c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src−/− relative to Src+/+ mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by ∼60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src−/− osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

We investigated the effects of dietary zinc deficiency on skeletal metabolism in an animal model. Thirty 21-d-old male Sprague-Dawley rats were fed for 28 d either a zinc-deficient (ZD) diet (1 mg zinc/kg) or a normal diet ad libitum (AL, 50 mg zinc/kg) or in the same quantity as the ZD (pair-fed, PF). Only in the ZD group were general physical signs of zinc deficiency observed. Compared with the AL and PF rats, ZD rats showed significantly lower mean values in ponderal growth rate, femur weight and length, circulating levels of insulin-like growth factor-I, bone mechanical properties and concentration of zinc and, on histomorphometry, a decrease in the thicknesses of the overall growth plate and hypertrophic cartilage. In contrast, although bone volume was significantly lower in the ZD and PF rats than in the AL rats, no difference was observed between the ZD and PF rats. Osteoclast surface/bone surface and osteoclast number/bone surface ratios were significantly greater in PF rats than in the other two groups and not different in ZD and AL rats. Collectively, these data indicate that zinc deficiency has profound effects on the skeletal system of growing rats. In particular, the effects of zinc deficiency on bone growth and mass are the result of the reduced activity of the growth plate, likely mediated by impairment in the insulin-like growth factor-I system. We did not demonstrate an effect on bone mass via increased bone resorption.

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 ...

Bone marrow (BM) fibrosis is a feature of severe hyperparathyroidism. Consistent with this observation, mice expressing constitutively active parathyroid hormone (PTH)/PTH-related peptide receptors (PPR) in osteoblasts (PPR*Tg) display BM fibrosis. To obtain insight into the nature of BM fibrosis in such a model, a double-mutant mouse expressing constitutively active PPR and green fluorescent protein (GFP) under the control of the type I collagen promoter (PPR*Tg/GFP) was generated. Confocal microscopy and flow cytometry revealed the presence of a cell population expressing GFP (GFP(+)) that was also positive for the hematopoietic marker CD45 in the BM of both PPR*Tg/GFP and control animals. This cell population was expanded in PPR*Tg/GFP. The existence of cells expressing both type I collagen and CD45 in the adult BM was confirmed by IHC and fluorescence-activated cell sorting. An analysis of total RNA extracted from sorted GFP(+)CD45(+) cells showed that these cells produced type I collagen and PTH/PTH-related peptide receptor and receptor activator for NF-κB mRNAs, further supporting their features of being both mesenchymal and hematopoietic lineages. Similar cells, known as fibrocytes, are also present in pathological fibroses. Our findings, thus, indicate that the BM is a permissive microenvironment for the differentiation of fibrocyte-like cells and raise the possibility that these cells could contribute to the pathogenesis of BM fibrosis.

The ADAMs (a disintegrin and metalloprotease) contribute to various biological functions including the development of tissues by taking part in cell-cell and cell-matrix interactions. We previously found that ADAM15 is prominently expressed in osteoblasts and to a lesser extent in osteoclasts. The aim of this study was to investigate a possible function of ADAM15 in bone. Adult ADAM15(-/-) mice displayed an increase in bone volume and thickness with an increase in the number and activity of osteoblasts, whereas osteoclasts were apparently unaffected. We found an increase in proliferation, alkaline phosphatase (ALP) staining and nodule deposition, and mineralization in cultures of ADAM15(-/-) osteoblasts compared to wild-type osteoblasts. We also observed an increase in β-catenin immunoreactivity in the nucleus of ADAM15(-/-) osteoblasts compared to wild-type, whereas β-catenin in the membrane/cytoplasm compartment appeared to undergo increased degradation. Furthermore, cyclin D1 and c-Jun, known downstream targets of β-catenin and effectors of cell activation, were found up-regulated in absence of ADAM15. This study indicates that ADAM15 is required for normal skeletal homeostasis and that its absence causes increased nuclear translocation of β-catenin in osteoblasts leading to increased osteoblast proliferation and function, which results in higher trabecular and cortical bone mass.