We established four types of mouse models exhibiting various speeds of OA progression. By applying a mouse genomics approach to the models, molecular backgrounds in various stages of OA development can be clarified.

Since interaction between bone and lipid metabolism has been suggested, this study investigated the regulation of bone metabolism by adiponectin, a representative adipokine, by analyzing deficient and overexpressing transgenic mice. We initially confirmed that adiponectin and its receptors were expressed in osteoblastic and osteoclastic cells, indicating that adiponectin can act on bone not only through an endocrine pathway as a hormone secreted from fat tissue, but also through an autocrine/paracrine pathway. There was no abnormality in bone mass or turnover of adiponectin-deficient (Ad-/-) mice, possibly due to an equivalent balance of the two pathways. In the culture of bone marrow cells from the Ad-/- mice, osteogenesis was decreased compared to the wild-type (WT) cell culture, indicating a positive effect of endogenous adiponectin through the autocrine/paracrine pathway. To examine the endocrine action of adiponectin, we analyzed transgenic mice overexpressing adiponectin in the liver, and found no abnormality in the bone. Addition of recombinant adiponectin in cultured osteoprogenitor cells suppressed osteogenesis, suggesting that the direct action of circulating adiponectin was negative for bone formation. In the presence of insulin, however, this suppression was blunted, and adiponectin enhanced the insulin-induced phosphorylations of the main downstream molecule insulin receptor substrate-1 and Akt. These lines of results suggest three distinct adiponectin actions on bone formation: a positive action through the autocrine/paracrine pathway by locally produced adiponectin, a negative action through the direct pathway by circulating adiponectin, and a positive action through the indirect pathway by circulating adiponectin via enhancement of the insulin signaling.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-κB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

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Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The ␣ subunit of Gs (Gs␣) is encoded by GNAS (Gnas in mice), and heterozygous Gs␣ inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gs␣ in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 (Gnas E2؊/E2؊ ) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice (Gnas ؉/E2؊ ). We generated chimeric mice containing wild-type cells and either Gnas E2؊/E2؊ or Gnas ؉/E2؊ cells. Gnas E2؊/E2؊ chondrocytes phenocopied PTH/PTHrP receptor (PPR) ؊͞؊ cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gs␣, one of several gene products that include Gnas exon 2, into Gnas E2؊/E2؊ cells prevented premature hypertrophy. Gs␣ mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal Gnas ؉/E2؊ growth plate chondrocytes, indicating biallelic expression of Gs␣ in these cells. Hypertrophy of Gnas ؉/E2؊ chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and Gnas ؉/E2؊ cells. These data suggest that Gs␣ is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gs␣ signaling in Gnas ؉/E2؊ chondrocytes.

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.