Down-regulation of JunB by RNAi in mouse muscles in vivo

The size of skeletal muscles, like that of all cells, is precisely regulated by intracellular signaling networks that determine the balance between overall rates of protein synthesis and degradation. Muscle fiber growth and protein synthesis are stimulated by the IGF1/Akt/mTOR pathway, and muscle wasting, as occurs with disuse, denervation, fasting, and various systemic diseases (e.g. cancer, sepsis) results from excessive protein breakdown in muscle and induction of a set of atrophy-related genes by the FoxO transcription factors. Here we show that the transcription factor JunB is also a major regulator of growth and atrophy of adult (post mitotic) muscle cells. We found that in atrophying myotubes, JunB protein was excluded from the nucleus and that decreasing JunB expression by RNAi in muscles of adults reduced fiber size. Furthermore, in normal muscles of adult mice JunB over-expression increased fiber diameter dramatically (up to 40% in 7 days), and stimulated protein synthesis, but unlike IGF-1 or insulin, JunB did not activate the Akt/mTOR pathway. Importantly, when JunB was transfected into denervated muscles to maintain its level high, fiber atrophy was prevented, and the induction of the critical atrophy-associated ubiquitin-ligases, atrogin-1 and MuRF-1, was greatly reduced. JunB inhibited their induction by impairing FoxO3 binding to their promoters and thus reduced the stimulation of protein breakdown by FoxO3. Thus JunB is important, not only in dividing populations, but also in mature skeletal muscle where it is required for the maintenance of muscle size and can induce rapid hypertrophy and block atrophy. Mouse adult tibialis anterior muscles were transfected with vectors expressing shRNA against JunB or scramble. 4 muscles were used for each condition. Mice were sacrificed 12 days later and total RNA was extracted. 1 μg of total RNA was amplified with the One Color Microarray Based Gene Expression kit (Agilent) following the manufacturer’s instructions. 1.6 μg of labeled target was hybridized on Agilent Whole Mouse Genome (4 × 44k) Oligo Microarray at 42° C. Data have been extracted with Agilent Future Extraction Software and normalized between experiments with the quantile method. Genes without positive and significant values on at least six experiments were filtered out. Data set of differentially expressed genes has been searched matching to the template profile based on Pearson correlation between dataset and template.