the inhibition of
Ca2+ entry across the plasma
membrane. These changes were not accompanied by decreased hormonal
binding (as judged from the PTH binding assay under the influence of
LPS).
The osteoblast is the main bone cell type that is primarily responsible
for bone formation. However, this cell also possesses receptors for
calciotropic hormones with bone-resorbing activity, and it is therefore
assumed that the osteoblast also serves as a mediator in the process of
bone resorption, which is ultimately carried out by the osteoclast (31,
32). Changes in cytosolic Ca2+ in
osteoblasts serve a main signaling pathway for calciotropic hormones
such as PTH and PGE2 (12, 57). The
pattern of
[Ca2+]i
response generated by these hormones in osteoblasts usually corresponds
to that found in other cell systems, namely, an acute instantaneous
IP3-mediated mobilization of
Ca2+ from intracellular stores
that is linked to the subsequent opening of a plasma membrane
Ca2+ channel (38, 41, 53). PKC,
which is activated during phosphatidylinositol breakdown by PTH and
PGE2, activates
Ca2+ channels in the plasma
membrane of osteoblasts including UMR-106 cells (59). Our data show a
close temporal relationship between the inhibitory effect of cytokines
on hormonally induced Ca2+
transients and the suppressive effect of cytokines on
IP3 generation. Thus the process
of Ca2+ mobilization from
intracellular stores may be attenuated because of inhibition of
phosphoinositol breakdown brought about by the cytokines. Furthermore,
the Ca2+ influx pathway mediated
by PKC activation was also attenuated by these cytokines.
The mechanism for the effect of IL-1, TNF-
, and LPS on
[Ca2+]i
metabolism in osteoblasts remains conjectural at this point. All three
cytokines have been shown to initiate a common spectrum of cellular
activities and to activate a number of second messengers (43). Several
downstream signals induced by these cytokines include the activation of
mitogen-activated protein kinases and phosphorylation of several
trascription factors (e.g., NF
B) (17, 28), arachidonate-dependent
kinase activity (42), and activation of the sphingomyelin/ceramide
pathway (25, 45). In spite of this large array of second messengers, it
has been established that activation of TK is a cardinal signal
transduction event mediating the biological responses of IL-1, TNF, and
LPS (1, 43, 52). Our data show that herbimycin A, a highly specific inhibitor of intracellular TK (51), abolished the inhibitory effect of
LPS on hormonally induced Ca2+
transients, suggesting a possible role of TK in mediating the effect of
cytokines on
[Ca2+]i
responses in osteoblasts. Recently, herbimycin A has been shown to
inhibit bone resorption, both in vivo and in vitro, through a direct
effect on osteoclast number and function (61). Therefore, the effect of
this drug, as demonstrated in our study, suggests that the TK pathway
can influence bone metabolism through an effect in osteoblasts as well.
The link between activation of TK and suppression of hormonally induced
Ca2+ signals remains speculative
at this point. It could be related to tyrosine phosphorylation of
certain phosphoinositides, which, it turn, could reduce the level of
substrate for IP3. In addition to
tyrosine phosphorylation, downstream signals, such as NF
B activation, could also be responsible for the observed biological responses. These possibilities await further investigations.
Theoretically, the inhibitory effect of cytokines on hormonally induced
Ca2+ responses could be explained
by an adaptation-like response, often described in relation to the
cytokines' effect, particularly the effect of LPS. The phenomenon of
adaptation refers to the observation that cells pretreated with low
doses of LPS become refractory to a subsequent LPS stimulation (29).
Therefore, it could be argued that the prolonged exposure of
osteoblasts to cytokines (24 h) is responsible for the downmodulation
of
[Ca2+]i
signals induced by PTH and PGE2.
We feel, however, that this is not the case for the following reasons.
1) The phenomenon of adaptation is
not universal, namely, not all cytokines are alike. Thus, although
tolerance may develop to LPS and TNF, such an effect has not been
demonstrated with IL-1 (33). In our study, however, all three agonists
(namely, IL-1, TNF, and LPS) manifested the same effect on
[Ca2+]i
responses. 2) In most systems, the
phenomenon of adaptation requires the binding of LPS to its receptor,
CD14, as well as LPS association with LPS binding protein (LBP) (4,
30). However, the serum-free conditions used in our study for
preincubation of the cells preclude the formation of LPS-LBP complex.
3) LPS adaptation has been mainly
demonstrated in macrophages, whereas other cell systems (e.g.,
osteoblasts, as used in our study) may not manifest this phenomenon
(24). Finally, LPS adaptation in macrophages has been shown to be
independent of TK activation (52). Therefore, our data showing that the
attenuation of
[Ca2+]i
signals by cytokines was abrogated by herbimycin A lend further support
to the notion that the observed effect is not part of an
adaptation-like response.
The physiological significance of the cytokines' effect on
agonist-induced
[Ca2+]i
rise is demonstrated by our data showing that LPS, while having no
effect of its own on cell mitogenic activity, potentiates the antimitotic effect of PGE2 and PTH
in osteoblasts. We and others (56, 57) have shown that the pleiotropic
functions of these hormones on bone metabolism can be explained on the
basis of antagonistic effects between the
Ca2+ and cAMP signaling pathways.
With respect to cell growth, the cAMP messenger system is antimitotic,
whereas
[Ca2+]i,
although having no effect of its own on cell growth, antagonizes the
cAMP antimitotic effect (56, 57). Thus agonists attenuating [Ca2+]i
response stimulated by PTH or PGE2
will potentiate the antimitotic effects of these hormones (57), whereas
agents enhancing
[Ca2+]i
response will mitigate the antimitotic effects of the hormones (56).
Our data, therefore, suggest that by attenuating
[Ca2+]i
rise induced by PGE2 and PTH
(which activate both second messengers), LPS further enhances the
antimitotic effect of these hormones. The enhancing influence of LPS on
the antimitotic effect of hormones could not be ascribed to increased
cAMP levels, since LPS does not modify cAMP production in osteoblasts
(Table 3).
Along these lines, cytokines that inhibited cAMP production by either
PTH or PGE2 in addition to their
attenuating effect on
[Ca2+]i
(Table 3) did not exert a potentiating antimitotic effect of that
particular hormone. Thus TNF-
did not influence the antimitotic effect of PTH, whereas IL-1
did not affect the antimitotic effect of
either PTH or PGE2. Again, this
finding is consistent with the notion that the effect of calciotropic
hormones (PTH, PGE2) on cell
growth depends on the interplay between the two cardinal second
messengers activated by these hormones, namely, cAMP and [Ca2+]i.
An inhibitory effect of cytokines (IL-1 and TNF-
) on hormonally induced cAMP production in osteoblasts, as described in our study, has
been described by other investigators as well (7, 21, 23, 44, 46).
NO,
[Ca2+]i,
and cytokines in osteoblasts.
NO has been recently identified as a messenger molecule regulating a
wide range of functions, which go over and beyond its effect as a
vasorelaxant (26). Among the "nonvascular" actions of NO, this
agent has been recently shown to affect bone metabolism. Thus cytokines
such as IL-1, TNF-
, LPS, and interferon-
induce NO production in
osteoblasts (13, 14). NO produced in bone exerts an inhibitory effect
on bone resorption mediated by osteoclasts (13, 27).
In view of these data, we explored the possibility that the effects of
TNF-
, IL-1
, and LPS on hormonally induced
Ca2+ transients in osteoblasts
were mediated by the
L-arginine/NO system. The
experiments described in Figs. 11 and 12 can be summarized as follows:
1) the NO system has a stimulatory,
rather than inhibitory, effect on
[Ca2+]i
responses triggered by the calciotropic hormones PTH and
PGE2 (i.e., an opposite effect to
that exerted by cytokines); 2) this effect of NO is mediated by cGMP and cGMP-dependent protein kinase; and
3) inhibition of NO synthesis in
osteoblasts (either by using L-arginine-free conditions or by
employing the NO synthase inhibitor L-NMMA) has an inhibitory effect
on hormonal-evoked
[Ca2+]i
signals. Furthermore, there was an additive inhibitory effect on
[Ca2+]i
when inhibition of NO synthesis was employed in the presence of
cytokines. Interestingly, the suppressive effects of
L-arginine-free conditions and
L-NMMA on
[Ca2+]i
were of greater extent in cells pretreated with LPS (Fig. 12, D and
d vs.
C and
c) than in cells not treated with
LPS (Fig. 12, B and
b vs.
A and
a). This difference is probably
related to a low ambient content of NO in osteoblasts when cells are
not preincubated with LPS. Also, during a 24-h incubation period, most
of the constitutively produced NO is probably degraded to NO
metabolites. On the other hand, in cells preincubated with LPS (as well
as other cytokines), NO production is stimulated (13, 14). Because NO
by itself augments hormonally induced [Ca2+]i
responses (Fig. 11), inhibition of NO production, under these circumstances, magnifies the attenuating effect of LPS on
[Ca2+]i.
All in all, the data presented in Figs. 11 and 12 indicate that the
effects of LPS and other cytokines on the
[Ca2+]i
signaling pathway in osteoblasts are not mediated by the
L-arginine/NO pathway. It
appears, rather, that induction of NO production by cytokines in
osteoblasts counterbalances the inhibitory effect of cytokines on
[Ca2+]i.
Our study adds to the gamut of cytokine and NO effects on bone
metabolism. TNF-
and IL-1
, which are produced by inflammatory cells in the proximity of osteoblasts (macrophages and monocytes), have
adverse effects on bone formation (9, 48) and stimulate osteoclastic
bone resorption (6, 37, 49). Both effects can be exerted through
binding of these cytokines to the osteoblast, which is the bone cell
mediating both bone formation and osteoclastic bone resorption (49,
50). TNF-
and IL-1 are also produced by osteoblasts and therefore
may exert an autocrine/paracrine effect on bone remodeling (15, 19). In
view of the cardinal role played by
[Ca2+]i
in bone remodeling (22, 40) (namely, elevated
[Ca2+]i
inhibits bone resorption), we speculate that attenuation of the
hormonally induced Ca2+ transients
potentiates the bone resorptive effect of calciotropic hormones such as
PTH and PGE2. In fact, a
synergistic bone resorptive activity of TNF-
, IL-1
, and LPS with
calciotropic hormones has been demonstrated (2, 35, 39). As shown in
our study, attenuation of the
[Ca2+]i
signaling pathway by cytokines also potentiates the antimitotic effect
of PTH and PGE2.
The effect of NO on
[Ca2+]i
in osteoblasts, as shown here, adds a new facet to the overall effects
of this messenger on bone metabolism. Thus far, NO has been shown to
have a direct effect on osteoclast shape and function (i.e., inhibition
of osteoclastic bone resorption) (13, 27). Our study points to a direct
effect of NO on osteoblasts as well. The origin of NO may be the
osteoblast itself or endothelial cells in marrow blood vessels.
Consistent with the notion of osteoblast-osteoclast "cross talk,"
stimulation of the hormonally induced
[Ca2+]i
signals in the osteoblast by NO may account for the inhibitory effect
of this second messenger on osteoclastic bone resorption (22, 40).
It is noteworthy that contrary to the direct effect of NO on
osteoclasts, which is not mediated by cGMP (27), the stimulatory effect
of NO on hormonally induced Ca2+
transients in osteoblasts could be mimicked by permeable cGMP analogs
and could be abrogated by using inhibitors of soluble guanylate cyclase
and inhibitors of cGMP-dependent protein kinase. It appears, therefore,
that NO acts independently on osteoclasts and osteoblasts by activating
different signal transduction pathways in each cell type.
In summary, our data show that inflammatory cytokines and NO affect
hormonally stimulated
[Ca2+]i
responses in opposite directions. These changes may bear relevance for
the biological functions of cytokines and NO in bone (namely, a
catabolic effect exerted by cytokines and an antiresorptive effect
played by NO). The stimulation of NO production by cytokines in
osteoblasts may downmodulate the detrimental effects of cytokines in
bone. This may be relevant to states characterized by an excess of
local production of cytokines in bone, such as may be seen during
inflammation (e.g., rheumatoid arthritis) or metastatic bone disease.
 |
ACKNOWLEDGEMENTS |
We thank Michal Bross and Ruby Snyder for excellent secretarial
assistance in the preparation of the manuscript.
 |
FOOTNOTES |
This work was supported by an institutional grant from the National
Kidney Foundation of Southern California.
Address for reprint requests: J. Green, Dept. of Nephrology, Rambam
Medical Center, Haifa 31096, Israel.
Received 20 October 1997; accepted in final form 20 February 1998.
 |
REFERENCES |
1.
Abu-Amer, Y.,
F. P. Ross,
J. Edwards,
and
S. L. Teitelbaum.
Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor.
J. Clin. Invest.
100:
1557-1565,
1997[Abstract/Free Full Text].
2.
Ago, J. M.,
P. P. Stashenko,
and
F. E. Dewhirst.
Osteoclast-activating factor and parathyroid hormone are synergistic in stimulating bone resorption.
J. Dent. Res.
65:
1645-1651,
1986.
3.
Akatsu, T.,
N. Takahashi,
N. Udagawa,
K. Imamura,
A. Yamaguchi,
K. Sato,
N. Nagata,
and
T. Suda.
Role of prostaglandins in interleukin-1-induced bone resorption in mice in vitro.
J. Bone Miner. Res.
6:
183-189,
1991[Medline].
4.
Bellezzo, J. M.,
R. S. Britton,
B. R. Bacon,
and
E. S. Fox.
LPS-mediated NF-
B activation in rat Kupffer cells can be induced independently of CD14.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G956-G961,
1996[Abstract/Free Full Text].
5.
Berridge, M. J.,
J. P. Heslop,
R. F. Irvine,
and
K. D. Brown.
Inositol triphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.
Biochem. J.
222:
195-201,
1984[Medline].
6.
Bertolini, D. R.,
G. E. Nedwin,
T. S. Bringman,
D. D. Smith,
and
G. R. Mundy.
Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factors.
Nature
319:
516-518,
1986[Medline].
7.
Blind, E.,
V. Knappe,
F. Raue,
J. Pfeilschifter,
and
R. Ziegler.
Tumor necrosis factor-
inhibits the stimulatory effect of the parathyroid hormone-related protein on cyclic AMP formation in osteoblast-like cells via protein kinase C.
Biochem. Biophys. Res. Commun.
182:
341-347,
1992[Medline].
8.
Butt, E.,
M. van Bemmelin,
L. Fischer,
U. Walter,
and
B. Jastorff.
Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates.
FEBS Lett.
263:
47-50,
1990[Medline].
9.
Canalis, E.
Interleukin-1 has independent effects on DNA and collagen synthesis in cultures of rat calvariae.
Endocrinology
118:
74-81,
1986[Abstract].
10.
Canalis, E.
Effects of tumor necrosis factor on bone formation in vitro.
Endocrinology
121:
1596-1604,
1987[Abstract].
11.
Canalis, E.,
T. McCarthy,
and
M. Centrella.
Growth factors and the regulation of bone remodeling.
J. Clin. Invest.
81:
277-281,
1988[Medline].
12.
Civitelli, R.,
I. R. Reid,
S. Westbrook,
L. V. Avioli,
and
K. A. Hruska.
Parathyroid hormone elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line.
Am. J. Physiol.
255 (Endocrinol. Metab. 18):
E660-E667,
1988[Abstract/Free Full Text].
13.
Clemens, W. G.,
M. Lowik,
P. H. Nibbering,
M. van de Ruit,
and
S. E. Papapoulos.
Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.
J. Clin. Invest.
93:
1465-1472,
1994[Medline].
14.
Damoulis, P. D.,
and
P. V. Hauschka.
Cytokines induce nitric oxide production in mouse osteoblasts.
Biochem. Biophys. Res. Commun.
201:
924-931,
1994[Medline].
15.
Dayer, J. M.,
B. Beutler,
and
A. Cerami.
Cachetin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts.
J. Exp. Med.
162:
2163-2168,
1985[Abstract].
16.
Ecarot-Charrier, B.,
F. H. Glorieux,
M. van der Rest,
and
G. Perreira.
Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture.
J. Cell Biol.
96:
639-643,
1983[Abstract].
17.
Geng, Y.,
J. Valbracht,
and
M. Lotz.
Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes.
J. Clin. Invest.
98:
2425-2430,
1996[Abstract/Free Full Text].
18.
Gowen, M.
Interleukin 1 and tumor necrosis factor.
In: Cytokines and Bone, edited by M. Gowen. Boca Raton, FL: CRC, 1992, p. 72-91.
19.
Gowen, M.,
K. Chapman,
A. Littlewood,
D. Hughes,
D. Evans,
and
G. Russell.
Production of tumor necrosis factor by human osteoblasts is modulated by other cytokines, but not by osteotropic hormones.
Endocrinology
126:
1250-1255,
1990[Abstract].
20.
Grynkiewicz, G.,
M. Peonie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3340-3450,
1985.
21.
Hanevold, C. D.,
D. T. Yamaguchi,
and
S. C. Jordan.
Tumor necrosis factor-
modulates parathyroid hormone action in UMR-106-01 osteoblastic cells.
J. Bone Miner. Res.
8:
1191-1200,
1993[Medline].
22.
Ivey, J. L.,
D. R. Wright,
and
A. H. Tashjian.
Bone resorption in organ culture. Inhibition by the divalent cation ionophores A23187 and X537A.
J. Clin. Invest.
58:
1327-1338,
1976[Medline].
23.
Katz, M. S.,
G. E. Gutierrez,
G. R. Mundy,
T. K. Hymer,
M. P. Caulfield,
and
R. L. McKee.
Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by downregulating parathyroid hormone receptors.
J. Cell. Physiol.
153:
206-213,
1992[Medline].
24.
Knopf, H. P.,
F. Otto,
R. Engelhardt,
M. A. Freudenberg,
C. Galanos,
F. Herrmann,
and
R. R. Schumann.
Discordant adaptation of human peritoneal macrophages to stimulation by lipopolysaccharide and the synthetic lipid A analog SDZ MRL 953: downregulation of TNF-alpha and IL-6 is paralleled by an upregulation of IL-1beta and granulocyte colony-stimulating factor expression.
J. Immunol.
153:
287-294,
1994[Abstract/Free Full Text].
25.
Kolesnick, R.,
and
D. W. Golde.
The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.
Cell
77:
325-328,
1994[Medline].
26.
Lowenstein, C. J.,
and
S. H. Snyder.
Nitric oxide, a novel biological messenger.
Cell
70:
705-707,
1992[Medline].
27.
MacIntyre, I.,
M. Zaidi,
A. S. M. Towhidul Alam,
H. K. Datta,
B. S. Moonga,
P. S. Lidbury,
M. Hecker,
and
J. R. Vane.
Osteoclastic inhibition: an action of nitric oxide not mediated by cGMP.
Proc. Natl. Acad. Sci. USA
88:
2936-2940,
1991[Abstract].
28.
Malinin, N. L.,
M. P. Boldin,
A. V. Kovalenko,
and
D. Wallach.
MAP3K related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1.
Nature
385:
540-544,
1997[Medline].
29.
Mathison, J. C.,
G. D. Virca,
E. Wolfson,
P. S. Tobias,
K. Glaser,
and
R. J. Ulevitch.
Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages.
J. Clin. Invest.
85:
1108-1118,
1990[Medline].
30.
Mathison, J.,
E. Wolfson,
S. Steinemann,
P. Tobias,
and
R. Ulevitch.
Lipopolysaccharide (LPS) recognition in macrophages: participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.
J. Clin. Invest.
92:
2053-2059,
1993[Medline].
31.
McSheehy, P. M. J.,
and
T. J. Chambers.
Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone.
Endocrinology
118:
824-828,
1986[Abstract].
32.
McSheehy, P. M. J.,
and
T. J. Chambers.
1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption.
J. Clin. Invest.
80:
425-429,
1987[Medline].
33.
Mengozzi, M.,
and
P. Ghezzi.
Defective tolerance to the toxic and metabolic effects of interleukin 1.
Endocrinology
128:
1668-1672,
1991[Abstract].
34.
Mulsch, A.,
A. Luckhoff,
U. Pohl,
R. Busse,
and
E. Bassenge.
LY 83583 (6-anilino-5,8-quinolinedione) blocks nitrovasodilator-induced cyclic GMP increases and inhibition of platelet activation.
Naunyn-Schmiedeberg's Arch. Pharmacol.
340:
119-125,
1989[Medline].
35.
Nanes, M. S.,
J. Rubin,
L. Titus,
G. N. Hendry,
and
B. Catherwood.
Tumor necrosis factor alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism.
Endocrinology
128:
2577-2582,
1991[Abstract].
36.
Partridge, N. C.,
D. Alcorn,
V. P. Michelangeli,
G. Ryan,
and
T. J. Martin.
Morphological and biochemical characterization of four clonal osteogenic sarcoma cells of rat origin.
Cancer Res.
43:
4308-4312,
1983[Abstract].
37.
Pfeilschifter, J.,
C. Chenu,
A. Bird,
G. R. Mundy,
and
G. D. Roodman.
Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclastlike cells in vitro.
J. Bone Miner. Res.
4:
113-118,
1989[Medline].
38.
Putney, J. W., Jr.
(Editor).
Advances in Second Messenger and Phosphoprotein Research. New York: Raven, 1992, vol. 26, p. 143-160.
39.
Raisz, L. G.,
K. Nuki,
C. B. Alander,
and
R. G. Craig.
Interactions between bacterial endotoxin and other stimulators of bone resorption in organ culture.
J. Periodont. Res.
16:
1-6,
1981[Medline].
40.
Raisz, L. G.,
C. L. Trummel,
and
H. Simmons.
Induction of bone resorption in tissue culture: prolonged response after brief exposure to parathyroid hormone or 25-hydroxycholecalciferol.
Endocrinology
90:
744-751,
1972[Medline].
41.
Randriamampita, C.,
and
R. Y. Tsien.
Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx.
Nature
364:
809-814,
1993[Medline].
42.
Rizzo, M. T.,
and
C. Carlo-Stella.
Arachidonic acid mediates interleukin-1 and tumor necrosis factor-alpha- induced activation of the C-Jun amino terminal kinases in stromal cells.
Blood
88:
3792-3800,
1996[Abstract/Free Full Text].
43.
Saklatvala, J.,
W. Davis,
and
F. Guesdon.
Interleukin 1 (IL-1) and tumor necrosis factor (TNF) signal transduction.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
351:
151-157,
1996[Medline].
44.
Schneider, H. G.,
E. H. Allan,
J. M. Mosely,
T. J. Martin,
and
D. M. Findlay.
Specific down-regulation of parathyroid hormone (PTH) receptors and responses to PTH by tumor necrosis factor-
and retinoic acid in UMR 106-06 osteoblast-like osteosarcoma cells.
Biochem. J.
280:
451-457,
1991[Medline].
45.
Schutze, S.,
T. Machleidt,
and
M. Kronke.
The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction.
J. Leukoc. Biol.
56:
533-541,
1994[Abstract].
46.
Shapiro, S.,
D. N. Tatakis,
and
R. Dziak.
Effects of tumor necrosis factor alpha on parathyroid hormone-induced increases in osteoblastic cell cyclic AMP.
Calcif. Tissue Int.
46:
60-62,
1990[Medline].
47.
Simpson, A. W.,
A. Stampfl,
and
C. C. Ashley.
Evidence for receptor-mediated bivalent-cation entry in A 10 vascular smooth muscle cells.
Biochem. J.
267:
277-280,
1990[Medline].
48.
Smith, D. D.,
M. Gowen,
and
G. R. Mundy.
Effect of interferon-
and other cytokines on collagen synthesis in fetal rat bone cultures.
Endocrinology
120:
2494-2499,
1987[Abstract].
49.
Thomson, B. M.,
G. R. Mundy,
and
T. J. Chambers.
Tumor necrosis factors
and
induce osteoblastic cells to stimulate osteoclastic bone resorption.
J. Immunol.
138:
775-779,
1987[Abstract/Free Full Text].
50.
Thomson, B. M.,
J. Saklatvala,
and
T. J. Chambers.
Osteoblasts mediate interleukin 1 responsiveness of bone resorption by rat osteoclasts.
J. Exp. Med.
164:
104-112,
1986[Abstract].
51.
Uehara, Y.,
and
H. Fukazawa.
Use and selectivity of herbimycin A as inhibitor of protein-tyrosine kinase.
Methods Enzymol.
201:
370-379,
1991[Medline].
52.
West, M. A.,
T. Bennet,
S. C. Seatter,
L. Clair,
and
J. Bellingham.
LPS pretreatment reprograms macrophage LPS-stimulated TNF and IL-1 release without protein tyrosine kinase activation.
J. Leukoc. Biol.
61:
88-95,
1997[Abstract].
53.
Xin, X.,
R. A. Star,
G. Tortorici,
and
S. Muallem.
Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx.
J. Biol. Chem.
269:
12645-12653,
1994[Abstract/Free Full Text].
54.
Yamaguchi, D. T.,
J. Green,
C. R. Kleeman,
and
S. Muallem.
Properties of the depolarization activated calcium and barium entry in osteoblast-like cells.
J. Biol. Chem.
264:
197-204,
1989[Abstract/Free Full Text].
55.
Yamaguchi, D. T.,
J. Green,
C. R. Kleeman,
and
S. Muallem.
Characterization of stretch-activated calcium permeating channels in the osteosarcoma cell line UMR-106.
J. Biol. Chem.
264:
4383-4390,
1989[Abstract/Free Full Text].
56.
Yamaguchi, D. T.,
J. Green,
C. R. Kleeman,
and
S. Muallem.
Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.
Cell Calcium
12:
609-622,
1991[Medline].
57.
Yamaguchi, D. T.,
T. J. Hahn,
T. G. Beeker,
C. R. Kleeman,
and
S. Muallem.
Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells.
J. Biol. Chem.
263:
10745-10753,
1988[Abstract/Free Full Text].
58.
Yamaguchi, D. T.,
T. J. Hahn,
A. I. Klein,
C. R. Kleeman,
and
S. Muallem.
Parathyroid hormone activated calcium channels in an osteoblast-like clonal osteosarcoma cell line: cAMP-dependent and cAMP-independent calcium channels.
J. Biol. Chem.
262:
7711-7718,
1987[Abstract/Free Full Text].
59.
Yamaguchi, D. T.,
C. R. Kleeman,
and
S. Muallem.
Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106.
J. Biol. Chem.
262:
14967-14973,
1987[Abstract/Free Full Text].
60.
Yanaga, F.,
A. Masayoshi,
K. Toshitaka,
and
H. Masato.
Signal transduction by tumor necrosis factor
is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.
J. Biol. Chem.
267:
5114-5121,
1992[Abstract/Free Full Text].
61.
Yoneda, T.,
C. Lowe,
C. H. Lee,
G. Gutierrez,
M. Niewolna,
P. J. Williams,
E. Izbicka,
Y. Uehara,
and
G. R. Mundy.
Herbimycin A, a pp60src-c tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.
J. Clin. Invest.
91:
2791-2795,
1993[Medline].
Am J Physiol Cell Physiol 274(6):C1686-C1698
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