Foxo1 Activation Promotes Proapoptotic Gene Expression In Differentiated Skeletal Muscle In Vitro

The winged-helix transcription factor FoxO1 (forkhead box, class O) plays an important role in the regulation of cell cycle, cell growth and cell survival. Recent findings have demonstrated attenuated sarcomeric alpha actinin expression and loss of myotube formation in differentiated skeletal muscle following FoxO1 activation in vitro. The mechanisms responsible for mediating the changes in myotube structure, however, remain to be elucidated. PURPOSE To determine the gene expression changes in differentiated skeletal muscle following FoxO1 activation in skeletal myotubes in vitro. METHODS FoxO1-estrogen receptor (FoxO1-ER) fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OHT) were stably transfected in C2C12 skeletal myoblasts using the pBABE retroviral system. FoxO1-ER transfected or control myoblasts (empty construct) were differentiated into myotubes in low serum and maintained for 4 days. On the 4th day following serum deprivation, total RNA was isolated from skeletal myotubes following treatment with either vehicle (control; 0.1% ethanol final concentration) or 4-OHT (500 nM) for 12 hours. 15 μg of labeled cRNA per sample was hybridized to mouse gene chips, washed and gene expression analyzed performed. All conditions were performed in triplicate and pooled for subsequent analysis. Semi-quantitative RT-PCR was performed for conformational analysis on several genes of interest demonstrating induction or repression following FoxO1 activation. Amplification products were normalized to the housekeeping gene GAPDH. RESULTS Gene expression analysis revealed that a total of 55 genes demonstrated significant changes (+/- 2 fold log ratio change; 20 induction, 35 repression) following 12h treatment with 4-OHT, including several genes associated with apoptosis, cytoskeletal maintenance, proteolysis, and cell size regulation. RT-PCR analysis on follistatin (1.6 fold induction), growth arrest and DNA-damage-inducible 45⊓ (2.6 fold induction), ubiquitin specific protease 2 (2.2 fold induction), and metalloproteinase-13 (undetectable transcript levels following 4-OHT treatment) confirmed the findings of the gene expression analysis. CONCLUSION These initial findings indicate that transcriptional activation of FoxO1 may impact skeletal muscle growth and/or repair by regulating genes responsible for maintaining myotube organization and survival. Supported by NIH grants T32 HL07692-12 (TJM), AR45671 (KAE), and DK-41430 (TGU) and the Department of Veterans Affairs Merit Review Program (TGU).