Calpain Is Required for Normal Osteoclast Function and Is Down-regulated by Calcitonin^*

Reagents and Antibodies

Salmon calcitonin was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Calphostin C, prostaglandin E₂, minimum essential medium, α modification (α-MEM), Igepal CA-630, and fetal bovine serum (FBS) were from Sigma. Calpeptin, MDL28170, PD151746, (R_p)isomer of 8-bromo-adenosine 3′,5′-cyclic monophosphorothioate ((R_p)-8-Br-cAMPs), H-89, and anti-m-calpain antibody were purchased from Calbiochem. Anti-μ-calpain antibody was purchased from Biomol (Plymouth Meeting, PA). Anti-talin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-filamin antibody was purchased from Chemicon International (Temecula, CA). Anti-Pyk2/CAK antibody was purchased from BD Biosciences. Anti-cleaved caspase-3 antibody was purchased from New England Biolabs, Inc. (Beverly, MA). Both anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Fisher. Boc-Leu-Met-7-amino-4-chloromethyl-coumarin (Boc-LM-CMAC) was purchased from Molecular Probes, Inc. (Eugene, OR).

Osteoclasts and Osteoclast-like Cells

Rabbit osteoclasts and osteoclast-like cells (OCLs) were generated as described elsewhere (44) with modifications. Briefly, the long bones and scapulae of neonatal New Zealand White rabbits (60–90 g) were dissected free of soft tissue and minced in α-MEM with 0.55 mg/liter sodium bicarbonate and 10 mm HEPES (pH 7.10). The bone fragments were then allowed to settle, the cell suspension was collected, and the cells were pelleted by centrifugation at 1500 × g for 5 min at 4 °C. To isolate mature osteoclasts, the cell pellets were resuspended in medium supplemented with 10% FBS, aliquoted (150 μl) onto coverslips in 6-well dishes, and cultured for 1 h at 37 °C in humidified air with 5% CO₂. Then 2 ml of medium supplemented with 10% FBS was added to each well. To produce rabbit OCLs, the resuspended cells were cultured in medium supplemented with 10% FBS and 10⁻⁸ m 1,25-dihydroxyvitamin D₃ (Proskelia, Romainville, France) for 9 or 10 days. The medium was changed every other day. Cells were cultured in medium with 0.1% FBS for 4 h before stimulation. Murine OCLs were produced by coculturing calvarial osteoblasts with bone marrow cells, as described elsewhere (45) and used immediately for Western blotting or immunofluorescence analysis, tartrate-resistant acid phosphatase (TRAP) staining or migration, retraction, and bone resorption assays as described below. All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee.

Immunoprecipitation and Immunoblotting

Prior to treatment, rabbit and murine cocultures were incubated for 18 h in medium containing 0.5% FBS. The serum-starved OCLs were then purified by peeling away the layer of less adherent, mainly stromal cells. The OCLs were stimulated with CT or other agents in the absence of the cocultured mesenchymal cells at 37 °C. The OCLs were lysed in 1% Igepal CA-630, 150 mm NaCl, 50 mm Tris-HCl (pH 8.0), 5 mm EGTA, 1 mm phenyl-methanesulfonyl fluoride, 10 mm NaF, 0.4 mm Na₂VO₄, and 10 μg/ml leupeptin and aprotinin. The lysates were processed for immunoprecipitation and incubated with specific antibodies and protein A- or protein G-Sepharose, and the immune complexes were washed with mRIPA, containing 5 mm EGTA, 1 mm phenylmethanesulfonyl fluoride, 10 mm NaF, 0.4 mm Na₂VO₄, and 10 μg/ml each aprotinin and leupeptin. The samples were then processed for immunoblotting, as described elsewhere (46).

Calpain Activity Assay

Authentic osteoclasts were plated on glass coverslips for 24 h. They were then loaded with 30 μm Boc-LM-CMAC for 1 h at 37 °C. After loading, images of the cells were obtained before and after CT addition. Calpain-catalyzed cleavage of Boc-LM-CMAC creates a fluorescent product; fluorescence correlates with calpain activity (47–49).

Immunofluorescence Analysis

Authentic osteoclasts on coverslips were fixed in phosphate-buffered saline (PBS) containing 3.7% formaldehyde for 10 min at room temperature and washed in PBS. Coverslips for actin labeling were extracted in ice-cold acetone for 3–5 min and returned to PBS. All other coverslips were permeabilized in 0.05% saponin for 30 min. The coverslips were blocked in 5% normal goat serum for 30 min and then incubated in the appropriate primary antibody for 2 h, washed in PBS, incubated for 1 h in the appropriate fluorescein- or rhodamine-conjugated secondary antibody, and washed. Those cover-slips used for actin labeling were incubated in a 1:40 dilution (in PBS) of rhodamine phalloidin (Molecular Probes) for 20 min and washed. All coverslips were mounted in FluorSave. Cells were examined using a scanning laser confocal imaging system (MRC-600; Bio-Rad). Images were recorded, composite images were compiled, and image enhancements were performed using Adobe Photoshop 6.0.

Cell Retraction Assay

Authentic rabbit osteoclasts were plated on glass or dentine slices for 24 h and then incubated for 30 min with vehicle or calpain inhibitors as described under “Results.” Cells were then fixed (inhibitor only) or treated with CT for 20 min before fixing (CT only, inhibitor plus CT). The fixed cells were stained with toluidine blue or TRAP-stained (50, 51), and the area of at least 70 osteoclasts/ group was measured with a computerized system for histomorphometry (Osteometrics, Atlanta, GA).

Cell Migration Assay

Cell migration was analyzed as described elsewhere (52) with modifications. Authentic murine osteoclasts or OCLs were plated on semipermeable membranes coated with vitronectin in Boyden chambers (CHEMICON) and incubated for 18 h with and without calpain inhibitors as described under “Results” and then fixed and stained for TRAP. The TRAP-positive cells on the bottom side of the membrane were counted. Data values are expressed as a ratio relative to the values obtained from the untreated control chambers.

In Vitro Bone Resorption

Authentic murine or rabbit osteoclasts or murine OCLs were plated on dentine slices and cultured for 24 h. They were then incubated with or without CT or calpain inhibitors for an additional 16 h and then fixed and stained with toluidine blue. The pit area was measured with a computerized system for histomorphometry (Osteometrics, Inc., Atlanta, GA).

Histology and Histomorphometry

μ-Calpain^−/− mice and genetically matched wild type mice were sacrificed by cervical dislocation at 5 and 10 weeks of age after being previously injected intraperitoneally with calcein (30 mg/kg), 7 and 2 days before sacrifice. Bone specimens were fixed in 4% formalin and then embedded in methylmethacrylate as described (52). 5-μm sections were cut and stained either with toluidine blue or by the Von Kossa method for staining calcified tissues (52). 10-μm sections were cut from the same samples and coverslipped unstained for dynamic measurements. Histomorphometric analysis was carried out with an Osteomeasure system (Osteometrics, Inc.), using standard procedures (53) and in blind fashion. Tibial sections were measured in the proximal metaphysis beginning 340 μm below the chondro-osseous junction. Tibial cortical thickness and periosteal mineral appositional rates were measured beginning 680 μm from the chondro-osseous junction on the anterofibular side. Serum levels of pyridinoline cross-links (collagen type I fragments resulting from proteolytic digestion), a marker of osteoclastic bone resorption, were measured using an ELISA kit from Nordic Biosciences (Copenhagen, Denmark). At least six animals per group were examined. Statistical analysis was performed using analysis of variance, with p values less than 0.05 accepted as significant; error bars represent ± S.D.

Osteoclast Survival

OCL survival was measured as reported previously (45). OCLs were generated by coculturing bone marrow cells from μ-calpain^−/− mice and genetically matched wild type mice with calvarial osteoblasts from neonatal CD-1 mice. Once the OCLs were formed, the calvarial cell layer was removed by collagenase/dispase treatment. Some plates were immediately stained for TRAP. The remaining cells were cultured in μ-MEM and 10% FBS with vehicle, calpain inhibitors, or CT for various times up to 24 h. The OCLs were then fixed and TRAP-stained. The numbers of TRAP-positive multinucleated cells were determined and expressed as a percentage of the TRAP-positive multinucleated cells present at the time when the calvarial cell layer was removed.

Both μ-Calpain and m-calpain Are Expressed in Osteoclasts and Localize in Podosomes

Calpain is known to modulate spreading (54) and motility (8, 55) of cells that form focal adhesions by cleaving several focal adhesion proteins, cytoskeletal proteins, and downstream effectors, including talin, focal adhesion kinase, Pyk2, Src, Rac1, and RhoA, and thereby regulating focal complex assembly/disassembly (8, 56). We hypothesized that calpains might therefore regulate the turnover of the podosomes and consequently osteoclast motility and bone resorption. Western blotting of lysates of OCLs generated in vitro revealed the presence of both μ-calpain and m-calpain (Fig. 1, A and B). In addition, immunofluorescence analysis showed that high levels of both μ-calpain (Fig. 1, C and D) and m-calpain (Fig. 1, E and F) were associated with the F-actin-rich belt of podosomes in authentic osteoclasts from both rabbits (Fig. 1, C and E) and mice (Fig. 1, D and F). Calpain was enriched throughout the region between the individual podosomes (Fig. 2C).

Talin, Filamin A, and Pyk2 Are Present in the Podosome Belt and Are Constitutively Cleaved by Calpain in Osteoclasts

The calpain substrates talin and filamin A are actin-binding proteins that associate with focal adhesion structures (14, 57–59), and talin has been shown to localize to podosomes in osteoclasts and other highly motile cells (60, 61). Immunofluorescence confirmed that talin is associated with podosomes (Fig. 2A) and showed that filamin A is also expressed in the osteoclasts and localized in and around podosomes (Fig. 2B) in a manner similar to calpain (Fig. 2C). Pyk2, another calpain substrate protein (21), is a nonreceptor tyrosine kinase related to focal adhesion kinase that is highly enriched in the podosomes of osteoclasts (39, 62) and plays a key role in the Src-dependent regulation of osteoclast adhesion and motility following the activation of α_vβ₃ integrin (46, 63, 64). Since podosomes form and disappear at a much higher rate than focal adhesions (5–7), we reasoned that if calpain-catalyzed proteolysis of these or other podosome-associated proteins contributes to the regulation of podosome turnover, and thus to the regulation of osteoclast attachment, motility, and spreading, it might be possible to detect calpain-generated cleavage products from these proteins in osteoclasts. Western blotting of OCL lysates with antibodies against talin (Fig. 3A), filamin A (Fig. 3B), or Pyk2 (Fig. 3C) detected all three proteins as well as less intense bands below the parent proteins of sizes consistent with reported cleavage products (15, 21, 65). Pretreatment of the OCLs with the calpain inhibitors calpeptin or MDL28170, which inhibit calpains by binding to the catalytic site (66), or PD151746, which inhibits calpains by binding to the calcium-binding site (67), reduced the amount of the lower bands in all three blots, strongly suggesting that one or more calpains are cleaving podosome-associated proteins in osteoclasts.

Calpain Is Required for Normal Osteoclast Spreading and Motility

Attachment, spreading, and motility of cells that form focal adhesions require the binding of attachment proteins, typically integrins, to the extracellular matrix and the subsequent engagement of the cytoskeleton by the integrin intracellular domain. The tension generated by these interactions leads to cell locomotion (68, 69). Since the calpain-catalyzed cleavage of cytoskeletal and attachment-related proteins regulates the assembly and disassembly of adhesion complexes (54) and cell motility (17, 55, 70, 71), we examined the effects of calpain inhibitors on osteoclast spreading and motility.

To determine if calpain promotes osteoclast spreading, authentic osteoclasts were isolated from rabbits or mice, cultured for 24 h, and then treated with calpain inhibitors or with CT as a positive control and fixed as described under “Experimental Procedures.” Both CT and the calpain inhibitors induced a prompt cellular retraction of rabbit (Fig. 4A) and mouse (not shown) osteoclasts plated on either glass (not shown) or dentine (Fig. 4A) to 30–50% of the area of untreated cells, suggesting that calpain activity is required for the normal spreading of osteoclasts. Similar results were obtained with three different calpain inhibitors (calpeptin, MDL28170, and PD151746). The addition of both CT and a calpain inhibitor together induced no more retraction than either agent alone.

The effect of calpain inhibitors on osteoclast spreading suggested that calpain might also play a role in osteoclast motility. To address this question, osteoclasts were plated on vitronectin-coated semipermeable membranes in Boyden chambers, incubated with or without CT (positive control) or calpain inhibitors for 16 h, and analyzed for the amount of migration across the membrane (Fig. 4B) as described under “Experimental Procedures.” Data were normalized to untreated controls. Treatment with calpain inhibitors caused a reduction of cell motility that was comparable with that induced by CT and, as was true of cell spreading, there was no additive reduction of motility when CT and a calpain inhibitor were both present.

To examine the specific role of μ-calpain in osteoclast migration, we analyzed the migration of OCLs generated from precursors obtained from μ-calpain^−/− mice (72) and genetically matched wild type animals. The μ-calpain^−/− OCLs exhibited less motility than the wild type controls but more motility than the CT-treated wild type OCLs (Fig. 4C). However, in contrast to the inability of CT to further reduce the motility of osteoclasts treated with calpain inhibitors (Fig. 4B), treatment with CT further reduced the motility of the μ-calpain^−/− OCLs. These results, together with the more complete inhibition obtained with different calpain inhibitors, suggest that osteoclast locomotion is also promoted by calpains other than μ-calpain, most likely m-calpain, and that CT down-regulates all of the calpains that are blocked by the chemical inhibitors.

Calpain Is Required for Normal Osteoclast Bone Resorbing Activity

Bone resorbing activity is dependent on osteoclast motility (73). Thus, reducing motility by inhibiting calpain might reduce bone resorption. To test this, authentic rabbit osteoclasts were isolated as described under “Experimental Procedures,” plated on dentine slices, and incubated for 16–18 h in the presence or absence of calpain inhibitors or CT as a positive control, as indicated (Fig. 5A). The calpain inhibitors reduced pit formation by 80–90%, at least as much as the CT-induced inhibition. There was no additive effect when the osteoclasts were treated with combinations of CT and calpain inhibitors. To examine the specific role of μ-calpain, we analyzed the resorbing activity of μ-calpain^−/− OCLs plated on dentine slices for 16–18 h (Fig. 5B). Consistent with the results obtained with the rabbit osteoclasts, CT reduced the bone resorbing activity of the wild type murine OCLs by about 75%. The resorption by the μ-calpain^−/− OCLs was about half of the resorption by the wild type OCLs, a significant reduction from the wild type activity but not as great a reduction as that achieved by treating the wild type OCLs with CT or by treating the rabbit osteoclasts with either CT or the calpain inhibitors. In contrast to the lack of an additional effect of CT on the bone resorbing activity of rabbit osteoclasts treated with calpain inhibitors, however, treatment of the μ-calpain^−/−OCLs with CT induced a further reduction in the bone resorbing activity, suggesting again that at least one other effector that is inhibited by the calpain inhibitors and CT, most likely m-calpain, also contributes to osteoclast bone resorbing activity.

Calcitonin Modulates Calpain Activity in Osteoclasts

The similar effects of CT and calpain inhibitors on osteoclast retraction, motility, and bone resorbing activity, together with the lack of any additive effect of the two agents on these functions, suggested that CT might act at least in part by inhibiting calpain. Calpain activity can be inhibited by either PKC or PKA (43), both of which are activated downstream of the CTR (29, 34, 36, 37). Thus, CT could inhibit calpain activity in osteoclasts and thereby modulate osteoclast attachment, motility, and bone resorbing activity. To determine whether CT affected calpain activity in osteoclasts, rabbit osteoclasts were incubated in the presence of 30 μm Boc-LM-CMAC, a membrane-permeable fluorogenic calpain substrate, for 1 h, during which time the fluorescence increased in the osteoclasts and contaminating cells as a consequence of constitutive calpain activity. Following the administration of CT, the fluorescence in the osteoclasts decreased, in contrast to the continued increased fluorescence in contaminating cells, particularly erythrocytes, that do not express CT receptors (Fig. 6A), consistent with the hypothesized down-regulation of osteoclast calpain activity by CT.

Since calpain inhibitors blocked the constitutive fragmentation of filamin, talin, and Pyk2 in osteoclasts (Fig. 3), we asked whether CT also affected the proteolysis of talin and filamin in a similar manner. To address the question, rabbit OCLs were treated with 10⁻⁸ m CT for various times, and the lysates were blotted with antibodies against filamin and talin (Fig. 6B). As noted before, both the full-length proteins and high MW cleavage products were detected in untreated OCLs. CT treatment transiently reduced the amounts of the fragments, which were largely absent at 5 min after the addition of CT and present again by 10–20 min, providing further evidence that CT inhibits calpain activity in osteoclasts.

PKC Mediates the CT-induced Inhibition of Calpain

The CTR signals through several effectors that are known to inhibit calpain in other cell systems, including PKA and PKC (29, 34, 36, 37). In order to determine whether either PKA or PKC mediate the CT-induced decrease in calpain activity, we examined the effect of PKC and PKA inhibitors on the CT-induced inhibition of the constitutive cleavage of talin. Mouse OCLs were treated with 10⁻⁹ m CT for 5 min in the absence and in the presence of calphostin C (25, 50, or 100 nm), H89 (50 or 100 nm) or (R_p)-8-Br-cAMPS (1 or 5 μM) and then lysed and processed for Western blotting with anti-talin antibody. Three high molecular weight proteins were detected in lysates from untreated OCLs. CT treatment significantly reduced the lowest band but had little effect on the upper two bands. Calphostin C dose-dependently prevented the CT-induced disappearance of the talin cleavage product (Fig. 7A), whereas the PKA inhibitors had little effect (Fig. 7B), suggesting that CT acts via PKC but not PKA to inhibit calpain activity.

The Absence of μ-Calpain Causes Bone Loss in Mice

Based on the ability of calpain inhibitors to reduce osteoclast spreading, motility, and bone resorbing activity in vitro, the decreased motility and bone resorbing activity of μ-calpain^−/− OCLs, and the CT-induced inhibition of calpain, we hypothesized that the μ-calpain^−/− mice would have increased bone mass due to decreased bone resorption. Contrary to our prediction, however, Von Kossa staining of sections of tibias from 10 week-old μ-calpain^−/− mice and genetically matched wild type mice revealed a marked decrease in the trabecular bone volume of the secondary spongiosa of the μ-calpain^−/− bones (Fig. 8A). Histomorphometric analysis of 5- and 10-week-old μ-calpain^−/− and wild type mice confirmed this observation. Trabecular bone volume (Fig. 8B) was markedly decreased at 10 weeks (but not at 5 weeks) in the μ-calpain^−/− mice due to diminished trabecular number (Fig. 8, C and D). No difference was observed in the cortical bone thickness at either age (Fig. 8E). Both osteoclast numbers and the amount of bone surface covered by osteoclasts were strongly increased by 30–60% in μ-calpain^−/− mice relative to wild type controls at both 5 and 10 weeks (Fig. 8, F and G), whereas no difference was observed in either osteoblast surface or number at either age (Fig. 8, H and I).

Bone formation was assessed in the same mice by examining fluorochrome labels incorporated during mineralization. There were no significant differences in either mineralizing surface or mineral apposition rate at either 5 or 10 weeks (Fig. 8, J and K). As a result, bone formation rate was also unchanged (data not shown).

To further assess whether bone resorption was increased in the absence of μ-calpain, we assayed the serum level of pyridinoline cross-links produced from collagen type I digestion, a well established serum marker of bone resorption, in 6-week-old μ-calpain^−/− mice and found that this parameter was moderately increased (Fig. 8L). Given the marked elevation in the number of osteoclasts, the moderate increase in cross-links is consistent with the reduced bone resorbing activity of individual μ-calpain^−/− OCLs that we observed in vitro. The results suggest that the reduced activity of the individual osteoclasts partly compensates for the greater number of osteoclasts, resulting in the slowly progressing osteopenia that was seen at 10 weeks but not at 5 weeks.

Inhibition or Disruption of Calpain Prolongs the Survival of OCLs

The increased number of osteoclasts in the spongiosa of μ-calpain^−/−mice could be the consequence of either increased osteoclast differentiation or increased osteoclast survival. We therefore examined the effect of deleting μ-calpain on the formation of TRAP-positive multinucleated OCLs in the in vitro coculture system. Similar numbers of OCLs were formed in the cocultures of bone marrow cells from wild type and μ-calpain^−/−mice (Fig. 9A), indicating that μ-calpain is not required for normal osteoclast differentiation and suggesting that the increased numbers of osteoclasts observed in the μ-calpain^−/− mice was not due to increased recruitment. On the other hand, both the absence of μ-calpain and the pharmacological inhibition of calpain prolonged the survival of the OCLs following the removal of the supporting calvarial stromal cells (Fig. 9, B and C). The increased survival of the μ-calpain^−/− OCLs may explain the increased number of osteoclasts observed in the μ-calpain^−/− mice. CT prolonged the survival of the OCLs in the absence of supporting stromal cells to the same degree as the presence of calpain inhibitors. Interestingly, CT and the calpain inhibitors were more effective at prolonging the survival of the OCLs than the absence of the μ-calpain gene was, suggesting again that these agents are affecting more than μ-calpain in osteoclasts.

Calpains are reported to trigger apoptosis in various cell types by cleaving and activating caspase-3 (24–26), and our laboratory has shown that caspase-3 is cleaved during OCL apoptosis (74). We therefore examined the effect of CT on the presence of cleaved caspase-3 in wild type OCLs cultured in the absence of supporting stromal cells (Fig. 9D) and found that CT inhibited the production of activated caspase-3. We also examined the rate of appearance of cleaved caspase-3 in wild type and in μ-calpain^−/−OCLs (Fig. 9E). Cleaved caspase-3 appeared in both the wild type and the μ-calpain−/− OCLs after the supporting stromal cell layer was removed, but the level of cleaved caspase-3 was higher in the wild type OCLs at 12 and 18 h, which would be predicted to promote the more rapid apoptosis of the wild type osteoclasts.