Calpain is required for normal osteoclast function and is down-regulated by calcitonin

Abstract

Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both mu-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of mu-calpain(-/-) osteoclast-like cells were also reduced, indicating that mu-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from mu-calpain(-/-) mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the mu-calpain(-/-) bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.

FIGURE 1. Osteoclasts express both μ-calpain and m-calpain

Western blotting of lysates of purified rabbit (A) or mouse (B) OCLs with antibodies against μ-calpain and m-calpain, respectively, identified immunoreactive bands at the predicted relative mobilities. Double immunofluorescence staining of authentic rabbit (C and E) or mouse (D and F) osteoclasts for F-actin (red) and μ-calpain (C and D) or m-calpain (E and F) (green) revealed that both calpains are present and distributed throughout the cell, with high levels seen in the peripheral F-actin-rich belt of podosomes. Scale bars, 5 μm.

FIGURE 2. Talin and filamin A are enriched in the osteoclast podosome belt

Double immunofluorescence staining of actin (red) and talin (A), filamin A (B), or μ-calpain (C) (green) revealed that both proteins localize in the actin-rich podosome ring. Talin was primarily localized in a ring surrounding the F-actin-containing podosome core, as previously described (3), whereas filamin and μ-calpain were more evenly distributed throughout the region between the individual podosomes. Scale bars, 2 μm.

FIGURE 3. Calpain constitutively cleaves talin, filamin A, and Pyk2 in osteoclasts

Western blotting of lysates of untreated rabbit (A and B, left panels) or mouse (A and B, right panels; C) OCL with antibodies against talin (A), filamin A (B), or Pyk2 (C) detected major bands at the predicted relative mobilities of the two proteins and less intense bands just below the parent protein (C lanes). Pretreatment of OCLs with membrane-permeable calpain inhibitors that block the catalytic site (calpeptin (CP; 40 μg/ml), MDL28170 (MDL; 50 μm)) or the calcium-binding site (PD151746 (PD); 100 μm)) largely eliminated the lower bands, indicating that calpain constitutively cleaves talin, filamin A, and Pyk2 in osteoclasts.

FIGURE 4. Calpain promotes osteoclast spreading and migration

A, rabbit osteoclasts were plated on dentine slices and cultured for 24 h and then treated with 10⁻⁸ m CT and/or with 40 μg/ml calpeptin (CP), 50 μm MDL28170 (MDL), or 100 μm PD151746 (PD), as indicated, for 20 min. The cells were prepared, and the areas of the cells were analyzed as described under “Experimental Procedures.” *, p < 0.01 relative to untreated control. B, to test the effects of calpain inhibitors on osteoclast migration, osteoclasts were cultured as described under “Experimental Procedures” for 16 h on semipermeable membranes in Boyden chambers in the presence of 10⁻⁹ m CT and/or the calpain inhibitors CP (40 μg/ml) and MDL (50 μm) as indicated. The membranes were stained for TRAP as described under “Experimental Procedures,” and the TRAP-positive cells on the bottom surface of the membranes were counted. *, p < 0.01 relative to untreated controls (C). C, the migration assay was performed with OCLs generated from bone marrow cells obtained from μ-calpain^−/− mice and genetically matched wild type animals, as described under “Experimental Procedures,” in the presence or absence of 10⁻⁹ m CT. *, p < 0.01 relative to the untreated WT. #, p < 0.01 relative to the untreated μ-calpain^−/− OCLs.

FIGURE 5. Bone resorption requires calpain activity

A, rabbit osteoclasts were plated on dentine slices and incubated for 18 h in the presence of calpain inhibitors (40 μg/ml CP, 50 μm MDL) and/or CT (10⁻⁹ m) as a positive control, as indicated. *, p < 0.05 relative to untreated cells. B, OCLs were generated from bone marrow cells obtained from μ-calpain^−/− mice and genetically matched wild type animals as described under “Experimental Procedures.” Aliquots of OCL preparations were transferred onto dentine slices and cultured for an additional 12 h in the presence or absence of 10⁻⁹ m CT. *, p < 0.01 relative to untreated WT; #, p < 0.05 relative to untreated μ-calpain^−/− OCLs.

FIGURE 6. CT inhibits calpain activity in osteoclasts

A, rabbit osteoclasts were preincubated with 30 μm Boc-LM-CMAC for 1 h and then treated with 10⁻⁸ m CT. Fluorescence images were taken before and after the addition of CT as indicated. The fluorescence intensity of the multinucleated osteoclasts decreased after the addition of CT. The progressive increase in the fluorescence in red blood cells and fibroblast-like cells (arrows) was not affected by the presence of CT. B, rabbit OCLs were treated with 10⁻⁸ m CT for the indicated times, and the lysates were blotted with antibodies against filamin and talin. CT transiently inhibited the constitutive cleavage of filamin and talin in OCLs.

FIGURE 7. Calcitonin-induced PKC activity inhibits calpain

OCLs were generated from murine bone marrow. Prior to treatment with CT and the inhibitors, OCLs were cultured for 18 h in 0.5% FBS. The supporting stromal cell layers were removed, and the OCLs were pretreated for 30 min with vehicle, calphostin C (CalC; 25, 50, and 100 nm), H-89 (50 and 100 nm), (R_p)-8-Br-cAMPS (Rp; 1 μM), or PD151746 (PD; 100 μm) followed by CT (10⁻⁹ m) for 5 min as indicated. Western blotting with anti-talin antibody was performed to observe the effect of PKA and PKC inhibitors on the CT-induced inhibition of talin cleavage. The membranes were stripped and reprobed for actin to show relative sample loading. A, three high molecular weight bands were observed in untreated cells. Treatment with PD151746 or CT largely eliminated the lowest band. The PKC inhibitor calphostin C dose-dependently blocked the CT-induced inhibition of talin cleavage. B, in contrast, neither PKA inhibitor (H-89 or (R_p)-8-Br-cAMPS) reversed the CT-induced inhibition of the talin cleavage. There was no effect of calphostin C, H-89, or (R_p)-8-Br-cAMPS alone on the presence of the talin cleavage product (not shown).

FIGURE 8. Bones of μ-calpain^−/− mice are progressively osteopenic

A, Von Kossa staining of tibial proximal metaphyses showing decreased trabeculation of the secondary spongiosa in bones from μ-calpain^−/− (CI⁻) mice. B–E, structural histomorphometry of tibial proximal metaphyses. B, BV/TV, trabecular bone volume; C, TbN, trabecular number; D, TbTh, trabecular thickness; E, CBTh, cortical bone thickness. F–K, cellular and dynamic histomorphometric parameters in tibial proximal metaphyses. F, OcS/BS, osteoclast surface/ bone surface; G, NOc/BP, number of osteoclasts/bone perimeter; H, ObS/BS, osteoblast surface/bone surface; I, NOb/BP, number of osteoblasts/bone perimeter; J, MS/BS, mineralizing surface/bone surface; K, MAR, mineral apposition rate. *, p < 0.05 relative to age-matched WT; **, p < 0.01 relative to age-matched WT. L, serum levels of collagen-derived pyridinoline cross-links.

FIGURE 9. Reducing calpain activity enhances the survival of OCLs

A, OCLs were generated from bone marrow obtained from μ-calpain^−/− (CI⁻/⁻) and genetically matched wild type mice. After removal of the supporting stromal cell layer, the cells were fixed and stained for TRAP, and the TRAP-positive multinucleated cells were counted. The numbers in the WT and μ-calpain^−/− cultures were similar at the time when the stromal cells layers were removed. B, OCLs were incubated in μ-MEM, 10% FBS for 12 h after removing the supporting stromal cell layer and then stained for TRAP and counted. The number of viable OCLs remaining in the WT and μ-calpain^−/− cultures after 12 h is shown as a percentage of the number of cells when the stromal cell layer was removed (T₀). *, p < 0.01 relative to the WT cells. C, wild type murine OCLs were generated in coculture. The supporting stromal cell layers were removed, and the OCLs were further incubated in the presence or absence of CT (10⁻⁹ M) or calpain inhibitors (40 μg/ml CP; 50 μm MDL) or dimethyl sulfoxide alone (D; 1 μl/ml) for 12 h and then TRAP-stained and counted. *, p < 0.05 relative to untreated controls. D, Western blotting of cleaved caspase-3 in WT OCLs treated with 10⁻⁹ m CT and untreated controls. E, wild type (+) and μ-calpain^−/− (−) OCLs were cultured for the indicated times in the absence of the supporting stromal cell layer. The time-dependent change in cleaved caspase-3 was detected by Western blotting with anti-cleaved caspase-3 antibody. The membrane was stripped and reprobed for actin to show relative sample loading.