Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro.
Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2-10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C(2)C(12) myotubes with the calcium ionophore A23187. Cotransfection of C(2)C(12) myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C(2)C(12) myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.
Allen
David L
DL
Dept. of Integrative Physiology, Univ. of Colorado, Boulder, Campus Box 354, Boulder, CO 80309, USA. allendl@colorado.edu
Uyenishi
Jill J
JJ
Cleary
Allison S
AS
Mehan
Ryan S
RS
Lindsay
Sarah F
SF
Reed
Jason M
JM
D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D023421:Models, Animal; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism; D010805:Physical Conditioning, Animal / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C511512:DSCR1 protein, mouse; C491779:Elf4 protein, mouse; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D050778:NFATC Transcription Factors; D012333:RNA, Messenger; D014157:Transcription Factors; D019703:Calcineurin