Cyclosporin A treatment upregulates Id1 and Smad3 expression and delays skeletal muscle regeneration.
The molecular signaling pathway linked to muscle regeneration has not yet been identified. Previously, we demonstrated that mice treated with cyclosporin A (CsA), a calcineurin inhibitor, failed to regenerate normally after muscle damage. Using reverse transcription (RT)-PCR, Western blot and immunohistochemical analysis, we investigated whether the amounts of nuclear factor of activated T cells (NFAT), myocyte-enhancer factor 2 (MEF2), the MyoD family, Id-1, and Smad3 change in the regenerating muscle after CsA treatment. Adult male ICR mice were subjected to a bupivacaine injection into the tibialis anterior muscle, and were treated with either CsA (25 mg/kg) or vehicle once daily. They were killed at 1, 2, 4, 6, 9 and 14 days post injury. RT-PCR analysis did not show a significant difference in MEF2s, MyoD and myogenin mRNA levels in the regenerating muscle in either placebo- and CsA-administered mice. In contrast, a significant increase in MRF4 mRNA was seen in CsA-administered mice compared to the placebo-treated mice at 4 and 9 days post surgery. In CsA-treated mice, the level of Id1 mRNA was elevated at day 9 relative to the placebo-treated mice. After 6 days, the CsA-treated mice possessed more abundant proliferating cell nuclear antigen (PCNA) and cyclin D1 protein in many satellite cells and/or myoblast-like cells in the regenerating muscle. The amount of myostatin, TGF-beta2 and Smad3 mRNA and proteins was increased more markedly in the mice treated with CsA. After 9 days, many satellite cells and/or myoblasts showed apparent co-localization of both MyoD and Smad3 in CsA-, but not in placebo-, treated mice. Our results demonstrated that CsA treatment upregulates Id1 and Smad3 expression and delays skeletal muscle regeneration in vivo.
Department of Legal Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-hirokoji, Kamigyo-ku, 602-8566 Kyoto, Japan. ksakuma@koto.kpu-m.ac.jp
Kunihiro
K
Sakuma
Ryuta
R
Nakao
Wataru
W
Aoi
Shuichiro
S
Inashima
Takahiko
T
Fujikawa
Miyuki
M
Hirata
Mamoru
M
Sano
Masahiro
M
Yasuhara
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006131:Growth Inhibitors / Q000494:pharmacology; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics*; D064326:MEF2 Transcription Factors; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017570:MyoD Protein / Q000378:metabolism; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism; D055435:Myostatin; D050778:NFATC Transcription Factors / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012038:Regeneration / Q000187:drug effects* / Q000502:physiology; D032496:Satellite Cells, Skeletal Muscle / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051900:Smad3 Protein / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
D056741:Cyclin D; D016213:Cyclins; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D064326:MEF2 Transcription Factors; C578039:Mef2b protein, mouse; C525912:Mstn protein, mouse; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D018006:Myogenic Regulatory Factors; D055435:Myostatin; D050778:NFATC Transcription Factors; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D016212:Transforming Growth Factor beta; C063403:myogenic factor 6; D016572:Cyclosporine