Ubc9 promotes the stability of Smad4 and the nuclear accumulation of Smad1 in osteoblast-like Saos-2 cells.
Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate within the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from human bone marrow to identify the proteins interacting with Smad4. Two full-length cDNA clones for Ubc9 were identified, and the potential functions of Ubc9 were investigated. To determine the role of Ubc9 in the BMP signaling pathway, the endogenous transcription of Ubc9 in the human osteoblast cell line Saos-2 was silenced using siRNA. The expression of BMP-induced transcription factors, including Runx2, Dlx5, Msx2, and Osterix, was examined using real-time reverse transcription polymerase chain reaction (qRT-PCR), and the protein expression of Smad4, Smad1, phosphorylated Smad1, and BMP type I receptors was determined by Western blotting. The subcellular localization of Smad1 and Smad4 was observed using immunofluorescence staining after Ubc9 silencing. To determine whether Smad4 is sumoylated in vitro, recombinant Smad4 was purified and sumoylated Smad4 was visualized using Western blotting. The mRNA expression of various transcription factors was markedly inhibited after Ubc9 silencing. The protein levels of Smad4 and phosphorylated Smad1 decreased in a dose-dependent manner according to the amount of siRNA applied. Gene silencing also decreased the nuclear accumulation of Smad1 and Smad4. The sumoylation assay showed that sumoylated Smad4 is present and dependent on Ubc9 in vitro, which was confirmed by pretreatment with Senp2, a SUMO-protease. These results suggest that Ubc9 promotes the stability of sumoylated Smad4. Furthermore, the expression of key transcription factors, phosphorylated Smad1 protein, and the nuclear accumulation of Smad1 and Smad4 are inhibited by Ubc9 silencing. Thus, Ubc9 plays an important role in the up-regulation of the BMP signaling pathway.
Shimada
Koichi
K
Department of Periodontology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan. shimada-ki@dent.nihon-u.ac.jp
Suzuki
Naoto
N
Ono
Yoichi
Y
Tanaka
Koji
K
Maeno
Masao
M
Ito
Koichi
K
D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025842:SUMO-1 Protein / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000235:genetics / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000502:physiology*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; C100596:DLX5 protein, human; D018398:Homeodomain Proteins; C084119:MSX2 protein; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; C497399:SMAD1 protein, human; C497796:SMAD4 protein, human; D025842:SUMO-1 Protein; D051898:Smad1 Protein; D051901:Smad4 Protein; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D044763:Ubiquitin-Conjugating Enzymes; C498211:BMPR1A protein, human; C498214:BMPR1B protein, human; D052005:Bone Morphogenetic Protein Receptors, Type I; D000098843:Ubiquitin-Conjugating Enzyme UBC9