Inflammatory cytokines (IL-1alpha , TNF-alpha ) 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
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Abstract
Introduction
Methods
Results
Discussion
References

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)-1alpha , tumor necrosis factor (TNF)-alpha , 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-1alpha , TNF-alpha , 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-1alpha ; tumor necrosis factor-alpha ; lipopolysaccharide; nitric oxide; intracellular calcium

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha and IL-1beta , which have similar biological potency, as well as TNF-alpha , 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-alpha and LPS induce nitric oxide (NO) production in osteoblastic cells. This effect is enhanced by IL-1alpha (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-alpha , IL-1alpha , 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-alpha 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-1alpha , TNF-alpha , 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
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -MEM. Cells were pelleted by centrifugation at 800 g for 5 min. The yielded cells were plated in alpha -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)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> × <FENCE><FR><NU>(R − R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB> − R)</DE></FR></FENCE> × <FR><NU>Sf<SUB>2</SUB></NU><DE>Sb<SUB>2</SUB></DE></FR>
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-1alpha was purchased from Genzyme (Cambridge, MA). TNF-alpha 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
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Abstract
Introduction
Methods
Results
Discussion
References

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-alpha , LPS, and IL-1alpha 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-alpha (100 ng/ml), LPS (10 ng/ml), or IL-1alpha (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-1alpha 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.

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-alpha , LPS, IL-1alpha , 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-alpha , 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-alpha (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.

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-alpha (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-alpha 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-1alpha showed a similar effect to that of TNF-alpha (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-alpha (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.

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-alpha (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-1alpha (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-alpha , LPS, or LPS + TNF-alpha 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.

The experiments described in Figs. 1-4 indicate that IL-1alpha , TNF-alpha , 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 (bullet ) or 10 ng/ml LPS (open circle ). 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.

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-alpha (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.

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-alpha , LPS, and IL-1alpha 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.

                              
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Table 1.   Effect of cytokines on hormonally induced Ca2+ transients in cultured calvarial osteoblasts

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-1alpha (10 ng/ml) and TNF-alpha (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-alpha and LPS on PTH (10-8 M)-induced Ca2+ transients was dose dependent. The effect of TNF-alpha 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-1alpha (data not shown). The maximal effect of IL-1alpha 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-alpha 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.

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-alpha and IL-1alpha 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.

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-1alpha , TNF-alpha , and LPS on phosphatidylinositol metabolism. Figure 9 describes the effect of LPS and IL-1alpha 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-1alpha (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-1alpha -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-1alpha -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-alpha 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)-1alpha (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.

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.

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).

                              
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Table 2.   Effect of LPS on PTH- and PGE2-induced antimitotic response in calvarial osteoblasts

In contrast to the effect of LPS, IL-1alpha did not modulate the hormonally (PGE2, PTH) induced antimitotic effect. TNF-alpha (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-alpha and IL-1alpha affect hormonally induced cAMP generation in addition to their effect on [Ca2+]i. Table 3 describes the effect of LPS, TNF-alpha , and IL-1alpha 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-1alpha downregulated cAMP production by both receptor (PTH, PGE2) and postreceptor mechanisms. TNF-alpha inhibited PTH-mediated cAMP generation while having no effect on cAMP production mediated by PGE2, cholera toxin, or forskolin.

                              
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Table 3.   Effect of cytokines on agonist-induced cAMP production

Role of NO in mediating the effects of cytokines on [Ca2+]i. IL-1, TNF-alpha , 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.

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.

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-1alpha and TNF-alpha . 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha , TNF-alpha , 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-alpha , 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., NFkappa 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 NFkappa 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-alpha did not influence the antimitotic effect of PTH, whereas IL-1alpha 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-alpha ) 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-alpha , LPS, and interferon-gamma 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-alpha , IL-1alpha , 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-alpha and IL-1alpha , 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-alpha 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-alpha , IL-1alpha , 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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