title: Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-kappa B signaling.
abstract: In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of I kappa B, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of I kappa B protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-kappaB-binding activity in L6 cells and prevented the ubiquitination of I kappa B proteins in the I kappa B- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-kappa B pathway.
authors.affiliation: Department of Nutrition, The University of Tokushima School of Medicine, Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Hirasaka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nikawa
authors.forename: Louis
authors.initials: L
authors.lastname: Yuge
authors.forename: Ibuki
authors.initials: I
authors.lastname: Ishihara
authors.forename: Akira
authors.initials: A
authors.lastname: Higashibata
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ishioka
authors.forename: Atsuko
authors.initials: A
authors.lastname: Okubo
authors.forename: Takashi
authors.initials: T
authors.lastname: Miyashita
authors.forename: Naoto
authors.initials: N
authors.lastname: Suzue
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ogawa
authors.forename: Motoko
authors.initials: M
authors.lastname: Oarada
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Kishi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D019927:Cyclin E / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000166:cytology; D009132:Muscles / Q000166:cytology; D018008:Myogenin / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D014893:Weightlessness
chemical_list: D019927:Cyclin E; D020672:I-kappa B Proteins; C503803:Myog protein, rat; D018008:Myogenin; D016328:NF-kappa B; D012333:RNA, Messenger; D025801:Ubiquitin; D018995:Myosin Heavy Chains; D010100:Oxygen