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Endocrinology Vol. 141, No. 8 2995-3005
Copyright © 2000 by The Endocrine Society

ARTICLES

A Common Downstream Signaling Activity of Osteoclast Survival Factors That Prevent Nitric Oxide-Promoted Osteoclast Apoptosis1

Kazuhiro Kanaoka, Yasuhiro Kobayashi, Fumio Hashimoto, Tomoki Nakashima, Mitsue Shibata, Kazuhide Kobayashi, Yuzo Kato and Hideaki Sakai

Department of Orthodontics (K.Ka., Y.Ko., F.H., K.Ko.), Department of Pharmacology (T.N., M.S., Y.Ka., H.S.), Nagasaki University School of Dentistry, Nagasaki 852-8588, Japan

Address all correspondence and requests for reprints to: Hideaki Sakai, DDS., Ph.D., Department of Pharmacology, Nagasaki University School of Dentistry, 1–7-1, Sakamoto, Nagasaki 852-8588, Japan. E-mail: h-sakai{at}net.nagasaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment with NO-releaser NOC18 significantly promoted apoptosis in murine osteoclast-like cells, with a transient increase in caspase-3-like protease activity. In contrast, the apoptosis was protected against by caspase inhibitors, most efficiently with the broadly acting caspase specific inhibitor z-Asp-CH2-DCB, indicating involvement of multiple caspases in progression of the apoptosis. Among osteoclast survival factors examined, calcitonin completely protected against morphologically defined-apoptosis and the increase of caspase-3-like protease activity. The effect of calcitonin was mimicked by treatment of cells with (Bu)2cAMP and forskolin, and abolished by protein kinase-A inhibitor H-89. Independently from the PKA activation, colony stimulating factor-1, interleukin-1ß and the receptor activator of NF-{kappa}B ligand also protected against the apoptosis but were less effective than calcitonin. All survival factors investigated inhibited conversion of procaspases-3 and -9 to their mature forms in the cells. Thus, downstream antiapoptotic signaling activity from each factor overlapped in inhibition of caspases. However, how this was attained seemed to be different from each other. Typically, only colony stimulating factor-1 up-regulated expression of endogenous caspase inhibitor protein, X-linked inhibitor of apoptosis (XIAP), in the osteoclast-like cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VIABILITY of most mammalian cells depends on survival factors that activate signal transduction pathways that suppress apoptosis (1). Osteoclasts, bone resorbing multinuclear giant cells of hematopoietic origin, must be included among these cells for the following reasons. Mature osteoclasts do not synthesize DNA and have no mitotic activity, hence, further proliferation before death is not possible (2). The mode of death in cultured osteoclasts satisfies the morphological and biochemical criteria of apoptosis (3, 4). Onset of osteoclast apoptosis is known to be regulated by osteoclast survival factors, such as colony stimulating factor (CSF)-1 (also called M-CSF), interleukin (IL)-1 and the receptor activator of NF-{kappa}B ligand (RANKL) (also called OPGL, TRANCE, and ODF) (5, 6, 7, 8).

These survival factors are expressed in osteoblastic-stromal cells adjacent to osteoclasts and each bind to distinct receptors on osteoclasts or their precursors. Although these factors can prevent spontaneous osteoclast apoptosis, the major intracellular signaling pathways seemed to independent. Both RANKL and IL-1 activate NF-{kappa}B and c-Jun N-terminal kinase (JNK) via TRAF family members (7, 8), whereas CSF-1 induces activation of several kinases through binding SH2 containing proteins, including phosphatidylinositol-3 kinase, to autophosphorylated CSF-1 receptor, c-fms (9, 10). Besides these local factors, calcitonin, a calciotropic hormone, has been reported to protect against spontaneous osteoclast apoptosis (11). Calcitonin activates both protein kinase-C and protein kinase-A (PKA) through a G protein-coupled calcitonin receptor. Thus, a complexity exists in the whole network of anti-apoptotic signaling activity in osteoclasts, and the molecular basis of how each osteoclast survival factor exerts its function is almost unknown.

In this study, we tried to find a common intracellular signaling activity delivered from several distinct antiapoptotic stimuli for osteoclasts. To this end, we first established a reproducible experimental system to observe osteoclast apoptosis. To date, many reagents such as bisphosphonate, vitamin K2, estrogen, glucocorticoid, inhibitors of NF-{kappa}B, inhibitors of vacuolar-type ATPase and TGF-ß1 have been shown to promote osteoclast apoptosis (12, 13, 14, 15, 16, 17, 18). In this study, we used the NO-releaser NOC18 (19). NO has been suggested to be a mediator of bone turnover with dual roles. Constitutive production of small amounts of NO seems to be necessary for normal osteoclast function. In contrast, a high concentration of NO strongly inhibits bone resorption, both in organ cultures and in cultures of isolated osteoclasts (20). The effect of antibone resorption by NO could be due to promoting apoptosis in osteoclasts because high concentrations of NO releasers have been reported to induce apoptosis in many cell types (21). In a preliminary experiment, we found that NOC18 efficiently promoted apoptosis, as determined by morphological means, in osteoclast-like cells developed by in vitro coculture of murine calvaria-derived osteoblastic cells and bone marrow cells. Most of the apoptotic cells lost substrate attachment and were released into culture media. Because of its efficiency and high reproducibility to promote osteoclast apoptosis, we performed further experiments using NOC18.

We first characterized NO-promoted apoptosis in murine osteoclast-like cells. Next, we examined the effects of various osteoclast survival factors on NO-promoted apoptosis to elucidate (1) the extent of the antiapoptotic action among the survival factors, and (2) a common downstream activity delivered from these factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
NOC18 was purchased from Dojin (Kumamoto, Japan). Disodium dihydrogen (cycloheptylamino)-methylene-1,1-bisphosphonate (incadronate, which is also known as YM175) was kindly provided by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). Acetyl-Asp-Glu-Val-Asp-4-methylcoumaryl-7-amide (Ac-DEVD-MCA), Carbobenzoxy-Asp-CH2-[(2,6-dichlorobenzoyl)oxy]methane (z-D-CH2-DCB) and calcitonin gene-related peptide (CGRP) were obtained from Peptide Institute Inc. (Osaka, Japan). z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk) and z-Ile-Glu-Thr-Asp-fmk (z-IETD-fmk) were obtained from Enzyme System Products (Livermore, CA). (Bu)2cAMP and 8-chlorophenylthio-cAMP (8-CPT-cAMP) were purchased from Sigma (St. Louis MO). The following reagents were from each company: z-Leu-Pro-Asp-CH2-DCB (z-LPD-CH2-DCB), Phoenix Pharmaceuticals, Inc. (Mountain View, CA); forskolin, Wako Life Science (Osaka, Japan); H-89, BIOMOL Research Laboratories, Inc. (Plymouth, PA; Pronase E, Calbiochem (La Jolla, CA); collagenase (type 3), Worthington Biochemical Corp. (Freehold, NJ), respectively. All other reagents used were of analytical grade.

Preparation of murine osteoclast-like cells and application of NOC18
Murine osteoclast-like cells were developed on collagen gel plates by the coculture of murine calvaria-derived osteoblastic cells and bone marrow cells in the presence of 1,25-(OH)2D3 (1 x 10-8 M, kindly provided from Teijin Co., Japan) and prostaglandin E2 (1 x 10-6 M, Sigma) according to the method described by Jimi et al. (8). On day 6 of the coculture, part of the stromal cells were removed by 0.002% Pronase E/0.02% EDTA treatment. Osteoclast-like cells together with stromal cells left behind were detached from plates by 0.1% collagenase treatment. The pronase treatment was indispensable to reduce clumping of cells. The cells were resuspended in fresh {alpha}-minimum essential medium ({alpha}MEM, Life Technologies, Inc., Gaithersburg, MD) containing 10% FCS, and replaced onto dentine disks set in wells of 96-well plates or replated in 8-well plastic LAB-TEK chamber slides (Nalge Nunc International, Naperville, IL). Before replating, 50 µl of the suspension was spotted onto a plastic dish ({phi} = 35 mm) and incubated for 15 min. Within this period, osteoclast-like cells were attached to the dish and could be counted. About 400 osteoclast-like cells were plated in each well. After 2 h of preincubation, few unattached cells were removed by a brief rinse with culture media ({alpha}MEM/10% FCS). The dentine disks were further transferred to wells of 48-well plates. The purity of osteoclast-like cells in the mixed cell population was about 3%. At bench side, NOC18 was dissolved at 20 mM with ice-cold culture media, filter sterilized, and rapidly diluted to the desired final concentration with culture media. Four hundred microliters of the NOC18 containing media was added to each well containing preset osteoclasts and incubated for up to 48 h. The effects of caspase inhibitors and osteoclast survival factors, such as synthetic eel-calcitonin (elcatonin, a kind gift from Asahi Chemical Industry Co. Tokyo, Japan), recombinant murine IL-1ß (Upstate Biotechnology, Inc. Lake Placid, NY), recombinant murine CSF-1 (R&D Systems, Minneapolis, MN), recombinant human RANKL (PeproTechEC, London, UK), were examined by adding these factors together with NOC18.

Preparation of rabbit osteoclasts
Primary mature osteoclasts were prepared from male rabbit (Japan White, ~110 g: Biotech, Saga, Japan) long bones according to our previous report (22). Briefly, the unfractionated bone cells were seeded on a collagen gel for 2.5 h. Stromal cells were removed by digestion with 0.002% actinase E (Kaken Pharmaceutical Co., Kyoto, Japan) and 0.01% collagenase (Type 3: Worthington Biochemical Corp.), followed by repeated washing with PBS. Mature osteoclasts remained behind but were finally detached from the gel with 0.1% collagenase treatment. The average purity of prepared osteoclasts was 91.3 ± 3.8% (mean ± SD, n = 3). The cells were resuspended in {alpha}MEM/10% FCS and about 400 osteoclasts were replaced onto a dentine disk set in a well of a 96-well plate. Further proceedings were the same as that performed for murine osteoclast-like cells.

Morphological analysis
Most of the cells committed to the apoptosis in the incubation lost substrate attachment and were released into the culture media. After incubation, culture media were collected and cell supports (dentine disks or plastic bottom) were briefly washed with PBS. The media and washing solutions were combined, then cells in the solution were fixed by adding an equal volume of 8% paraformaldehyde (PFA)/PBS and kept for 1 h on ice. The fixed cells were trapped onto nitrocellulose membranes set in a 96-well vacuum device (ATTO, Tokyo, Japan) and stained for tartrate-resistant acid phosphatase (TRAPase) activity followed by nuclear staining with Hoechst 33258 (1 µg/ml in PBS) (18, 22). Attached cells on cell supports were fixed with 4% PFA/PBS for 1 h on ice and subjected to TRAPase staining and Hoechst staining. The stained cells, both on nitrocellulose membranes and on cell supports, were examined under a fluorescence microscope (BH-2, Olympus Corp., Tokyo, Japan).

Some cell preparations were subjected to a single-cell gel electrophoresis assay (Comet assay, Trevigen, Gaithersburg, MD) to detect DNA fragmentation (23). Briefly, cells were fixed with methanol, embedded in 0.4% low-temperature melting agarose, and put on a glass slides. The immobilized cells in the agarose were subjected to electrophoresis at 1 V/cm for 5 min, and the DNA was stained with SYBR Green. Fragmented nuclear DNA migrated from the cells after electrophoresis and exhibited a comet-like pattern.

Apoptotic osteoclasts were identified by the criteria defined by Hughes et al. (18), i.e. by the presence of chromatin condensation and/or nuclear fragmentation in osteoclasts also showing cytoplasmic concentration and/or fragmentation. Necrotic osteoclasts were defined as the cells with nuclear and cytoplasmic swelling and pallor without nuclear disintegration. Percentage of apoptotic osteoclasts was defined as:

Measurement of caspase-3-like protease activity
Murine osteoclast-like cells in the mixed cell population were prepared as described above and replated on plastic plates (60 mm diameter). The cells were treated with 600 µM NOC18 in the presence or absence of survival factors. After a proper chase time, culture media were collected and plates were briefly rinsed with PBS. Cells in the media and washing solutions (detached cells) were collected by centrifugation at 700 x g for 5 min. Stromal cells on the plates were removed by pronase treatment (6). Osteoclast-like cells remaining on the plates and the detached cells were combined and lyzed in a cell lysis buffer (1% Triton-X100/1 mM dithiothreitol/50 mM KCl/5 mM EDTA/20 µM cytochalasin B/2 mM phenylmethylsulfonyl fluoride/1 µg/ml leupeptin/1 µg/ml pepstatin/10 µg/ml antipain/3 µg/ml chymostatin/10 mM HEPES, pH 7.5). Ac-DEVD-MCA hydrolase activity in the cell lysates was measured according to our previous report (24). Free MCA generated after incubation was detected with excitation at 380 nm and emission at 460 nm. One unit (U) of the enzymatic activity was defined as the amount of enzyme releasing 1 pmol MCA in 1 min.

Immunoblotting
Cell lysates of osteoclast-like cells were prepared as described above, and the protein concentration of each sample was measured with micro BCA protein assay reagent (Pierce Chemical Co., Rockford, IL). The samples were denatured in SDS-solubilizing buffer (2% SDS/100 mM dithiothreitol/10% glycerol/0.0025% bromophenol blue in 62.5 mM Tris-HCl, pH 6.8) and loaded to a 12% SDS-PAGE gel. Twenty micrograms of lysate protein were applied to each lane. After SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane, and immunostained with anti-procaspase-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-procaspase-3 (Upstate Biotechnology, Inc. Lake Placid, NY) anti-procaspase-9 (MBL, Nagoya, Japan), anti-XIAP (MBL, Nagoya, Japan), anti-cIAP1 (H-83, Santa Cruz Biotechnology, Inc.), anti-cIAP2 (H-85, Santa Cruz Biotechnology, Inc.), or anti-ß-actin antibody (Sigma), respectively, as previously described (22). The antigenic sites were detected with an enhanced ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Statistical analysis
The statistical differences among groups were evaluated using one-way ANOVA. Fisher’s protected least significant difference (Fisher’s PLSD) was used to identify differences between the groups when ANOVA indicated that a significant difference (P < 0.05 or P < 0.01) existed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Promotion of osteoclast apoptosis by NOC18
Murine osteoclast-like cells were generated by an in vitro murine co-culture method on collagen gels. The mixed cell populations were replated on dentine disks or the plastic bottom in 8-well LAB-TEK chamber slides and cultured for up to 48 h in {alpha}MEM/10% FCS in the presence or absence of NOC18. The osteoclast-like cells in the mixed cell population used in the experiments were shown in Fig. 1aGo.



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  		Figure 1. Morphology of normal and apoptotic murine osteoclast-like cells treated with NOC18. a–d: Double staining of TRAPase and Hoechst 33258. a, Murine osteoclast-like cells in a mixed cell population plated on plastic; b, a normal osteoclast-like cell on a dentine disk; c, an apoptotic cell on a dentine disk; d, an apoptotic cell appearing in culture media. Cells in media were fixed, trapped onto nitrocellulose membranes and stained. The apoptotic cells showed picnotic cell nuclei retaining significant TRAPase activity in the cytoplasm. e, Comet assay of an apoptotic cell appeared in cultured media. Fragmented nuclear DNA migrated laterally from the cell nuclei. Bar, 50 µm.

 
In the control culture, some osteoclast-like cells were spontaneously committed to apoptosis, showing cytoplasmic contraction and small condensed nuclei with fragmented DNA, as revealed by TRAPase/Hoechst staining and by Comet assay, respectively (data not shown). In the first-16 h of the control culture, about 20% of total osteoclast-like cells cultured on the plastic bottom were committed to apoptosis, whereas about 5% of cells cultured on dentine disks were apoptotic. Differences in the viability of osteoclasts depending on supporting materials was also previously noted (11, 12). The spontaneous apoptosis is committed to independently of endogenous NO production because NO-synthetase (NOS) inhibitor, L-arginine-methyl ester had no effect on its onset (data not shown).

Addition of NOC18 significantly promoted commitment to apoptosis in the osteoclast-like cells in a dose-dependent manner. The morphology of the apoptotic cells were shown in Fig. 1Go, which was indistinguishable from spontaneous apoptosis. The apoptotic cells showed contracted cytoplasm with sufficient TRAPase activity and small condensed nuclei (Fig. 1Go, c and d) in which nuclear DNA was fragmented as shown by Comet assay (Fig. 1eGo). The effect of NOC18 was observed at concentrations above 300 µM and about 85% of the osteoclast-like cells cultured on dentine disks were committed to apoptosis at 500 µM (Fig. 2aGo). Cells on dentine disks were slightly more susceptible to NO-induced apoptosis. Four hundred micromolar NOC18 induced apoptosis in more than 60% osteoclast-like cells cultured on dentine disks in the first 16 h-culture, whereas 600~800 µM of NOC18 was required on plastic to obtain the same effect (compare Fig. 2Go, panels a and b). Most of the osteoblastic cell population on dentine disks or on plastic were intact below 600 µM of NOC18. Less than 5% of the total osteoclast-like cell population showed a necrotic morphology, with irregular cell shape, swelling of nuclei and with faint TRAPase activity in this culture system (data not shown). Higher concentrations of NOC18 above 800 µM induced necrotic cell death, in which cells were swollen and irregularly deformed (data not shown). It should be noted that most of apoptotic osteoclast-like cells, after NOC18 treatment, lost substrate attachment and were released into the culture media. The percentage of total apoptotic cells increased in a time dependent manner as shown in Fig. 2cGo.



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  		Figure 2. Kinetics of NO-promoted apoptosis in murine osteoclast-like cells. a and b, The osteoclast-like cells in mixed cell populations were cultured on dentine disks (a) or on plastic LAB-TEK chamber slides (b) for 16 h in the presence of various concentration of NOC18. Cells were stained for TRAPase and Hoechst 33258, and apoptotic cells were counted under a fluorescence microscope. NOC18 potently induced apoptosis in osteoclast-like cells. Among apoptotic osteoclasts, the majority were found in culture media (the unattached cell population, shown in the dark gray box), and the remainder were found on cell supports (the attached population, shown in the light gray box). c, Time course of appearance of apoptotic osteoclast-like cells. Cells were cultured on dentine or on plastic for up to 48 h in the presence of NOC18 at the concentration indicated in the figure. Each experiment was repeated three to four times in quadruplicate wells and expressed as the mean ± SD. (*, P < 0.05; **, P < 0.01 against nontreated cells).

 
In the subsequent study, osteoclast-like cells cultured on dentine disks were mainly used for morphological observations, whereas cells cultured on plastic plates (60 mm diameter) were subjected to analysis of caspase activity or immunoblotting, because of large scale cell availability.

Involvement of caspases
Accumulating lines of evidence show that appearance of caspase-3-like protease activity is a common feature of apoptosis (25, 26, 27). To test this in the osteoclast-like cell apoptosis promoted by NO, we measured caspase-3-like protease activity using Ac-DEVD-MCA as the substrate. After exposure to 600 µM of NOC18, the activity was rapidly increased up to 8 h and decreased afterward (Fig. 3Go). Only 23% of osteoclast-like cells showed apoptotic morphology at 8 h, as shown in Fig. 2Go, but the value was continuously increased afterward. Thus, activation of caspase-3-like protease in osteoclasts preceded the morphological changes, as observed in many other cell populations (25, 26, 27).



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  		Figure 3. Time course of Ac-DEVD-MCA hydrolase activity in murine osteoclast-like cells treated with NOC18. Murine osteoclast-like cells were cultured on plastic plates (60 mm diameter) in the presence of 600 µM NOC18 for 3, 8, 16, and 36 h. Stromal cells on plates were removed with pronase treatment at each time point. The purified osteoclast-like cell population and cells recovered in culture media were combined and lyzed. Protease activity in the cell lysates was measured using Ac-DEVD-MCA which is relatively specific to caspase-3, but also cleaved by other caspases such as caspases-2, and -7. Results are expressed as the mean ± SD of three independent experiments.

 
The significance of caspase in the osteoclast apoptosis was also examined by coincubation of cells with several caspase inhibitors. All inhibitors used in this study protected the cells from apoptosis as shown in Fig. 4Go. Both z-DEVD-fmk (100 µM) and z-IETD-fmk (100 µM), which are relatively specific for caspases-3 and 8, respectively, reduced the percentage of morphologically fined apoptosis from about 60% to 35%. The broadly acting caspase specific inhibitor z-D-CH2-DCB (100 µM) was most effective (22%), although the process of apoptosis was not completely inhibited. The data may imply that NO-promoted apoptosis includes both caspase-dependent and -independent mechanisms or a limitation of the inhibitors to penetrate cytoplasm and interact with their targets. ZnSO4, which was recently shown to inhibit the caspase cascade (28), also significantly prevented osteoclast apoptosis.



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  		Figure 4. Effect of various caspase inhibitors on NO-promoted apoptosis in murine osteoclast-like cells. Cells were cultured on dentine disks for 16 h in the presence of NOC18 (400 µM) and various caspase inhibitors. Apoptosis was detected by morphological means. z-D-CH2-DCB, a broadly acting caspase specific inhibitor, was most effective among caspase inhibitors to inhibit the NO-promoted apoptosis. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD. (*, P < 0.05; **, P < 0.01 against nontreated cells)

 
Effects of calcitonin and survival factors
By now, several factors have been shown to maintain osteoclast survival. We first examined the effect of calcitonin and its related factors (Fig. 5aGo). Concomitant incubation of cells with calcitonin for 16 h markedly protected against NO-promoted apoptosis of osteoclast-like cells in a dose-dependent manner. The percentage of apoptotic cells was decreased to less than 8% at greater than 10-10 calcitonin. CGRP also exerted protective effects. The effect of calcitonin was mimicked by the membrane permeable cAMP analog (Bu)2cAMP (1 mM) or the activator for adenylate cyclase, forskolin (10 µM). Thus, activation of PKA strongly antagonizes NO-promoted apoptosis in osteoclast-like cells, not only preventing spontaneous osteoclast apoptosis (11). Although less effective than 16 h treatment, an initial 4 h treatment with calcitonin provided highly significant antiapoptotic action on osteoclast-like cells exposed to NOC18 (Fig. 5bGo).



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  		Figure 5. Effects of calcitonin and calcitonin-related factors on NO-promoted apoptosis in murine osteoclast-like cells. a, Cells were cultured on dentine disks for 16 h in the presence of NOC18 (400 µM) together with various concentrations of calcitonin or calcitonin-related factors. b, Cells were exposed to NOC 18 (400 µM) for 16 h while calcitonin at the indicated dose was present for only the initial 4 h of incubation. Apoptosis was detected by morphological means. Calcitonin showed a potent antiapoptosis effect on NO-promoted apoptosis in murine osteoclast-like cells. The effect was mimicked by CGRP, (Bu)2cAMP, and forskolin. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD. (*, P < 0.05, **, P < 0.01 against nontreated cells). CT, Calcitonin.

 
We next examined the effect of factors known to maintain survival of osteoclast-like cells. CSF-1, and RANKL effectively protect the cells from NO-promoted apoptosis in a dose-dependent manner, whereas IL-1ß was effective at the highest concentration tested (Fig. 6Go). We could not obtain plateau effects even at the highest concentration used, however, these factors seemed to be less effective than calcitonin when used at the same concentration. CSF-1 (500 ng/ml {cong}2 x 10-8 M), IL-1ß (500 ng/ml {cong} 4 x 10-8 M) and RANKL (200 ng/ml {cong} 7 x 10-9 M) were not as effective as that of 10-11 M calcitonin (compare Figs. 5Go and 6Go), although a combination of CSF-1 (500 ng/ml) and RANKL (200 ng/ml) was comparable to that of 10-11 M calcitonin (Fig. 6dGo).



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  		Figure 6. Effects of IL-1ß, CSF-1, and RANKL on NO-promoted apoptosis in murine osteoclast-like cells. Cell were cultured on dentine disks for 16 h in the presence of NOC18 (400 µM) and various amounts of IL-1ß (a), CSF-1 (b), RANKL (c), or a combination of CSF-1 and RANKL (d). Apoptosis was detected by morphological means. Each factor could circumvent NO-promoted apoptosis in osteoclast-like cells, although to a lesser extent than calcitonin. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD. (*, P < 0.05, **, P < 0.01 against nontreated cells).

 
The antiapoptotic effects of the survival factors were well correlated with the extent of inhibition of caspase-3-like protease activity, as shown in Fig. 7Go. Cell lysates used were prepared from osteoclast-like cells incubated for 8 h in the presence of NOC18 (600 µM) concomitant with various factors. Calcitonin, CGRP, (Bu)2cAMP, and 8-CPT-cAMP potently reduced the activity to a negligible level. The effect of calcitonin was abrogated by co-incubation with PKA inhibitor H-89, again indicating involvement of PKA in prevention of osteoclast apoptosis (Fig. 7aGo). Treatment of cells with calcitonin (10-8 M) for an initial 4 h significantly reduced the caspase-3-like protease activity (Fig. 7bGo), though to a lesser extent than incubation for 8 h. This was well correlated with the antiapoptotic effect of the short time calcitonin treatment (Fig. 5bGo).



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  		Figure 7. Effects of osteoclast survival factors on NO-induced Ac-DEVD-MCA hydrolase activity in murine osteoclast-like cells. Cells were cultured on plastic plates for 8 h in the presence of NOC18 (600 µM) and various indicated factors. a, Cells were coincubated with calcitonin and calcitonin-related factors. b, Calcitonin (10-8 M) was added for only the initial 4 h. c, Cells were coincubated with IL-1ß, CSF-1, or RANKL. Ac-DEVD-MCA hydrolase activity was measured as in Fig. 3Go. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD. (*, P < 0.05, **, P < 0.01 against nontreated cells).

 
IL-1ß, CSF-1, and RANKL also reduced the activation of caspase-3-like protease, but to a lesser extent than calcitonin or CGRP. At the concentration used, CSF-1 was the most effective cytokine employed, which was well correlated to the effectiveness of CSF-1 in prevention of apoptosis detected by morphological means compared with that of IL-1ß and RANKL (Fig. 6Go). The combination of CSF-1 and RANKL was more potent than a single factor.

We tested if NOC18 promote apoptosis in purified authentic rabbit osteoclasts. When rabbit osteoclasts were cultured on dentine disks in the presence of 400 µM NOC18, apoptosis was significantly promoted (Fig. 8Go). Moreover, NO-promoted rabbit osteoclast apoptosis was significantly protected by calcitonin, CSF-1, and RANKL (Fig. 8Go). If compared with NO-promoted apoptosis in murine osteoclast-like cells, a difference existed in (1) that more than 95% of apoptotic rabbit osteoclasts were found in the unattached cell population; (2) the effect of calcitonin was less than that observed in murine osteoclast-like cells.



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  		Figure 8. Effects of osteoclast survival factors on NO-promoted apoptosis in rabbit osteoclasts. Cells were cultured on dentine disks for 16 h in the presence of 400 µM NOC18 and various osteoclast survival factors indicated. Apoptosis was detected by morphological means. NOC18 potently promoted apoptosis in rabbit osteoclasts. The NO-promoted apoptosis was significantly protected with calcitonin, CSF-1, and RANKL. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD (*, P < 0.05, **, P < 0.01 against nontreated cells).

 
We also examined if the antiapoptotic action accompanied with inhibition of caspase-3-like protease activity by calcitonin, CSF-1 and RANKL is relevant to protection of apoptosis induced by a reagent other than NOC18. Incadronate (YM175), a nitrogen-containing bisphosphonate, has been shown to induce apoptosis in osteoclasts in vivo (29). In initial experiments, we found that incadronate at concentrations of 10~100 µM significantly promoted apoptosis in in vitro developed murine osteoclast-like cells (data not shown). Further experiments were performed three times with a single dose at 30 µM. When the cells were cultured on dentine disks in the presence of 30 µM incadronate, apoptosis was significantly promoted (Fig. 9aGo). The presence of calcitonin, CSF-1, and RANKL significantly protected the cells from the incadronate-promoted apoptosis, which was well correlated to the extent of inhibition of caspase-3-like protease activity in the cells by these factors (Fig. 9bGo).



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  		Figure 9. Effects of osteoclast survival factors on incadronate (YM175)-promoted apoptosis in murine osteoclast-like cells. a, Cells were cultured on dentine disks for 16 h in the presence of 30 µM incadronate and various osteoclast survival factors indicated. Apoptosis was detected by morphological means. Incadronate potently promoted apoptosis, which was significantly protected with calcitonin, CSF-1, and RANKL. Each experiment was repeated three times in quadruplicate wells and expressed as the mean ± SD (*, P < 0.05, **, P < 0.01 against nontreated cells). b, Cells were cultured on plastic plates for 8 h in the presence of 30 µM incadronate and various osteoclast survival factors. Ac-DEVD-MCA hydrolase activity was measured as in Fig. 3Go. Calcitonin, CSF-1, and RANKL significantly protected incadronate-induced Ac-DEVD-MCA hydrolase activity. Results are expressed as the mean ± SD of three independent experiments.

 
Conversion of procaspase-3 and levels of IAPs
Several caspases including caspases-2, -3, and -7 can liberate fluorochrome MCA from Ac-DEVD-MCA (30). Therefore, measurement of caspase activity using this substrate could not specify which caspase was affected by survival factors. To clarify this, we examined activation of each caspase by performing immunoblotting to detect the proform. It is generally accepted that caspase is synthesized as a proteolytically inert proenzyme. The enzyme gains its proteolytic activity after conversion from the proform to the mature form by autocatalytic or heterocatalytic processing to remove propeptides (30).

We have tried commercially available antibodies against procaspases-3, -2, and -9 and could obtain each corresponding protein in murine osteoclast-like cell lysates. Caspases-2 and -9 are classified as initiator caspases, whereas caspase-3 is an effector caspase downstream from them. As control apoptotic cells, we used calvaria-derived osteoblastic cells which were treated with UV to induce apoptosis (leftmost 2 lanes in Fig. 10aGo). During apoptosis, the band corresponding to procaspase-2 was unchanged (Fig. 10aGo, top panel). In contrast, the amount of procaspase-3 in osteoclast-like cells was decreased after exposure to NOC18. The reduction of procaspase-3 was restored by coincubation of the cells with caspase inhibitor z-D-CH2-DCB, calcitonin, CSF-1, and RANKL (Fig. 10aGo, second panel). This indicated that these factors prevent osteoclast apoptosis by affecting a site upstream of caspase-3. The amount of procaspase-9, which is activated through mitochondrial permeability transition (27), was slightly reduced by NO-treatment of osteoclast-like cells (Fig. 10aGo, third panel). The level of procaspase-9 was restored by calcitonin, CSF-1, and RANKL, but not by z-D-CH2-DCB.



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  		Figure 10. Immunoblotting analyses for procaspases-2, -3, -9, XIAP and cIAP2. a, Changes in apoptosis-related proteins in murine calvaria-derived osteoblastic cells (Osteoblasts) and murine osteoclast-like cells (Osteoclasts) committed to apoptosis. The osteoblastic cells were irradiated with 300 J/m2 UV and cultured in {alpha}MEM/10% FCS. More than 90% of the irradiated osteoblastic cells showed apoptotic morphology at the end of 24-h culture. Murine osteoclast-like cells were cultured on plastic plates in the presence of 600 µM NOC18 for the indicated time. In some cultures, cells were concomitantly incubated with z-D-CH2-DCB (100 µM), calcitonin (CT, 10-8 M), CSF-1 (500 ng/ml), RANKL (200 ng/ml), or RANKL (200 ng/ml) plus CSF-1 (500 ng/ml) for 24 h. Cell lysates were prepared as described in the Fig. 3Go legend and each 20 µg of cellular protein was subjected to SDS-PAGE, followed by immunoblotting for procaspases and IAPs shown. Detection was carried out with ECL kit. Cellular levels of procaspases-3, -9, XIAP and cIAP2 were decreased after induction of apoptosis, but were, in most cases, nearly sustained by osteoclast survival factors. The level of procaspase-2 was unchanged. b, CSF-1 increased the cellular level of XIAP in osteoclast-like cells in a dose-dependent manner. Osteoclast-like cells were cultured on plastic plates in the presence of various concentrations of CSF-1. Cell lysates were prepared after 24 h of culture and each 20 µg of cellular protein was subjected to immunoblotting for XIAP (upper panel) and ß-actin (lower panel). Exposure time of ß-actin blot to film was one-third of that of XIAP. c, CSF-1 increased cellular level of XIAP in osteoclast-like cells in a time-dependent fashion. Osteoclast-like cells were cultured on plastic plates in the presence of CSF-1 (250 ng/ml). Cell lysates were prepared at each time point and each 20 µg of cellular protein was subjected to immunoblotting for XIAP and ß-actin. Both proteins were detected on the same blot. Results of a–c are representative of three separate experiments, respectively, in which similar results were obtained.

 
We also tested whether changes were observed in levels of the endogenous inhibitors of apoptosis protein family, XIAP and cIAP2 (31, 32), in the osteoclast-like cells. So far, we could not detect cIAP1 in the osteoclast-like cells using commercially available anti-cIAP1 antibodies (data not shown). The cellular level of XIAP was gradually decreased during progression of apoptosis, but sustained by treatment of the cells with survival factors, although the effect of RANKL was marginal. Interestingly, the level of XIAP in the osteoclast-like cells treated with CSF-1 was apparently more than that in cells before incubation (Fig. 10aGo, fourth panel). The cellular level of cIAP2 was also slightly decreased during progression of apoptosis but sustained by treatment of the cells with survival factors including RANKL.

To further confirm the effect of CSF-1 in increasing XIAP, we cultured murine osteoclast-like cells on plastic plates in {alpha}MEM/10% FCS supplemented with different amounts of CSF-1 (Fig. 10bGo). Because the cells were committed to spontaneous apoptosis on the plastic, the XIAP level was decreased after 24 h incubation without CSF-1. The level of XIAP was apparently increased by CSF-1 treatment in a dose-dependent manner. A plateau effect was observed at a concentration of 250 ng/ml. The time course of XIAP induction by CSF-1 was also examined (Fig. 10cGo). At 8 h of incubation, CSF-1 treated osteoclast-like cells possessed more XIAP than untreated cells. To verify the accuracy of the amount of protein applied to each lane, ß-actin was immunologically detected on the same blot shown in Fig. 10cGo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis has been suggested as the key mechanism controlling the functional longevity of osteoclasts (33). To date, many researchers have noticed that isolated mature osteoclasts from bone are fragile and easily committed to apoptosis (2, 3, 4). This apoptotic death process is significantly promoted when serum in media is depleted or the cells are treated with various agents (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). In this study, we used NOC18, a slow NO releaser, to promote osteoclast apoptosis. One mole of NOC18 produces 2 mol of NO with a half-life of 21 h at pH 7.4 (37 C) (19). Most of our experiments were performed with 400 µM of NOC18. Theoretically, about 25 µM of NO was produced in the first 1 h at this concentration. This concentration of NO is quite high and may be hard to obtain in vivo. However, the morphological changes in osteoclasts undergoing induced and spontaneous apoptosis are indistinguishable.

Previously, van’t Hof and Ralston showed NO donors S-nitroso-acetyl-penicillamine and sodium nitroprusside at concentrations up to 500 µM were not sufficient to induce apoptosis in mature murine osteoclast-like cells developed in a coculture system, although precursors of osteoclasts were significantly affected (34). The contrast between our and Ralston’s results could be derived from differences in reagents used, cell culture conditions employed and the evaluation system used. In the present study, we performed morphological evaluation of cells both on solid substrate and in culture media, according to a proposal by Hughes et al. (18). As we showed here, more than 70% (average in all experiments) of the apoptotic cells lost substrate adhesion and were recovered in the media, therefore, evaluation of cells in media was necessary in our study. NOC18 promoted apoptosis in purified authentic rabbit osteoclasts, of which influences from other bone cells are minimized. Based on this, we considered that NO directly acted on mature osteoclasts to commit to apoptosis. However, as for osteoclast-like cells in the mixed cell population, we cannot exclude the possibility that some of the indirect action of NO is mediated through effects on stromal cells.

The mechanism by which NO induces apoptosis is not fully clarified. However, among various possibilities, the influence of NO on mitochondria could be an inducer of apoptosis (19, 21, 35). NO affects mitochondria in three principal ways: 1) reversible inhibition of respiration; 2) irreversible inactivation of mitochondrial enzymes; and 3) induction of the mitochondrial permeability transition. Involvement of the mitochondrial permeability transition in NO-induced apoptosis was supported in cells transfected with bcl-2, which were highly resistant to treatment with various NO-releasers and also to endogenously generated NO, compared with wild-type cells (36, 37). Bcl-2 prevents pore formation in mitochondria and protects mitochondrial permeability transition (38). The mitochondrial permeability transition induces a leak of cytochrome c from mitochondria and leads to the subsequent conversion of procaspase-9 to the mature form, via activation of the Apaf-1 molecule (26, 27). The activated caspase-9 processes downstream caspases, such as procaspases-3 and -7, which results in the breakdown of key enzymes and cellular structures leading to apoptosis. Because osteoclasts are rich in mitochondria and possess procaspases-3 and -9, mitochondrial permeability transition could be a pathway to evoke apoptosis in osteoclasts by NO. Supporting this notion, conversion of procaspases-3 and -9 during apoptosis was shown in the present study (Fig. 10aGo).

Calcitonin, to our surprise, almost completely protected the murine osteoclast-like cells from morphologically defined apoptosis after treatment with NOC18. Although to a lesser extent, the protective effect was also observed in NO-promoted apoptosis in authentic rabbit osteoclasts. The difference of the effect of calcitonin between the two cell types could be derived from (1) species differences, and (2) the amount of contaminating stromal cells. At this point, the mechanism how stromal cells are involved in the NOC18 promotion of osteoclast apoptosis is not clear and will be further investigated by us in future experiments.

It was shown, for the first time, that calcitonin potently inhibited the appearance of caspase-3-like protease activity in murine osteoclast-like cells treated with NOC-18. Similarly, inhibition of caspase activity by calcitonin was also observed in incadronate-promoted murine osteoclast-like cell apoptosis. Involvement of PKA in the prevention of apoptosis by calcitonin was confirmed using cAMP analogs, forskolin, and H-89, an inhibitor of PKA. Activation of PKA has also been shown to protect or retard the onset of apoptosis in Fas-treated primary hepatocytes, smooth muscle cells transfected with the c-myc gene and in in vitro-cultured human neutrophils that are spontaneously committed to apoptosis (39, 40, 41, 42). Thus, antiapoptotic action through activation of PKA seems to regulate a fundamental step in commitment of apoptosis in certain cell populations including osteoclasts, but not in all cell types (43). Our data showed that PKA lies at least in a site upstream of activation of procaspase-9 and procaspase-3. Concomitant treatment of cells with calcitonin for only an initial 4 h highly significantly protected the cells from NO-promoted apoptosis, which correlated with the inhibition of caspase-3-like protease activity (Figs. 5bGo and 7bGo). This means that inhibition of an initial transient increase of caspase activity may be critical to prevent apoptosis and the anti-apoptotic action of calcitonin is fairly long lasting. Stabilization of mitochondria could be a candidate to explain the antiapoptotic action of PKA because recent studies show that PKA is involved in phosphorylation and inactivation of BAD (44, 45). Phosphorylation of BAD by PKA results in an increase in the relative amount of functional Bcl-2 or Bcl-XL, both of which are dominant members in prevention of mitochondrial permeability transition.

CSF-1 also gave protection against NO-promoted osteoclast apoptosis, although not with the potency of calcitonin. Immunoblotting data showed CSF-1 sufficiently prevented NO-induced conversion of procaspases-3 and -9, indicating that the CSF-1 interaction point lies upstream of these enzymes. For a long time, CSF-1 has been suggested as being an osteoclast survival factor (5, 6), However, the actual effector of the antiapoptotic action under CSF-1 signaling was obscure. Here, we demonstrated for the first time that CSF-1 caused an increase in the antiapoptotic protein XIAP in osteoclasts. XIAP is a direct proteinaceous inhibitor of caspase-3 and caspase-7 (31). Recently, XIAP has been shown to block proteolytic processing of procaspase-9 induced by mitochondrial permeability transition (46). These lines of evidence support our understanding that CSF-1 induces an increase of XIAP and leads to inhibition of conversion of procaspase-9 and subsequent activation of procaspase-3. Of course, CSF-1 may evoke other signaling activities to inhibit caspase activation other than via XIAP. Whether the increase of XIAP by CSF-1 is brought about by transcriptional activation or protein stabilization is now under investigation in our laboratory. Unlike XIAP, cIAP2 was not induced by CSF-1.

The importance of the NF-{kappa}B pathway in osteoclast survival has been proposed by several researchers as inhibition of the NF-{kappa}B activation significantly promotes osteoclast apoptosis (7, 16). Both IL-1 and RANKL activate NF-{kappa}B and JNK, via their association with TRAF molecules (47, 48). Because NO partly inhibits NF-{kappa}B activity by modulating SH-groups in the molecule (21, 35), restoration of NF-{kappa}B activity by IL-1 and RANKL could explain their prevention of NO-promoted apoptosis in osteoclast-like cells. However, how NF-{kappa}B activity promotes survival of osteoclasts is unknown. RANKL has been shown to induce the survival of dendritic cells, which was mediated by up-regulation of Bcl-XL (49). In contrast to dendritic cells, Jimi et al. (8) showed that RANKL did not induce the expression of Bcl-XL in murine osteoclast-like cells. We also examined changes in the Bcl-XL level during NO-induced apoptosis and the effect of RANKL on the Bcl-XL expression in the osteoclast-like cells. Immunoblotting analysis showed that the expression of Bcl-XL was unchanged (Kanaoka et al., unpublished observation). Thus, up-regulation of Bcl-XL did not seem to be a major downstream activity of RANKL in osteoclast-like cells. Activation of the NF-{kappa}B pathway has been shown to induce expression of IAPs, including XIAP, in tumor necrosis factor-{alpha}-treated human endothelial cells (50). However, RANKL did not apparently increase the amount of IAPs in murine osteoclast-like cells in our study. RANKL apparently had an additive effect on inhibition of caspase-3-like protease activity by CSF-1 (Fig. 7bGo), but not on the increase of XIAP levels induced by CSF-1 (data not shown). Thus, it was likely that each downstream signaling activity to inhibit activation of caspases by RANKL and CSF-1 was almost independent.

In summary, we found that osteoclast survival factors protect against NO-promoted apoptosis in murine osteoclast-like cells, to a different extent. A part of their downstream activities is focused, at least, on inhibition of the activation of the caspase cascade, although how this is accomplished seems to vary among the factors.


   Footnotes
 
1 This investigation was supported in part by Grants-in-Aid (Nos. 09470404, 10557166 and 10771017) for scientific research from the Ministry of Education, Science, and Culture, Japan. Back

Received November 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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