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In vitro assays revealed that COX-2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX-2 inhibitors similarly suppressed bone destruction in adjuvant-induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment. Introduction: Cyclooxygenase (COX)-2 and carbonic anhydrase II (CA II) are known to play important roles in the differentiation of osteoclasts and the activity of mature osteoclasts, respectively. Because several COX-2 selective agents were recently found to possess an inhibitory potency against CA II, this study compared the bone sparing effects of COX-2 selective agents with and without the CA II inhibitory potency. Materials and Methods: Osteoclast differentiation was determined by the mouse co-culture system of osteoblasts and bone marrow cells, and mature osteoclast activity was measured by the pit area on a dentine slice resorbed by osteoclasts generated and isolated from bone marrow cells. In vivo effects on arthritic bone destruction were determined by radiological and histological analyses of hind-paws of adjuvant-induced arthritic (AIA) rats. Results: CA II was expressed predominantly in mature osteoclasts, but not in the precursors. CA II activity was inhibited by sulfonamide-type COX-2 selective agents celecoxib and JTE-522 similarly to a CA II inhibitor acetazolamide, but not by a methylsulfone-type COX-2 inhibitor rofecoxib. In vitro assays clearly revealed that celecoxib and JTE-522 suppressed both differentiation and activity of osteoclasts, whereas rofecoxib and acetazolamide suppressed only osteoclast differentiation and activation, respectively. However, bone destruction in AIA rats was potently and similarly suppressed by all COX-2 selective agents whether with or without CA II inhibitory potency, although only moderately by acetazolamide. Conclusions: Suppression of osteoclast differentiation by COX-2 inhibition is more effective than suppression of mature osteoclast activity by CA II inhibition for the treatment of arthritic bone destruction.

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (10 microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.

This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-κB signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-κB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IκB kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-κB pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-κB-mediated downregulation is essential for efficient osteoclast differentiation.

The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects.

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

To further approach the mechanisms of bone calcification, embryonic rat calvariae were observed at electron microscopic level by the means of fine structures and various cytochemical localizations, including nonspecific proteoglycan (PG) stained by cuprolinic blue (CB), decorin, chondroitin sulfate, hyaluronan, and alkaline phosphatase (ALP), as well as the elemental mapping of calcium (Ca) and phosphorus (P) by energy-filtering transmission electron microscopy (EFTEM). In the calvariae, calcification advanced as the distance from osteoblasts increased. Closer to the osteoblasts, the osteoid was marked by an abundance of CB-positive PGs around collagen fibrils. After crystallization within matrix vesicles, calcified nodules formed and expanded, creating a coherent calcified matrix. The sizes of CB-positive PG-like structures diminished as calcification proceeded. Although small CB-positive structures were accumulated in early stage-calcified nodules, they were localized along the periphery of larger calcified nodules. Cytochemical tests for decorin, chondroitin sulfate, and hyaluronan determined their presence in the areas around collagen fibrils of the osteoid, as well as in and around calcified nodules, whereas ALP was found in the matrix vesicles, as well as in and around the calcified nodules. Ca tended to localize at the PG sites, while P often mapped to the collagen fibril structures, in the uncalcified matrix. In contrast, Ca/P colocalization was visible in and around the calcified nodules, where ALP and smaller CB-positive structures were observed. The difference in the localization patterns of Ca and P in uncalcified areas may limit the local [Ca2+][PO4(3-)] product, leading to the general inhibition of hydroxyapatite crystallization. The downsizing of CB-positive structures suggested enzymatic fragmentation of PGs. Such structural alterations would contribute to the preservation and transport of calcium. ALP possesses the ability to boost local phosphate anion concentration. Therefore, structurally altered PGs and ALP may cooperate in Ca/P colocalization, thus promoting bone calcification.