title: Characterization of Osterix protein stability and physiological role in osteoblast differentiation.
abstract: Osterix (Osx/SP7) is a C2H2 zinc finger-containing transcription factor of the SP gene family. Osx knockout mice indicate that the gene plays an essential role in osteoblast differentiation and bone formation. However, the mechanisms involved in the regulation of Osx are still poorly understood. Here, we report a novel post-translational mechanism for the regulation of Osx in mammalian cells. We found that the stability of endogenous and exogenous Osx reduced after cycloheximide treatment. In cells treated with the proteasome inhibitors MG-132 or lactacystin, both endogenous and exogenous Osx protein expression increased in a time-dependent manner. Co-immunoprecipitation (Co-IP) assays showed that both endogenous and exogenous Osx were ubiquitinated. Six lysine residues of Osx were identified as candidate ubiquitination sites by construction of point mutant plasmids and luciferase reporter assays. Furthermore, we confirmed that K58 and K230 are the ubiquitination sites of Osx by Co-IP assays and protein stability assays. Moreover, the Osx K58R and K230R mutations promoted the expression of osteoblast differentiation markers (alkaline phosphatase, collagen I and osteocalcin) and enhanced osteogenic differentiation in C2C12 cells. Taken together, our data indicate that Osx is an unstable protein, and that the ubiquitin-proteasome pathway is involved in the regulation of Osx and thereby regulates osteoblast differentiation.
authors: Department of Developmental Genetics, Nanjing Medical University, Nanjing, People's Republic of China.
authors: Yanyan
authors: Y
authors: Peng
authors: Kaikai
authors: K
authors: Shi
authors: Lintao
authors: L
authors: Wang
authors: Jianlei
authors: J
authors: Lu
authors: Hongwei
authors: H
authors: Li
authors: Shiyang
authors: S
authors: Pan
authors: Changyan
authors: C
authors: Ma
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism / Q000502:physiology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010006:Osteoblasts / Q000166:cytology*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D059748:Proteolysis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D005819:Genetic Markers; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D000469:Alkaline Phosphatase