title: Ubc9 negatively regulates BMP-mediated osteoblastic differentiation in cultured cells.
abstract: SUMO (small ubiquitin-related modifier) modification (SUMOylation) has been reported to regulate various biological events such as cell-cycle progression, proliferation, and survival. Bone morphogenetic proteins (BMPs) play an important role in osteoblast differentiation and maturation. Although Smad4, which acts as a transcriptional factor in the BMP signaling, is a target of SUMOylation, the involvement of SUMOylation in osteoblast differentiation remains unclear. In this report, we demonstrated spatial expression patterns of SUMO proteins and Ubc9 (ubiquitin conjugating enzyme 9), which is a unique E2-SUMOylation enzyme, in mouse tibia. Furthermore, siRNA knockdown of Ubc9 enhanced osteoblastic differentiation induced by BMP2 in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells. Ubc9 knockdown elevated the BMP signaling transduction and reduced the expression of muscle-related genes in cooperation with BMP2. Finally, a luciferase assay using an Id1 (target gene of BMP signaling) reporter revealed that Smad4 mutants prevented from SUMOylation at their Lys158 possessed more potent transcriptional activity than wild-type Smad4. Taken together, these findings suggest that Ubc9 negatively regulates osteoblastic differentiation induced by BMP via, at least in part, SUMOylation of Smad4.
authors.affiliation: Department of Oral Histology, Matsumoto Dental University, 1780 Hirooka-gobara, Shiojiri, Nagano 399-0781, Japan. ayukita@po.mdu.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Yukita
authors.forename: Akihiro
authors.initials: A
authors.lastname: Hosoya
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ito
authors.forename: Takenobu
authors.initials: T
authors.lastname: Katagiri
authors.forename: Makoto
authors.initials: M
authors.lastname: Asashima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D058207:Sumoylation / Q000187:drug effects; D013977:Tibia / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D055396:Bone Morphogenetic Protein 2; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051785:Smad Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9