Figure 2.

Induction of NF-AT–mediated transcription in skeletal muscle cells. (A) Myotubes contain NF-AT proteins that are functionally active in response to known pharmacological inducers of NF-AT–mediated transcription in T cells. Primary mouse myotubes containing either the control (minimal IL-2 promoter only, □) or the NF-AT-responsive (triplex of the distal IL-2 gene NF-AT response element [NFRE] upstream of the minimal IL-2 promoter,▪) retroviral reporter plasmids were treated for 6 h with vehicle alone (basal), PMA (10 nM), or ionomycin (1 μM) alone or the two together (P+I). Some cultures were pretreated with cyclosporine A (CSA; 1 μM) for 1 h before addition of PMA and ionomycin to the medium. Luciferase values were subsequently measured. Luciferase activity is induced only in the cells containing the NF-AT response element upstream of the minimal IL-2 promoter. Both PMA and ionomycin stimulate NF-AT–mediated transcription in myotubes, but the two drugs together markedly syngerize. The P+I response is blocked in the presence of CSA. Each bar represents the mean ± SEM of two experiments, each performed in triplicate. (B) Drugs with different mechanisms of increasing intracellular calcium induce NF-AT– mediated transcription in myotubes. Primary mouse myotubes containing the NF-AT responsive reporter plasmid were treated with vehicle alone or different doses of thapsigargin (panel A), ryanodine (panel B), or ionomycin (panel C) in the presence of 10 nM PMA for 6 h before the measurement of luciferase activity. Cultures were either treated with these drugs alone (•) or together with 1 μM CSA (○). All three agents induce NF-AT–mediated transcription, but the maximum levels of induction differ, with ionomycin giving the greatest induction. Each point represents the mean ± SEM of three independent experiments, each performed in triplicate.