Depolarization-induced slow Ca2+ transients stimulate transcription of IL-6 gene in skeletal muscle cells

Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-1,4,5-trisphosphate (IP3)-mediated slow Ca2+ signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative RT-PCR, we have shown that K+ depolarization of myotubes induces a transient increase in IL-6 mRNA level, which peaks at 3-4 h and is independent of extracellular Ca2+. Inhibitors of IP3-dependent Ca2+ signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, decreased activation of IL-6 gene expression as did Ca2+ signals inhibitor BAPTA-AM, whereas ryanodine, a fast Ca2+ transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify activator protein-1 and NF-kappa B sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of IP3-mediated Ca2+ signals on IL-6 transcription in skeletal muscle cells