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 -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 -calpain ؊/؊ osteoclast-like cells were also reduced, indicating that -calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from -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 -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.Osteoclasts are large multinucleated cells of the monocyte-macrophage lineage that play a critical role in skeletal development and repair and in calcium homeostasis by resorbing mineralized cartilage and bone. They display a high degree of motility, which is required for normal bone resorbing activity. As in other highly motile cells, the integrinbased attachment complexes of osteoclasts are podosomes, which are structurally and functionally distinct from focal adhesions (1-6). Although many of the same proteins are present in both podosomes and focal adhesions, podosomes are notably more dynamic, undergoing assembly and disassembly within minutes (5-7). The rapid and cyclic podosome assembly and disassembly and their dynamic interaction with elements of the actin cytoskeleton are thought to be critical for the high motility of osteoclasts (7).Recently, calpains have been found to play crucial roles in the regulation of motility in cells such as fibroblasts that form focal adhesions (8). Calpains are a family of cytosolic cysteine proteases, many of which are Ca 2ϩ -dependent, that catalyze the limited cleavage of specific proteins during regulatory signaling (9, 10). The most studied members of the calpain family are the ubiquitously expressed -calpain ...

Necessity of inositol (1,4,5)-trisphosphate receptor 1 and μ-calpain in NO-induced osteoclast motility

“…Our study shows that -calpain is crucial for NO-induced osteoclast motility and that -calpain is regulated by a Ca 2+ signal that requires PKG1, Src and VASP. Our findings are consistent with results from previous studies showing calpain activity in osteoclasts (Lee et al, 2005;Hayashi et al, 2005;Marzia et al, 2006). Earlier research suggested calpain involvement in osteoclast differentiation and function (Lee et al, 2005;Marzia et al, 2006), but here we describe for the first time -calpain activation and Ca 2+ signaling in NO/cGMP-induced osteoclast motility.…”

Rho GTPases in osteoclasts: Orchestrators of podosome arrangement

“…Many proteins critical for podosome organization are regulated by calcium. For example, Pyk2 can be 14 activated by calcium (Sanjay et al, 2001) and calpains are Ca2+-dependent proteases critical for osteoclasts function (Marzia et al, 2006) that may cleave 3 integrin (Flevaris et al, 2007), RhoA (Kulkarni et al, 2002), Pyk2 (Marzia et al, 2006) and WASp (de la Fuente et al, 2007). Change in calcium concentration affects podosome organization of osteoclast (Miyauchi et al, 1990) and knocking out the calcium transporter TRPV5 generates inactive osteoclasts (van der Eedern et al, 2005).…”

Cortactin Has an Essential and Specific Role in Osteoclast Actin Assembly

“…In this study, WASp-deficient osteoclasts on bone showed a decrease in the area of bone surface resorbed, whereas other parameters, such as volume and depth of resorption pits, were unaffected. In addition, calpain, which can proteolyze the podosome proteins talin, Pyk2 and filamin, has been found to be important for osteoclast activity (Marzia et al, 2006). Dynamin localizes to osteoclast podosomes, and expression of dynamin mutants can inhibit osteoclast activity (Bruzzaniti et al, 2005).…”