title: RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts.
abstract: OBJECTIVE
  BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts.
  
  
  METHODS
  The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2.
  
  
  RESULTS
  Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus.
  
  
  CONCLUSIONS
  We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan. Electronic address: y-yoshi@cc.osaka-dent.ac.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoshikawa
authors.affiliation: Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yoshizawa
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Domae
authors.affiliation: SofSera, Osaka 594-1157, Japan.
authors.forename: Yohki
authors.initials: Y
authors.lastname: Hieda
authors.affiliation: Department of Oral and Maxillofacial Surgery 1, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Takeyama
authors.affiliation: Department of Geriatric Dentistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Shuitsu
authors.initials: S
authors.lastname: Hirota
authors.affiliation: Department of Geriatric Dentistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Akiyo
authors.initials: A
authors.lastname: Kawamoto
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Goda
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Tamura
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Kamada
authors.affiliation: Department of Geriatric Dentistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Komasa
authors.affiliation: Department of Oral and Maxillofacial Surgery 1, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Shosuke
authors.initials: S
authors.lastname: Morita
authors.affiliation: Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yamagata
authors.affiliation: Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ikeo
mesh_terms: D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D008024:Ligands; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018167:Receptors, Calcitriol / Q000378:metabolism; D015398:Signal Transduction; D051898:Smad1 Protein / Q000502:physiology*
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D008024:Ligands; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018167:Receptors, Calcitriol; D051898:Smad1 Protein; C497401:Smad1 protein, mouse
keywords: Bone morphogenetic protein-2; Osteoblast; Osteoclast; Receptor activator of nuclear factor κB ligand; Smad1; Vitamin D receptor