Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2alpha enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2alpha, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2alpha expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis development, and a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-kappaB (NF-kappaB) family member, as a potent inducer of HIF-2alpha expression. Hence, HIF-2alpha is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis.

Objective. To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis.Methods. Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes.Results. No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time.Conclusion. These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.Utilizing the differentiation and proliferation capabilities of stem cells, regenerative medicine attempts to treat irreversible organ failures that cannot be dealt with by conventional medical treatment. In the skeletal area, cartilage has a relatively poor regenerative capacity and, thus, may benefit most from regenerative medicine. Conditions such as osteoarthritis and congenital skeletal defects are apparent targets that have great medical and socioeconomic impact. To make cartilage regenerative medicine a reality, it is essential to know the conditions that are both necessary and sufficient for chondrogenesis.A number of factors have been shown to be vital for chondrogenesis. These factors include the sexdetermining region Y-type high mobility group box (SOX) family of transcription factors (1), insulin-like growth factor 1 (IGF-1) (2), fibroblast growth factor 2 (FGF-2) (3), Indian hedgehog (IHH) (4), bone morphoDr. Ikegawa

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.

To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.