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.
Salingcarnboriboon
R Ampornaramveth
RA
Department of Microbiology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
Pavasant
P
P
Noda
M
M
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C118387:Cblb protein, mouse; D050659:Core Binding Factor Alpha 1 Subunit; D015675:Osteocalcin; D050721:Proto-Oncogene Proteins c-cbl