Cbl-b enhances Runx2 protein stability and augments osteocalcin promoter activity in osteoblastic cell lines.

Cbl-b is a member of Cbl family of E3 ubiquitin (Ub) ligase. Besides the important role in ubiquitination process, other members of Cbl family have been suggested to show non-ubiquitination-related function in regulation of osteoblastic differentiation. However, the role of Cbl-b in regulation of osteoblastic function has not been known yet. To elucidate the role of Cbl-b in regulation of osteoblastic function, we examined its effects on Runx2, a master gene of osteoblastic differentiation. We co-expressed Cbl-b and Runx2 in osteoblastic cell lines and tested their effects on osteocalcin promoter activity together with the expression of Runx2 and its downstream genes. Luciferase assay demonstrated that Cbl-b synergistically enhances osteocalcin promoter activity in conjunction with the effect on Runx2. Co-transfection of Cbl-b and Runx2 further upregulated Runx2 protein levels without any alteration in Runx2 mRNA expression. The upregulation of Runx2 protein by Cbl-b was inhibited by the treatment with lactacystin, a specific inhibitor of the 26S proteasome. These results indicated that Cbl-b would control Runx2 protein levels at the post-translational event. Moreover, the upregulation of downstream genes of Runx2 such as osteocalcin and alkaline phosphatase mRNA was also observed. These data propose the involvement of Cbl-b in the regulation of osteoblast-related genes expression.

Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property.

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.