title: Cell differentiation and p38(MAPK) cascade are inhibited in human osteoblasts cultured in a three-dimensional clinostat.
abstract: A three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10(-3) G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogen-activated protein kinase (MAPK) cascades. In a normal 1 G condition, alkaline phosphatase (AlPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cells looked larger and bulged. AlPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38(MAPK) (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal-regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AlPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MAPK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phosphorylation.
authors.affiliation: Institute of Health Sciences, Faculty of Medicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. ryuge@hiroshima-u.ac.jp
authors.forename: Louis
authors.initials: L
authors.lastname: Yuge
authors.forename: Izumi
authors.initials: I
authors.lastname: Hide
authors.forename: Takanori
authors.initials: T
authors.lastname: Kumagai
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kumei
authors.forename: Sin'ichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Masamoto
authors.initials: M
authors.lastname: Kanno
authors.forename: Masanori
authors.initials: M
authors.lastname: Sugiyama
authors.forename: Katsuko
authors.initials: K
authors.lastname: Kataoka
mesh_terms: D002113:Calcification, Physiologic; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D050660:Core Binding Factors; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D010766:Phosphorylation; D011725:Pyridines / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014893:Weightlessness; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D050660:Core Binding Factors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009363:Neoplasm Proteins; D011725:Pyridines; D014157:Transcription Factors; D015675:Osteocalcin; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580