title: Rho-kinase limits BMP-4-stimulated osteocalcin synthesis in osteoblasts: regulation of the p38 MAP kinase pathway.
abstract: OBJECTIVE
  We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells.
  
  
  METHODS
  The levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR.
  
  
  RESULTS
  BMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4.
  
  
  CONCLUSIONS
  These results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan; Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kondo
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan; Department of Clinical Laboratory, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan. Electronic address: tokuda@ncgg.go.jp.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Tokuda
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Matsushima-Nishiwaki
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan; Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Kuroyanagi
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan; Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Mizutani
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kozawa
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Otsuka
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D055415:Bone Morphogenetic Protein 4 / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D047428:Protein Kinase Inhibitors; D015675:Osteocalcin; D054460:rho-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: BMP-4; MAP kinase; Osteoblast; Osteocalcin; Rho-kinase