Ubiquitin E3 ligases Atrogin-1 and MuRF1 protein contents are differentially regulated in the rapamycin-sensitive mTOR-S6K1 signaling pathway in C2C12 myotubes

Abstract

Muscle-specific ubiquitin E3 ligases, Atrogin-1 and MuRF1, are highly expressed in multiple conditions of skeletal muscle atrophy. The PI3K/Akt/FoxO signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. Evidence supporting this is largely based on stimuli by insulin and IGF-1, that activate anabolic signaling, including Akt and Akt-dependent transcription factors. However, Akt activation also activates the mammalian target of rapamycin complex 1 (mTORC1) which induces skeletal muscle hypertrophy. However, whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we confirmed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein content and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein content in C2C12 myotubes. Interestingly, inhibition of mTORC1 using a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein content, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671, and found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was not significantly altered. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by rapamycin-sensitive mTOR-S6K1 dependent signaling pathway.