title: Myosin VI reduces proliferation, but not differentiation, in pluripotent P19 cells.
abstract: BACKGROUND
  We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2'-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.
  
  
  RESULTS
  Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca(2+) ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.
  
  
  CONCLUSIONS
  These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School, Kanazawa, Ishikawa, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takarada
authors.forename: Miki
authors.initials: M
authors.lastname: Kou
authors.forename: Noritaka
authors.initials: N
authors.lastname: Nakamichi
authors.forename: Masato
authors.initials: M
authors.lastname: Ogura
authors.forename: Yuma
authors.initials: Y
authors.lastname: Ito
authors.forename: Ryo
authors.initials: R
authors.lastname: Fukumori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kokubo
authors.forename: Gabriela B
authors.initials: GB
authors.lastname: Acosta
authors.forename: Eiichi
authors.initials: E
authors.lastname: Hinoi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D008156:Luciferases; D051379:Mice; D018995:Myosin Heavy Chains / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D008156:Luciferases; D018995:Myosin Heavy Chains; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C489685:Hes5 protein, mouse; C089785:myosin VI; C022616:thiazolyl blue