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Molecular Endocrinology, doi:10.1210/me.2004-0161
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Molecular Endocrinology 19 (3): 771-780
Copyright © 2005 by The Endocrine Society

Unoccupied {alpha}vß3 Integrin Regulates Osteoclast Apoptosis by Transmitting a Positive Death Signal

Haibo Zhao, F. Patrick Ross and Steven L. Teitelbaum

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Steven L. Teitelbaum, M.D., Washington University School of Medicine, Department of Pathology and Immunology, Campus Box 8118, 660 South Euclid Avenue, St. Louis, Missouri 63110. E-mail: teitelbs{at}wustl.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell/matrix detachment is a general inducer of programmed cell death, an event mediated by loss of integrin/ligand association. Because {alpha}vß3 is the major integrin expressed by the osteoclast, we asked whether its occupancy promotes survival of the resorptive cell. Thus, we generated wild-type preosteoclasts and placed them on selective matrix proteins. Consistent with the posture that {alpha}vß3 occupancy promotes survival, preosteoclasts plated on native collagen, a matrix not recognized by the integrin, undergo apoptosis 4-fold faster than those on the {alpha}vß3 ligand, vitronectin. To further explore the role of {alpha}vß3 in osteoclast apoptosis, wild-type and ß3–/– preosteoclasts were suspended and apoptosis determined, with time. ß3–/– preosteoclasts, in suspension, undergo a rate of apoptosis only 40–60% of that of their wild-type counterparts, indicating that unoccupied {alpha}vß3 transmits a positive death signal that we find regulated by caspase-8. Attesting to specificity of the unoccupied integrin-transmitted death signal, apoptosis in the absence of {alpha}vß3 is mediated by capsase-9. We have shown that the resorptive defect of ß3–/– osteoclasts is rescued by wild-type ß3 cDNA but not by one bearing a S752P mutation. To determine whether the same holds true regarding osteoclast apoptosis, we constructed lentivirus vectors encoding green fluorescent protein, wild-type ß3, or ß3S752P. Once again, native ß3–/– preosteoclasts were protected against apoptosis. Similar to its effect on bone resorption, transduced wild-type ß3 normalizes the apoptotic rate of ß3–/– preosteoclasts. Unexpectedly, however, ß3S752P transductants also die at a rate indistinguishable from wild type. Thus, unoccupied {alpha}vß3 integrin regulates osteoclast apoptosis via a component of the integrin that is different than that regulating resorption.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INTEGRINS ARE HETERODIMERIC glycoproteins consisting of various combinations of {alpha}- and ß-subunits. In addition to promoting cell/matrix attachment, integrins, upon engagement by their respective extracellular ligands, transmit a diverse array of intracellular signals. These matrix-initiated signals impact a variety of cellular functions including survival, proliferation, gene transcription, and migration (1, 2).

The {alpha}vß3 integrin plays a fundamental role in many aspects of osteoclast biology and is progressively enhanced as precursor cells, in the form of bone marrow macrophages (BMMs), assume the osteoclast phenotype (3). Thus, the mature osteoclast contains an abundance of the heterodimer (4, 5). Attesting to the fundamental role {alpha}vß3 plays in the resorptive process, function blocking antibodies and peptidomimetic antagonists inhibit bone degradation in vitro and in vivo (6, 7). Most importantly, ß3 integrin knockout mice develop osteosclerosis due to osteoclast dysfunction (8). Studies employing these mutant animals document that a sequence of {alpha}vß3-regulated events such as osteoclast spreading, cytoskeletal reorganization, and cell migration precede and are essential for the polykaryon’s ultimate mission of bone resorption (9, 10).

Attachment of nontransformed cells is essential for survival, as loss of matrix recognition results in a form of apoptosis known as anoikis (11). Matrix-derived antiapoptotic signals are mediated by integrins, including {alpha}vß3, which inhibit death-related intracellular events such as caspase activation (11). Given the fact that integrins provide prosurvival signals, one would expect absence of such a receptor known to be functional in a particular cell to accelerate its death and, hence, promote its paucity. Thus, we were surprised that the number of osteoclasts in {alpha}vß3-deficient mice is substantially increased (8). To address this conundrum, we turned to the rate of programmed cell death in cells committed to the osteoclast lineage, which lack the {alpha}vß3 integrin. Consistent with observations of Stupack et al. (12), unoccupied {alpha}vß3 in wild-type cells transmits a positive death signal involving activation of caspases 8 and 3. On the other hand, preosteoclasts derived from ß3–/– mice exhibit prolonged survival and ultimately activate a caspase-9-regulated apoptotic pathway. Interestingly, unoccupied integrin-mediated apoptosis is independent of S752 in the ß3 cytoplasmic domain, a residue essential for all previously documented {alpha}vß3-mediated intraosteoclast signaling events (9). Thus, the abundance of osteoclasts present in mice lacking {alpha}vß3, reflects, at least in part, the absence of a death signal that is transmitted, in wild-type cells, by the unoccupied integrin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unoccupied {alpha}vß3 Integrin Induces Apoptosis in Preosteoclasts
To determine whether the {alpha}vß3 integrin regulates osteoclast apoptosis, we generated cells committed to the osteoclast phenotype (preosteoclasts) by culturing BMMs in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor {kappa}B (RANK) ligand (RANKL) (13). After 2 d, the cells express mRNA and protein of the osteoclast-specific marker cathepsin K (Fig. 1Go, A and B). Although virgin BMMs contain no ß3 mRNA or protein, they appear after 2 and 3 d, respectively, (Fig. 1Go, A and B). After 3 d of culture with M-CSF and RANKL, BMMs differentiate into mononuclear, tartrate-resistant acid phosphatase (TRAP)-positive preosteoclasts (Fig. 1CGo). Unlike mature osteoclasts, these precursors are easily lifted from culture dishes, lending themselves to analysis of events mediated by matrix attachment. Using this model, we asked whether {alpha}vß3 integrin occupancy affects osteoclast apoptosis. Thus, we cultured preosteoclasts on a matrix protein recognized by the integrin, namely vitronectin, or native collagen, which does not recognize {alpha}vß3. As measured by an ELISA that detects cytoplasmic histone-associated-DNA-fragments formed in apoptotic cells, preosteoclasts, plated on native collagen undergo apoptosis 4-fold faster than those plated on vitronectin (Fig. 2AGo). The fact that unoccupied {alpha}vß3 promotes preosteoclast death is underscored by the decreased amount of actin in total cell lysates derived from collagen as compared with vitronectin adherent cultures (Fig. 2BGo). To confirm that unoccupied {alpha}vß3 promotes preosteoclast programmed cell death, we performed another apoptosis assay using cell surface labeling of annexin V followed by flow cytometric analysis. In keeping with our ELISA-based data, the number of apoptotic cells, as determined by fluorescein isothiocyanate (FITC)-annexin V staining, is 8- to 9-fold greater in cells cultured on collagen as compared with those cultured on vitronectin (Fig. 2CGo).



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  		Fig. 1. Generation of Preosteoclasts from BMMs

Bone-marrow macrophages were prepared from 6- to 8-wk-old wild-type mice and cultured with 12 ng/ml M-CSF and 100 ng/ml RANKL. A, Expression of ß3 integrin and cathepsin K mRNA in d-1 to d-5 cultures was detected by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as PCR control. B, Expression of ß3 integrin and pro- and active (mat-) cathepsin K protein in d-1 to d-5 cultures was determined by immunoblot. Actin served as loading control. C, Pure populations of TRAP-expressing predominantly mononuclear cells appear within 3 d of culture.

 


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  		Fig. 2. Unoccupied {alpha}vß3 Integrin Promotes Preosteoclast Death

BMMs were cultured with 12 ng/ml M-CSF and 100 ng/ml RANKL for 3 d. Preosteoclasts were lifted and replated on type I collagen (COL) or vitronectin (VN) in serum free {alpha}-MEM with M-CSF and RANKL for 16 h. A, Apoptosis rate was measured by ELISA. The data represent the mean ± SD of four individual assays of 405 nm wavelength (***, P < 0.001; n =4). B, Actin in total cell lysates as detected by Western blotting. C, Preosteoclasts were cultured on COL or VN for 16 h and were then lifted with 0.02% EDTA/PBS. Apoptotic cells were stained by using the Annexin V-FITC Apoptosis Detection Kit and were analyzed by flow cytometry. The numbers in the upper right corner in each panel are percentages of FITC-annexin V-positive cells after the treatment. M1, Annexin V-FITC and propidium iodide negative; M2, annexin V-FITC positive and propidium iodide negative.

 
Preosteoclasts Lacking {alpha}vß3 Integrin Exhibit Enhanced Survival
The fact that engagement of an {alpha}vß3 ligand protects osteoclasts against apoptosis indicates that osteoclasts lacking the integrin should die at an accelerated rate. To determine whether this is the case, we cultured preosteoclasts in suspension, with time, and measured the rate of apoptosis. As shown in Fig. 3Go, apoptosis of nonadherent, wild-type preosteoclasts maximizes within 1–2 h. In contrast, ß3 integrin knockout cells have a rate of apoptosis only 40–60% of their wild-type counterparts after 1 and 2 h in suspension and require 4 h for cell death to maximize.



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  		Fig. 3. Apoptosis Is Attenuated in Preosteoclasts Lacking {alpha}vß3 Integrin

Wild-type (open bars) and ß3–/– (solid bars) preosteoclasts were cultured in suspension in serum free {alpha}-MEM with 12 ng/ml M-CSF and 100 ng/ml RANKL, and time and apoptosis rate was measured by ELISA. (**, P < 0.01 vs. wild type; ***, P < 0.001 vs. wild type; n =4).

 
ß3 Integrin Wild-Type and Null Preosteoclasts Undergo Apoptosis by Distinct Pathways
Apoptosis may be induced via the stress (intrinsic) or death receptor (extrinsic) signaling pathways (14). In the death receptor pathway, ligation of specialized receptors such as Fas and TNF receptor 1 activates initiator caspase-8 and, ultimately, executioner caspases such as caspase-3. In the stress pathway, cytochrome c is released from the mitochondrial intermembrane space. Cytochrome c and ATP bind Apaf-1 to form a multimeric complex that recruits and activates caspase-9, followed by the activation of executioner caspases (15).

To determine which of these pathways is involved in osteoclast apoptosis induced by unoccupied {alpha}vß3 integrin, we assessed the activity state of caspases-8,-9, and -3 in total cell lysates derived from wild-type and {alpha}vß3-deficient preosteoclasts maintained in suspension. Although caspase-8 is progressively activated, with time, in nonadherent, wild-type preosteoclasts, the caspase activity decreases in preosteoclasts lacking the integrin (Fig. 4AGo). No obvious activation of caspase-9 is observed in suspended wild-type cells, but the caspase is increasingly activated in ß3–/– preosteoclasts with time in suspension (Fig. 4AGo). Consistent with the delayed rate of suspension-induced apoptosis in ß3–/– preosteoclasts, activated executioner caspase-3 also appears and maximizes later in these cells.



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  		Fig. 4. Differential Caspase Activation and Cytochrome c Release in ß3+/+ and ß3–/– Preosteoclasts

ß3 Integrin wild-type and null preosteoclasts were cultured in suspension with time in serum-free {alpha}-MEM containing 12 ng/ml M-CSF and 100 ng/ml RANKL. A, The cells were lysed in 3-[(3-chloroamino-dopropyl)-dimethylamino]-1-propanesulfonate buffer, and activated caspase-8, -9, and -3 were detected by Western blotting. Pro-caspase-9 and actin served as loading control. B, The cells were resuspended in isotonic mitochondrial buffer and fractionated into heavy membrane fraction and cytosolic fraction (see Materials and Methods). The distribution of cytochrome c was detected by Western blotting. Actin served as loading control.

 
Because mitochondrial release of cytochrome c is critical for initiating the intrinsic apoptosis pathway, we assessed the intracellular distribution of this electron transporter. Wild-type or ß3-null preosteoclasts were cultured in suspension with time, and cytosolic and mitochondrial-enriched membrane fractions were isolated. Minimal mitochondrial cytochrome c is released into the cytosol of wild-type preosteoclasts (Fig. 4BGo). However, a dramatic increase in cytoplasmic cytochrome c occurs within 2 h of ß3–/– preosteoclast suspension (Fig. 4BGo).

Caspase-8, in addition to directly activating caspase-3, cleaves the proapoptotic Bcl-2 family member, Bid, in a cell-specific manner. Truncated Bid, in turn, facilitates cytochrome c mobilization. This indirect pathway provides cross-talk between the death receptor and mitochondrial pathways (16). We find, however, that the quantity of intact Bid is unchanged, with time, in both suspended wild-type and ß3–/– preosteoclasts (data not shown), suggesting that Bid cleavage does not occur. Thus, this signaling pathway is insignificant in integrin-mediated apoptosis in osteoclasts (17). The more rapid induction of apoptosis occurring in suspended wild-type preosteoclasts, as opposed to those lacking the integrin, probably reflects activation of the death receptor pathway. On the other hand, the more delayed cell death, extant in ß3 deleted cells, is likely a manifestation of activated mitochondrial signaling.

To further explore the possibility that the caspase-8/caspase-3 signaling mediates, at least in part, integrin-associated osteoclast apoptosis, we exposed collagen-plated wild-type preosteoclasts to various caspase specific inhibitors for 18 h. Supporting a central role for the extrinsic pathway in unoccupied {alpha}vß3-mediated cell death, the magnitude of apoptosis in wild-type preosteoclasts is reduced by caspase-3- or caspase-8- but not caspase-9-specific inhibitors (Fig. 5AGo). Next, collagen-plated ß3–/– preosteoclasts were similarly treated with caspase inhibitors. To maximize apoptosis, the cells were then cultured in suspension for 2 h. In this circumstance, the magnitude of apoptosis is attenuated by caspase-3 and -9 inhibitors but not by one that blocks caspase-8 (Fig. 5BGo). To validate these inhibitor studies, we constructed a retroviral vector carrying a mutated form of caspase-9 known to exert a dominant/negative (D/N) effect (12). Transduced BMMs were cultured with M-CSF and RANKL for 3 d and the resultant preosteoclasts were suspended for 2 h before apoptosis assay. Consistent with the inhibitor-based experiment, D/N caspase-9 does not impact wild-type cells but blunts ß3–/– preosteoclast apoptosis. Similar attempts using four distinct caspase-8 D/N constructs resulted in universal and rapid cell death, perhaps reflecting the enzyme’s recently described role in nuclear factor-{kappa}B activation (18). Taken together, these data indicate that wild-type and {alpha}vß3-deficient preosteoclasts undergo apoptosis by distinct mechanisms.



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  		Fig. 5. Effects of Caspase Inhibitors and D/N Caspase-9 on Preosteoclast Apoptosis

A, ß3+/+ Preosteoclasts, plated on collagen, were treated with 40 µM of caspase-3 inhibitor fmk-DEVD, the caspase-8 inhibitor fmk-IETD, and the caspase-9 inhibitor fmk-LEHD, for 18 h. The magnitude of apoptosis in wild-type cells was determined immediately thereafter by ELISA (**, P < 0.01 vs. control; n =4). B, To maximize apoptosis in the relatively resistant ß3–/– cells, they were first pretreated with 40 µM of caspase inhibitors for 16 h and cultured in suspension for 2 h with inhibitors before apoptosis assay (**, P < 0.01 vs. control; n =4). C, Empty vector (pMX) and D/N caspase-9 (DN-cas 9)-transduced BMMs were cultured with 12 ng/ml MCSF and 100 ng/ml RANKL for 3 d. Preosteoclasts were lifted and cultured in suspension for 2 h (**, P < 0.01 vs. pMX; n =4).

 
{alpha}vß3 Integrin-Induced Preosteoclast Apoptosis Is Not Mediated by ß3S752
To determine whether expression of wild-type ß3 rescues the attenuated apoptosis extant in cells lacking the integrin, we constructed lentivirus vectors encoding green fluorescent protein (GFP) or human wild-type ß3 (hß3), whose cytoplasmic domain is identical with that of the mouse. ß3–/– BMMs were transduced with each viral construct at an efficiency of 60–90%, as determined by fluorescent microscopy (Fig. 6AGo) or flow cytometry (Fig. 6BGo). Similar to alternative methods of viral transduction (9), lentiviral transduction of wild-type ß3 integrin rescues the cytoskeleton of ß3–/– osteoclasts (Fig. 6CGo). Importantly, whereas the GFP-bearing vector has no effect, the wild-type construct increases the apoptosis rate above that of native preosteoclasts (Fig. 6DGo).



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  		Fig. 6. Lentiviral Transduction of Human ß3 and ß3S752P Accelerates Preosteoclast Apoptosis

A, Wild-type BMMs or mature osteoclasts were transduced with a lentiviral vector containing GFP. Left panels, Phase contrast microscopy; right panels, fluorescent microscopy demonstrating high transfection efficiency in macrophages and mature osteoclasts. B, Flow cytometric analysis of transduced ß3–/– macrophages demonstrating equivalent expression of transduced wild-type ß3 and ß3S752P. C, Lentiviral transduction of human ß3 integrin but not ß3S752P rescues the spreading defect of ß3–/– osteoclasts. The top panels represent osteoclasts derived from ß3+/+ and ß3–/– marrow cells, transduced with empty vector. The bottom panels represent osteoclasts generated from ß3–/– marrow cells transduced with either human wild-type ß3 or ß3S752P. The red reaction product is TRAP. D, Transduction of ß3–/– preosteoclasts with hß3 or ß3S752P, but not GFP, increases preosteoclast apoptosis. ß3–/– preosteoclasts lentiviral transduced with indicated constructs were plated on collagen. After 16 h, the magnitude of apoptosis was determined by ELISA (***, P < 0.001 vs. ß3–/– GFP; {Delta}{Delta}{Delta}, P < 0.001 vs. ß3+/+; n =4).

 
We have shown that the human ß3 mutation, S752P, obviates all previously tested aspects of osteoclast function including differentiation, cytoskeletal organization, general intracellular signaling, and bone resorption (9, 10). We therefore asked whether such is the case with regard to integrin-mediated apoptosis. Unexpectedly, in light of the seemingly global role of S752 in osteoclast function, ß3S752P transductants die at a rate indistinguishable from those expressing the wild-type integrin (Fig. 6DGo). These data indicate that ß3S752 uniquely fails to regulate preosteoclast apoptosis.

Caspase-8 Associates with Unoccupied Wild Type {alpha}vß3 and {alpha}vß3S752P
If unoccupied {alpha}vß3 mediates its apoptotic effect in the osteoclast via caspase-8 activation, a reasonable hypothesis holds that the caspase would associate with the unliganded integrin. To address this issue, ß3–/– preosteoclasts, transduced with hß3 cDNA, were either cultured in suspension or on vitronectin-coated plates for 2 h. The cells were lysed and hß3 integrin was immunoprecipitated with an extracellular domain-specific antibody. As shown in Fig. 7Go, the integrin is equally expressed in adherent and nonadherent hß3-transduced preosteoclasts. As negative control, ß3 integrin is undetectable in lysates lacking the antibody or in mock-transduced knockout cells. To determine the magnitude of association of caspase-8 with {alpha}vß3, the ß3 immunoprecipitate was blotted with an anti-caspase-8 antibody. Consistent with a central role for caspase-8 in {alpha}vß3-mediated apoptosis, an abundance of the caspase, in the form of its zymogen (54–55 kDa), associates with the integrin in suspended, but not adherent, preosteoclasts. Because ß3S752P-transduced cells die at a rate indistinguishable from those expressing the wild type-integrin, one would expect caspase-8 to associate with the S752P mutant. In fact, caspase-8 binding to the mutant and wild-type integrins in suspended cells is indistinguishable.



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  		Fig. 7. Association of Caspase-8 with Unliganded ß3 Integrin

ß3–/–-Coated plates (A, lane 3) or cultured in suspension (S, lanes 2 and 4–5) for 2 h. Empty vector-transduced ß3–/– cells in suspension serve as control (lane 1). ß3 Integrin was precipitated with monoclonal antibody 7G2. The immunoprecipitates were separated by 8% SDS-PAGE and blotted with polyclonal anti-caspase-8 (upper panel) or 7G2 (lower panel). IgG-HC, IgG heavy chain.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Regardless of cause, osteoporosis reflects an enhancement of the bone-resorbing activities of osteoclasts relative to the bone-forming capacity of osteoblasts. Thus, the most established method of arresting this disease is to decrease the number or resorptive activity of osteoclasts. Because osteoclast number reflects the rates of precursor proliferation, differentiation, and programmed cell death, each event presents itself as a therapeutic target in the management of osteoporosis (19). In fact, regulation of osteoclast survival is a pivotal event in bone remodeling (20). Agents currently used to arrest osteoporosis such as estrogen (21, 22) and bisphosphonates (23, 24) inhibit resorption, at least in part, by inducing osteoclast apoptosis

When the osteoclast resorbs bone, it forms an isolated extracellular acidified microenvironment between itself and the juxtaposed skeletal matrix. Similarly, osteoclast differentiation depends upon recognition of bone by the polykaryon’s precursor cells. Thus, two essential components of the resorptive process, namely osteoclast recruitment and activation of the differentiated cell, depend upon cell/matrix attachment.

With the realization that the means by which osteoclasts and their precursors bind to bone are central to the resorptive process and, thus, potential therapeutic targets, attention has turned in recent years to characterizing plasma membrane-residing matrix attachment molecules. These studies establish that uncommitted osteoclast precursors in the form, for example, of BMMs contain an abundance of the integrin {alpha}vß5 (5). Upon exposure to RANKL and the appearance of osteoclast lineage-specific proteins, these cells cease to express {alpha}vß5, which is replaced by {alpha}vß3. This integrin mediates adhesion to bone and transduces cytoskeleton organizing signals (5, 25). The fact that mice deleted of the ß3 gene generate dysfunctional osteoclasts establishes {alpha}vß3 as central to the resorptive process (8). Furthermore, the capacity of organic mimetics of its ligand’s recognition sequence to arrest pathological bone loss in vivo (7, 26, 27) positions {alpha}vß3 as a promising antiosteoporosis target.

Disruption of matrix binding rapidly induces osteoclast apoptosis (Fig. 3Go and Ref. 28). Taken with observations made in other cells, the most common paradigm holds that anoikis-mediated osteoclast death represents loss of an integrin-mediated survival signal. This model therefore predicts that absence of {alpha}vß3 would accelerate osteoclast apoptosis and hence decrease osteoclast number in vivo. Unexpectedly, however, the number of osteoclasts in {alpha}vß3-deficient mice is increased 3.5-fold relative to their wild-type and heterozygous littermates. Interestingly, a similar paradox exists with regard to endothelial cells. Specifically, expression of {alpha}vß3 is enhanced in neoangiogenic blood vessels (29), and antagonists of the integrin suppress angiogenesis in vivo by inducing apoptosis of endothelial cells. Although these data suggest a role for {alpha}vß3 in angiogenesis (30), new blood vessel formation in ß3-integrin-null mice appears unimpaired (31) and, in some circumstances, enhanced (32).

Integrins transmit prosurvival signals when interacting with matrix-residing ligand, but there is emerging evidence that, in other circumstances, these receptors promote apoptosis (33). For example, integrin occupancy by nonimmobilized (i.e. soluble) ligand such as the snake venom echistatin induces endothelial cell apoptosis and does so before the cells detach from matrix (34). Although these inhibitor and targeted gene disruption studies appear contradictory, Stupack et al. (12) find that unligated integrins initiate a death pathway in several tumor lines and human endothelial cells. Consequently, antagonists that prevent ligation by matrix residing proteins would induce the integrin-mediated death pathway. Alternatively, disrupted integrin expression would remove this apoptotic trigger and promote survival.

Given that osteoclasts are rich in {alpha}vß3, we asked whether this newly identified integrin-mediated apoptosis pathway contributes the dramatic increase of osteoclast number in ß3-deficient mice (8). Preosteoclasts cultured on native collagen, which supports adhesion but fails to bind the integrin, undergo apoptosis 4-fold faster than those plated on the {alpha}vß3 ligand, vitronectin, establishing that unoccupied {alpha}vß3 promotes death of cells committed to the osteoclast phenotype. This notion is further underscored by the fact that targeted deletion of ß3 integrin prolongs preosteoclast survival, and reintroduction of {alpha}vß3 into these cells accelerates their death.

Caspases are aspartate-specific cysteine proteases that mediate apoptosis (35, 36, 37). They are synthesized as inactive precursors that are proteolytically processed to generate active enzymes inhibited by peptides derived from their substrate recognition sequences (38). Using specific antibodies and peptide inhibitors, we establish that caspase-8 is activated in preosteoclasts wherein {alpha}vß3 is unoccupied by matrix-residing ligand. In contrast, and consistent with their relative attenuation of cell death, caspase-8 remains comparatively dormant in nonattached in ß3–/– preosteoclasts. With time, as the magnitude of apoptosis of suspended ß3–/– preosteoclasts mirrors that of wild-type cells, activated caspase-9 appears in the mutant cells. Thus, apoptosis of cells bearing the unoccupied integrin is mediated by a death receptor pathway and the delayed apoptosis of osteoclasts in which {alpha}vß3 is absent, by stress signaling, both terminating in activation of effector caspase-3. Mirroring the situation in endothelial cells (12), an abundance of caspase-8 associates with unligated {alpha}vß3 integrin in preosteoclasts, substantiating a common mechanism by which the non-matrix-attached integrin generates a positive death signal. Finally and most interestingly, S752P, a mutation found in the human bleeding disorder Glanzmann’s thrombasthenia, fails to interfere with the integrin-mediated apoptosis. This finding suggests the components of the unligated ß3 subunit, which transmit its death signal, are uniquely distinct from other aspects of the integrin’s functions in osteoclasts such as multinucleation, adhesion, cytoskeleton reorganization, and bone resorption (9, 10).

Osteoclasts are terminally differentiated cells that undergo rapid apoptosis after the removal of trophic factors, such as M-CSF and RANKL. Recently, the bcl-2 family member Bim (39) and Fas, the death receptor of the TNF receptor family (40), have been implicated in the regulation of the cells’ death. Although M-CSF and RANKL are prosurvival cytokines, apoptosis of mature osteoclasts continues even in the presence of these agents (our unpublished observations and as reported in Ref. 41). This observation suggests that an intrinsic molecule(s) generated during osteoclast maturation may trigger apoptosis independent of M-CSF and RANKL. Given that {alpha}vß3 progressively increases with osteoclast differentiation and, in the mature resorptive cell, localizes in the ruffled membrane as well as in regions not juxtaposed to bone such as the basolateral membrane (42, 43), the integrin may serve as a biosensor that regulates apoptosis as a function of matrix ligand occupancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Bone Marrow Macrophages Isolation and Preosteoclast Culture
Primary BMMs were prepared as described previously with slight modification (13). Whole bone marrow was extracted from femora and tibia of 6- to 8-wk-old mice with {alpha}-MEM and cultured, overnight, in {alpha}-MEM containing 10% inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin ({alpha}10 medium). The nonadherent cells were collected by centrifugation and replated in a new 15-cm Petri dish in {alpha}10 medium with 1/10 vol of CMG 14-12 culture supernatant, which is equivalent to 130 ng/ml of recombinant M-CSF. Cells were incubated at 37 C in 6% CO2, 94% air for 4 d. Fresh media and M-CSF were supplemented every other day. Cells were washed with PBS, lifted with 1x trypsin/EDTA (Invitrogen, Carlsbad, CA) in PBS, and seeded at 1.5 x 106 cells per 10-cm dish. TRAP-expressing preosteoclasts were generated after 3-d culture of BMMs with 1/50 vol of CMG 14-12 culture supernatant and 100 ng/ml recombinant RANKL.

Osteoclast Apoptosis Assay
Preosteoclasts were lifted in 1x trypsin/EDTA (Invitrogen) for 10 min at 37 C. The reaction was stopped by 1x Trypsin Inhibitor Solution (Sigma, St. Louis, MO). Cells (5 x 105) were either replated in 6-well plates coated with type I collagen (Vitrogen; Cohesion Technologies, Palo Alto, CA) or 2 µg/ml vitronectin (Sigma), or suspended in a Teflon beaker (Nalgene, Lima, OH). Apoptosis rate was measured by using a Cell Death Detection ElisaPLUS Kit (Roche, Indianapolis, IN) or annexin V staining using Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA) and analyzed by flow cytometry.

RT-PCR
Total RNA was purified from d-1 to d-5 cells cultured with M-CSF and RANKL by using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNAs were synthesized from 1 µg of total RNA using the SuperScript First-Strand Synthesis System (Invitrogen) in a volume of 20 µl. The reaction mixture was adjusted to 100 µl with distilled H2O for PCR analysis. One microliter of these cDNAs was then amplified with primers specific for ß3 integrin (sense, 5'-TTACCCCGTGGACATCTACTA-3'; antisense, 5'-AGTCTTCCATCCAGGGCAATA-3'), cathepsin K (sense, 5'-GGAAGAAGACTCACCAGAAGC-3'; antisense, 5'-GTCATATAGCCGCCTCCACAG-3'), and glyceraldehyde-3-phosphate dehydrogenase (sense, 5'-ACTTTGTCAAGCTCATTTCC-3'; antisense, 5'-TGCAGCGAACTTTATTGATG-3'). After 20–35 cycles of 94 C (30 sec), 60 C (30 sec), and 72 C (30 sec), 10 µl of PCR products were separated on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.

Subcellular Fractionation
For detection the release of cytochrome c from mitochondria, subcellular fractionation was performed as previously reported (44). Briefly, preosteoclasts were harvested by centrifugation and resuspended in isotonic mitochondrial buffer (210 mM mannitol; 70 mM sucrose; 1 mM EDTA; 10 mM HEPES, pH 7.4; and 1x protease inhibitor cocktail complete from Roche). Cells were homogenized for 30 strokes with a Dounce homogenizer and passed through 27-gauge needles 30 times. Cell lysates were centrifuged at 500 x g for 5 min at 4 C to get rid of nuclei and unbroken cells. The supernatant was then centrifuged at 10,000 x g for 30 min at 4 C to obtain the heavy membrane pellet enriched for mitochondria, and the resulting supernatant was used as the cytosolic fraction. The fractions were lysed in RIPA buffer for Western blotting.

Western Blotting and Immunoprecipitation
Cultured cells were washed with ice-cold PBS and lysed in 1x cell lysis buffer or 3-[(3-chloroamino-dopropyl)-dimethylamino]-1-propanesulfonate buffer (Cell Signaling Technology, Beverly, MA). After incubation on ice for 30 min, the cell lysates were clarified by centrifugation at 15,000rpm for 20 min. Thirty micrograms of protein were subjected to 8 or 12% sodium dodecyl sulfate polyacrylamide gels and transferred electrophoretically onto nitrocellulose membrane. The filters were blocked in 5% milk/Tris-buffered saline-0.1% Tween 20 for 1 h and incubated with primary antibodies at 4 C overnight followed by probing with secondary antibodies coupled with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). The proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL). Goat anti-ß3 integrin polyclonal antibody was purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody against cathepsin K was purchased from Chemicon (Temecula, CA). Polyclonal antibodies against mouse active caspase-3, caspase-9, and cytochrome c were obtained from Cell Signaling Technology. Rabbit anti-caspase-8 antibodies were obtained from Pharmgene (San Diego, CA) and Santa Cruz Biotechnology, respectively.

For immunoprecipitation, BMMs were grown in two 15-cm dishes with M-CSF and RANKL for 3 d. Preosteoclasts were lifted and plated onto 2-µg/ml vitronectin-precoated dishes or cultured in suspension as described above. Cells were washed in cold PBS and lysed on ice in lysis buffer (20 mM HEPES, pH 7.2; 1% Triton X-100; 2 mM CaCl2; complete protease inhibitor cocktail from Roche; and 1 mM phenylmethylsulfonyl fluoride). Lysates were passed through a 25-gauge needle 10 times and incubated on ice for 30 min. The cell lysates were clarified by centrifugation at 15,000 rpm for 30 min. Eight hundred micrograms of protein were incubated with 2 µg monoclonal anti-human ß3-integrin antibody, 7G2, overnight with rotation. Protein A/G agarose was then added and incubated with rotation for 3 h at 4 C. Immunoprecipitates were washed three times in lysis buffer, and the beads were boiled in 2x sodium dodecyl sulfate sample buffer for 5 min. After centrifugation, proteins were separated by 8 or 10% sodium dodecyl sulfate polyacrylamide gels.

Retroviral Vectors Construction and Transduction
The lentivirus transfer vector and all packaging plasmids were kindly provided by Dr. Mark Sands (Washington University School of Medicine, St. Louis, MO). The full-length human ß3 integrin cDNA was cloned in the place of GFP into plasmid ppt-PGK-GFP. VSV-pseudotyped vectors were produced by transient four-plasmid transfection into 293T cells as previously described (45). Briefly, 5 x 106 293T cells were plated in a 10-cm culture dish 24 h before transfection in high-glucose DMEM (HyClone Laboratories, Logan, UT) containing 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 incubator. Medium was changed 2 h before transfection. Three and a half micrograms of the envelope plasmid pMD.G, 6.5 µg of pMGLg/p, and 2.5 µg of pRSV-REV were used for the transfection of one dish. The precipitate was formed by adding the plasmids to a final volume of 450 µl of 0.1x TE (1 mM Tris, pH 8.0; 0.1 mM EDTA) and 50 µl of 2.5 M CaCl2, mixing well, then adding dropwise 500 µl of 2x HEPES-buffered saline [281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4 (pH 7.12)] with vortexing and immediate addition of the precipitate to the cultures. The medium was refreshed after 16 h, and the conditioned medium was collected after another 24 h and cleared by filtering through 0.45-µm cellulose acetate filters. Titers of all vector preparations were determined by transducing NIH-3T3 cells with serial dilutions of vector supernatants, followed by the measurement of the expression of GFP and ß3 integrin (by fluorescence-activated cell sorting analysis) 2 d later. The titers of 1 x 106 to 10 x 106 U/ml infections were obtained for all vectors. For transducing marrow macrophages, fresh bone marrow cells were flushed from long bones of 6- to 8-wk-old mice. Red blood cells were lysed in 0.747% NH4Cl/0.017% Tris-Cl. Cells (5 x 106) were plated and cultured for 2 d with {alpha}-10 medium/1:10 CMG 14-12 in a 10-cm suspension culture dish. BMMs were then transduced with 3- to 5- ml viral supernatant plus 4µg/ml polybreven for 24 h.

The plasmids carrying the prodomain, C360S and R413G of human caspase-8, were kindly provided by Drs. Toshiyuki Miyashita (National Children’s Medical Research Center, Tokyo, Japan) (46) and Sug Hyung Lee (Catholic University of Korea, Seoul, Korea) (47), respectively. The D/N form of human caspase-9 with C287A point mutation was generously provided by Dr. G. S. Salvesen (The Burnham Institute, La Jolla, CA) (12). The mutants were first amplified by PCR and cloned into the pMX-puro retrovirus vector as described previously (48, 49). The expressing plasmids were transiently transfected into Plat-E packaging cells (50) using FuGENE 6 Transfection Reagent (Roche). Viruses were collected at 48 h after transfection. BMMs were infected with virus for 24 h in the presence of 10% CMG 14-12 culture supernatant and 4 µg/ml polybrene (Sigma). Cells were further selected in the presence of M-CSF and 2 µg/ml puromycin (Sigma) for 3 d before treatment.

Flow Cytometry
Two days after virus transduction, cells were washed with PBS and lifted by 1x trypsin/EDTA (Invitrogen). After centrifugation, cells were resuspended in blocking buffer (0.5% BSA/2 mM EDTA/PBS) and incubated on ice for 20 min. Surface human ß3 was labeled with 1 µg/ml monoclonal antibody 1A2 for 30 min on ice. Cells were then washed with blocking buffer twice and were incubated with FITC-labeled goat antimouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 20 min on ice. Cells were washed twice and resuspended in 500 µl blocking buffer. Flow cytometry was conducted on BD FACSCalibur system (BD Biosciences) with CellQuest software.

Statistics
Data are presented as mean ± SD.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (AR32788, AR46523, and AR48853 to S.L.T.; and AR46852 and AR48812 to F.P.R.).

First Published Online December 9, 2004

Abbreviations: BMM, Bone marrow macrophage; D/N, dominant/negative; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; hß3, human wild-type ß3; M-CSF, macrophage colony-stimulating factor; RANK, receptor activator of nuclear factor {kappa}B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase.

Received for publication April 16, 2004. Accepted for publication November 29, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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