From the
Departmenta of Orthopaedics and
Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, August 22, 2002 , and in revised form, March 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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NF-B proteins are a family of transcription factors expressed in most cell types, play an important role in the development of osteoclasts, and regulate immune and inflammatory responses (12, 13, 14, 15, 16). NF-
B proteins are also involved in protecting cells from undergoing apoptosis in response to cytokine treatment (17, 18, 19). These events include activation of anti-apoptotic gene products, such as cellular inhibitors of apoptosis (cIAPs) (19, 20, 21, 22, 23).
Diverse signal transduction complexes mediate stimulation of NF-B pathway (19). The inactive form of the transcription factor exists in the cytoplasm associated with regulatory proteins called I
B (24). Signals activate upstream I
B kinases, which in turn phosphorylate N-terminal moieties on I
B proteins. These events facilitate dissociation of the inhibitory protein and release of NF-
B, which then translocates to the nucleus, binds to DNA, and activates target genes (19, 24, 25). In osteoclast precursor cells, members of the TNF family (RANKL and TNF) are potent activators of the NF-
B pathway and strong inducers of osteoclastogenesis (7, 11, 26, 27). However, studies from knockout mice showed that in the absence of certain NF-
B molecules, the anti-apoptotic effect of NF-
B is hampered. Under these conditions, pro-inflammatory cytokines such as TNF exert a potent pro-apoptotic outcome (20, 22, 23, 28).
We have shown previously that TNF strongly augments RANKL-primed osteoclastogenesis utilizing mechanisms that involve NF-B activation (11). We have also shown that inhibition of NF-
B activation arrests osteoclastogenesis (29). In the current study, we examined the direct effect of TNF on osteoclasts in which the NF-
B pathway has been blocked. Our findings point out that administration of DN-I
B to osteoclasts and preosteoclasts arrests their differentiation and renders them unprotected by virtue of reduced TRAF6, Bcl-XL, and cIAP-1 expression. This process is further propagated by TNF. In this system, we document that several apoptotic factors, namely caspase 3, caspase 9, PARP, and Bax, are clearly activated. These findings suggest that TNF, in the presence of DN-I
B, induces osteoclast apoptosis through combined inhibition of cIAP-1 and activation of caspases.
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MATERIALS AND METHODS |
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AnimalsC3H/HeN male mice were purchased from Harlan Industries (Indianapolis, IN).
Cell CultureOsteoclast precursor cells in the form of bone marrow macrophages were isolated as previously described (27). Briefly, whole bone marrow of 46-week-old mice was collected and incubated in tissue culture plates at 37 °C in 5% CO2 in the presence of 10 ng/ml M-CSF (27). After 24 h in culture, the nonadherent cells were collected and layered on a Ficoll-Hypaque gradient. Cells at the gradient interface were collected, plated in -minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2 in the presence of 10 ng/ml M-CSF, and plated according to each experimental condition.
Osteoclast GenerationOsteoclasts were generated by culturing purified precursor cells in the presence of M-CSF and soluble RANKL (1020 ng/ml each) for 4 days. Bona fide osteoclasts develop on day 34 of culture at a point when cells are fixed and tartrate-resistant acid phosphatase-stained or subjected to further treatments. DN-TAT:IB was added at 5 ng/ml on day 3 of culture followed (after 2 h) by TNF (10 ng/ml). The cultures were then continued for an additional day, after which they were processed for immunostaining, immunoblots, or TUNEL assay.
ImmunostainingBone marrow macrophages were plated on multi-well coverslips in the absence or presence (1 h) of DN-TAT:IB. The cells were then fixed and stained with anti-HA antibody and detected with fluorescent secondary antibody.
ImmunoblottingTotal cell lysates were boiled in the presence of 2x SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 10% (w/v) SDS, 10% glycerol, 0.05% (w/v) bromphenol blue, distilled water) for 5 min and subjected to electrophoresis on 812% SDS-PAGE (30). The proteins were transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad) and incubated in blocking solution (10% skim milk prepared in phosphate-buffered saline containing 0.05% Tween 20) to reduce nonspecific binding. The membranes were washed with phosphate-buffered saline/Tween buffer and exposed to primary antibodies (1 h at room temperature), washed again four times, and incubated with the respective secondary horseradish peroxidase-conjugated antibodies (1 h at room temperature). The membranes were washed extensively (5 x 15 min), and an ECL detection assay was performed following the manufacturer's directions.
pTAT Construct and Protein CouplingIB
constructs were cloned into the pTAT-HA bacterial expression vector previously described by Nagahara et al. (31), which contains a six-histidine tag, for easy purification; an HA tag for detection followed by the TAT transduction domain; and finally the I
B sequence. The resultant plasmid, pTAT-I
B, was transformed into the DH5
strain of Escherichia coli. The transformants were screened initially by restriction enzyme mapping. A recombinant containing the correct restriction fragments was then sequenced on both strands. This plasmid was then used to express the TAT-coupled I
B
in the BL21 (DE3) strain of E. coli. Following a 46 h of induction, the cells were sonicated in 8 M urea, and the TAT-coupled I
B
was purified on a nickel-Sepharose column (Qiagen) and then applied to an ionic exchange column (Mono Q) in 4 M urea. To shock misfold the protein, the ionic exchange column was switched in one step from 4 M urea to aqueous buffer (20 mM HEPES). I
B
was eluted by stepping from 50 mM to 1 M NaCl followed by desalting on a PD-10 column (Pharmacia Corp.) into phosphate-buffered saline or 20 mM HEPES, pH 7.2, 137 mM NaCl and frozen in 10% glycerol at 80 °C. pTAT-coupled and misfolded proteins remain highly concentrated and resistant to freeze-thaw denaturation and readily enter the cells upon incubation. Once in hand, the coupled TAT proteins were added to cultured marrow cells without the aid of transfection agents.
TdT-mediated dUTP-Biotin Nick End Labeling (TUNEL) Assay The cells were fixed with paraformaldehyde and washed at room temperature with Tris-buffered saline. The slides were then covered with 0.1% H2O2 in Tris-buffered saline for 30 min at room temperature in a closed container to quench endogenous peroxidase activity. After washing the slides were then covered with TdT reaction buffer to rinse out Tris-buffered saline. The solution was then removed, and 25 µl of TdT/digoxigenin-dUTP mix was added to each well. The slides were incubated for 4560 min at 37 °C in a humidified container. Detection was performed by incubating slides with anti-digoxigenin primary antibody solution for 1 h at room temperature in a closed container followed by incubation with horseradish peroxidase-conjugated anti-sheep secondary antibody for 1 h at room temperature. Color was then developed by incubating slides with substrate working solution for 1020 min at room temperature. The slides were then transferred to Methyl Green solution and incubated for 1 min followed by sequential washes with 70, 95, and 100% ethanol and two washes with Xylene. The slides were mounted and visualized by light microscope.
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RESULTS |
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DN-IB Localizes to Cytoplasmic and Nuclear Compartments and Inhibits NF-
B ActivationFollowing protein expression and purification, the deleted I
B
protein was added to osteoclast cultures for 2 h. The cells were then fractionated to separate cytoplasmic and nuclear fractions. Immunoblots using anti-HA antibody show that pTAT-I
B is present in both fractions and most abundant in the cytoplasmic compartment (Fig. 1B). The protein is abundant in cytosols and nuclei within 1 h, and its levels remain elevated up to 24 and 8 h, respectively (Fig. 1B). The relative short time of protein retention in the nuclei may be due to nuclear transport and clearance of the protein from this compartment.
Next we examined whether the newly transduced DN-IB is active. To accomplish this goal, we tested whether DN-I
B is capable of inhibiting TNF-induced NF-
B activation. To this end, cells in the absence or presence of DN-I
B (60 min) were challenged with TNF for 30 min. At the end of incubation cells were processed for nuclear extraction, and NF-
B activity was measured by electrophoretic mobility shift assay. The results depicted in Fig. 1C clearly show that DN-I
B potently inhibits TNF-induced activation of NF-
B.
DN-IB Induces Apoptosis of Osteoclast Precursors (Macrophages) by TNFMarrow-derived macrophages are the precursor cells that in the presence of RANKL and M-CSF undergo osteoclastogenesis in an NF-
B-dependent process. We have shown previously that specific inhibition of NF-
B activation arrests osteoclastogenesis (29). However, inhibition of the transcription factor was not investigated in TNF-stimulated precursor cells that have not been primed with RANKL and hence do not have high NF-
B activity. Thus, we asked whether DN-I
B inhibits NF-
B stimulation by TNF in osteoclast precursor cells. Our findings indicate that TNF alone exerts morphological changes typical for inflammatory-primed cells, such as spreading and cytoplasmic granulation (Fig. 2b). Limited morphological changes, such as cellular shrinkage and DNA condensation, were observed when DN-I
B alone was added to macrophages (Fig. 2c, arrow), suggesting that the inhibitory protein likely initiated DNA condensation and morphological change by merely interfering with NF-
B availability. More importantly, in the presence of DN-I
B, TNF exerts a potent apoptotic signal on macrophages evident by TUNEL assay (Fig. 2d). The data demonstrate that under combined treatment with both proteins, the cells exhibit all of the major morphological hallmarks of apoptosis, including, shrinkage, membrane shredding, DNA condensation, and fragmentation.
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DN-IB Renders Osteoclasts Sensitive to Apoptosis by TNF NF-I
B is an anti-apoptotic factor essential for osteoclastogenesis and in part mediates the osteoclastogenic properties of TNF. Therefore, we asked whether inhibition of NF-
B alters the TNF pro-osteoclastogenic response by RANKL-primed precursors. As we have published previously (11, 29), TNF mounts a strong osteoclast response when added to preosteoclast cultures (Fig. 3A, panel a). Interestingly, however, administration of DN-I
B to primed osteoclast cultures significantly inhibits osteoclast formation. More importantly, TNF in the presence of DN-I
B caused a marked reduction and shrinkage of osteoclasts typical for apoptotic processes (Fig. 3A, panel d). The apoptotic effect is obvious as shown by chromatin condensation (compare nuclei in panels c and f) and overall cytoplasmic shredding (compare panels b and e).
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The inhibitory effect of DN-IB on osteoclast formation was obvious when multi-nucleated tartrate-resistant acid phosphatase-positive osteoclasts were counted (Fig. 3B). The data demonstrate that although TNF causes an approximate 5-fold increase in osteoclast number, inclusion of DN-I
B for the same time period (1-day) led to complete inhibition of cytokine-induced osteoclastogenesis. In fact, osteoclast formation in the presence of DN-I
B and TNF was 68% lower than control (p < 0.005) and a statistically insignificant 18% below control when treated with DN-I
B alone.
Osteoclast Programmed Death by DN-IB and TNF Is Mediated at Least in Part by Caspase 3, Caspase 9, and PARP TNF, based on precise and cell-specific machinery, induces pro- and anti-apoptotic signals. Evidence from our studies as well as others indicates that under normal circumstances TNF activates NF-
B in primed osteoclast precursors and supports survival of osteoclasts (33, 34, 35, 36). Inhibition of NF-
B, however, diverts TNF signaling toward a pro-apoptotic avenue. To investigate the mechanisms underlying TNF-mediated apoptosis of osteoclast precursors, RANKL-primed cells were treated with TNF in the absence or presence of the NF-
B inhibitor, I
B
. The cells were then lysed and screened for caspases, the mediators of apoptosis. Our data show that caspases 3 and 9 and PARP are activated in cells treated with DN-I
B and TNF (Fig. 4). Activation is evident by cleavage of the precursor zymogen yielding the active subunits relevant to each protein. We also observed activation of caspase 9 in the presence of DN-I
B alone, although this activation was not evident in the downstream proteins, namely caspase 3 and PARP. No evidence of activation of caspases 7 and 8 was observed.
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DN-IB Facilitates TNF Induction of Apoptosis by Blocking and/or Reducing Expression of Key Signaling and Pro-survival MoleculesTo further examine the mechanism underlying this DN-I
B-dependent, TNF-induced apoptosis, we turned to investigate parallel pathways known to play essential roles in signaling cell survival and death. Bax, Bcl-2, and Bcl-XL are members of the Bcl/Bax family that differentially regulate apoptosis. Our findings point out that TNF and DN-I
B significantly increase the expression of Bax (Fig. 5A). Specifically, TNF and DN-I
B increased Bax expression levels by 5- and 8-fold, respectively, as measured by densitometric analysis. Combined addition of both proteins resulted in slight increase in Bax expression (10-fold) compared with control. In addition, although not affecting basal level expression, DN-I
B inhibits TNF induction of Bcl-XL expression (Fig. 5B). No effect on Bcl-2 was observed (not shown). Thus, an increased ratio of Bax/Bcl-XL supports apoptosis and further substantiates caspase-mediated apoptosis.
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Osteoclasts, similar to other cells, possess survival machinery inducible by cytokines and various growth factors. Given that cIAP protein is central in protecting cells, we examined expression of this protein in osteoclasts treated with various combinations of TNF and DN-IB. The data depicted in Fig. 6A indicate that TNF induces expression of cIAP-1 by 23- and 18-fold compared with Tat and Tat+I, respectively. The TNF-induced increase was partially blocked (74%) by DN-I
B. We also examined the expression of TRAF2 and TRAF6; the latter factor is essential for osteoclastogenesis, and both proteins are required for osteoclast signaling. Although no obvious differences in TRAF2 expression were observed (not shown), DN-I
B reduced TRAF6 expression levels in the absence or presence of TNF (Fig. 6B).
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DISCUSSION |
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These events point to NF-B as a centerpiece of the pathoosteoclastogenic response. Switching this transcription factor off not only arrests the pathologic process but in the presence of preexisting pro-inflammatory cytokines, such as TNF, would lead to adverse effects, namely cell death and reduction of osteoclast numbers. Our data confirm this notion. In this regard, a potent osteoclastic response exerted by TNF (Fig. 3) turns deadly in the presence of DN-I
B. The most likely scenario according to these data is that DN-I
B forms a complex with NF-
B and inhibits its activation as evident by the gel shift analysis. This complex resists phosphorylation by upstream kinases because it lacks all of the potential N-terminal phosphorylation sites. This action of arresting NF-
B activation compromises NF-
B-mediated anti-apoptotic events, thus tilting the balance of TNF action toward induction of apoptosis. This proposition is supported by our finding that DN-I
B and TNF combined treatment activates caspases 3 and 9, PARP, and Bax and causes DNA condensation and cytoplasmic shredding (Figs. 3 and 4). Our findings are in agreement with previous reports in which adenoviral delivery of DN-I
B (super-repressor) to chemoresistant tumors in mice sensitizes these cells to undergo apoptosis in response to TNF or other therapeutic agents resulting in tumor regression (39).
NF-B regulates the expression of a wide range of pro-inflammatory cytokines, such as interleukin-1, interleukin-6, and TNF, all known as pro-osteoclastogenic factors. Attenuation of NF-
B activation often leads to a decline in the expression of these cytokines, as we and other have reported in previous studies (33, 35). Thus, it is conceivable that in the absence of NF-
B-directed endogenous cytokine support for osteoclast differentiation, proliferation, and survival, TNF action is averted toward an apoptosis-susceptible osteoclast.
DN-IB, in the absence of TNF, appears to have little or no effect on certain pro-apoptotic factors. This is evident in the case of caspase 3 and PARP activation (Fig. 4). The requirement of TNF to further potentate osteoclast death is likely due to the complexity of the osteoclast survival machinery. Namely, NF-
B unrelated pathways such as extracellular signal-regulated kinase and Akt cascades might be able to maintain survival of osteoclasts under physiological conditions (40, 41). These signaling pathways, however, may not be able to withstand a direct TNF-transmitted apoptotic signal in the absence of NF-
B. In support of this speculation, it is clear that in the presence of TNF, direct administration of DN-I
B seems to facilitate programmed death of osteoclasts. In this regard, it has been shown that treatment of isolated osteoclasts with antisense oligonucleotides to the p65 and p50 subunits of NF-
B or with NF-
B inhibitory agents such as pyrralidinedithiocarbamate- and tosylphenylalanyl chloromethyl ketone-induced apoptosis (42, 43). Nonetheless, our observations indicate that DN-I
B, in the absence of TNF, is a poor inducer of osteoclast apoptosis. It appears likely that NF-
B inhibition alone compromises cellular protection mechanisms and sets the stage for cytokine-driven programmed cell death. These speculations are in agreement with recent findings by Xing et al. (43), which reported that although p50 and p52 NF-
B subunits are required for osteoclast formation, the absence of these subunits does not cause further apoptosis of osteoclasts (43). However, in this model neither the apoptotic outcome of stimulation with TNF nor the role of other key NF-
B molecules, such as p65, were investigated. Thus, although it is likely that under unstimulated conditions, p50 and p52 may not exert an anti-apoptotic effect, other NF-
B members, particularly p65, might be essential for osteoclast survival. Furthermore, genetic adaptation of NF-
B-null cells to acquire other compensatory mechanisms in the knockout mouse should be considered.
Programmed cell death is governed by complex pathways that intersect negatively and positively at various levels and compartments. To further elucidate the mechanism underlying DN-IB/TNF-induced apoptosis, we investigated the role of Bcl/Bax family in osteoclast death. Traditionally, these proteins regulate the pro-apoptotic cytochrome c/Apaf1 pathway, whereby Bax activates and Bcl-XL inhibits this pathway. Clearly, the increased ratio of Bax/Bcl-XL supports apoptosis and complements caspase activation.
NF-B protects cells by induction of various gene products. Cellular inhibitors of apoptosis have immerged as central to transmitting NF-
B-related anti-apoptotic signals. Furthermore, cIAPs play a rate-limiting role in NF-
B activation because they are required for upstream activation of the transcription factor in a positive feedback loop. Moreover, a direct anti-apoptotic effect of these proteins over caspases 3 and 9 has been established (44, 45, 46). We find that combined treatment of osteoclasts with DN-I
B and TNF reduces cIAP-1 expression. In what appears as consequential events, NF-
B activation and TRAF6 protein expression, another integral protein required for osteoclastogenesis, are reduced. Thus, it is conceivable that insufficient levels of cIAP-1 are no longer able to maintain caspases 3 and 9 under control, which leads directly to intensification of the apoptotic event. A plausible explanation for reduced levels of cIAP-1 in the presence of DN-I
B stems from recent findings implicating caspases as partially responsible for cleavage of cIAPs (46, 47).
In other studies apoptosis of osteoclasts was achieved by exposure to osteoprotegrin, a soluble TNF receptor family member and RANKL decoy molecule (36). This protein acts upstream by interfering with RANK/RANKL binding. It is unlikely that osteoprotegrin plays a part in the DN-IB/TNF-mediated apoptosis of osteoclasts that we describe in this study. This notion is supported by the facts that 1) we used a relatively osteoblast-devoid pure population of osteoclast precursors that do not express or secrete osteoprotegrin and 2) RANKL was added exogenously at levels that permit osteoclastogenesis and was maintained continuously throughout the duration of the culture.
In summary, we have used a convenient tool, namely a TAT fusion system that permits efficient delivery of DN-IB into osteoclasts and their precursors. Our data indicate that DN-I
B attenuates NF-
B activation and in the presence of TNF, which alone is a pro-osteoclastogenic factor, leads to osteoclast apoptosis. The mechanism of this cell death includes reduction in cIAP-1 expression leading to activation of caspases and up-regulation of Bax/Bcl-XL ratio. These finding may provide the basis for designing strategies to block bone loss because of heightened osteoclast activity.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Washington University School of Medicine, Dept. of Orthopedic Surgery, One Barnes Hospital Plaza, 11300 West Pavilion, Campus Box 8233, St. Louis, MO 63110. Tel.: 314-362-0335; Fax: 314-362-0334; E-mail: abuamery{at}msnotes.wustl.edu.
1 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-B ligand; TNF, tumor necrosis factor; TRAF, TNF receptor associate factor; cIAP, cellular inhibitor of apoptosis; DN-I
B, dominant-negative IkB; HA, hemagglutinin; PARP, poly(ADP-ribose)polymerase; TUNEL, TdT-mediated dUTP-biotin nick end labeling.
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REFERENCES |
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