Based on developmental fate and function, cartilage tissue is broadly classified into transient cartilage (e.g. growth plate, GP) and permanent cartilage (e.g. articular cartilage, AC). The former eventually disappears and is replaced by bone during the endochondral ossification process, whereas the latter retains its permanency. Osteo(chondro)clasts, multinucleated giant cells of the monocyte/macrophage lineage, are selectively induced in the GP during endochondral ossification and play central roles in the resorption of cartilagenous matrices. The aim of this study was to investigate the factors determining the GP-specific recruitment of osteo(chondro)clasts. We especially focused on the expression pattern of the receptor activator of NF-kappaB ligand (RANKL), an essential factor for osteo(chondro)clast differentiation, and on that of epigenetic and transcriptional factors affecting RANKL gene expression. Knee joints of male BALB/c mice aged 8 weeks were dissected and subjected to immunohistochemical analysis using anti-RANKL, Runx2, Dlx5 and Msx2 antibodies. The methylation status of the mouse RANKL gene promoter in both the GP and the AC was analyzed by sodium bisulfite mapping using microdissected mouse tissue. The expression of BMP-2, -3, -4, -6 and type X collagen mRNA was examined by in situ hybridization (ISH). At the boundary between the calcifying cartilage and the hypertrophic chondrocytes of the GP, RANKL-expressing chondrocytes overlapped those expressing Runx2, Dlx5 and Msx2, near numerous osteo(chondro)clasts. Although similar BMP-2 and -4 expression was observed in chondrocytes in both the GP and the AC as well as in maturing osteoblasts, a rather restricted BMP-6 expression pattern was observed in resting and proliferating chondrocytes in the GP. On the other hand, sodium bisulfite mapping showed that mostly non-CpG methylation was similarly scattered in a non-specific manner in chondrocytes in the GP and the AC. Taken together with the fact that putative Runx2 binding elements are located in the RANKL promoter, our data suggest that Runx2, an essential transcription factor for skeletal development, is also a key regulator of RANKL expression in chondrocytes in the GP. Furthermore, a selective and sequential expression of a subset of BMP and of transcription factors may define the expression pattern of RANKL through Runx2.
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