Muscle is a highly dynamic and plastic tissue that adapts to varying loading conditions by changes in muscle mass and fiber type composition. Muscle mass is mainly regulated by changes in protein synthesis and protein degradation. A decrease in protein synthesis and or an increase in protein degradation will lead to a reduction in protein and eventually muscle mass, which is called muscle atrophy. Most proteins are degraded via two proteolytic systems: the ubiquitin proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Muscle RING-finger-1 (MuRF1) is an E3 ligase considered a key mediator of UPS-mediated muscle atrophy. Recently, our group demonstrated that the transcription factor EB (TFEB) is involved in muscle remodeling by directly activating Trim63/MuRF1 expression and described the protein kinase D1 (PKD1)/histone deacetylase 5 (HDAC5)/TFEB axis as regulator of muscle atrophy. Because TFEB belongs to the microphthalmia family (MiTFE, TFEB, TFE3) of transcription factors, we hypothesized that TFEB shares its transcriptional activity towards Trim63/MuRF1 with the MiTF-family members. Similarly, class IIa HDACs and PKDs belong to respective families with close structural, regulatory, and functional properties. Therefore, I investigated the interaction and functional significance of these three families. I demonstrated that class IIa HDACs physically interact with and directly inhibit the activity of TFEB and TFE3 towards Trim63/MuRF1. I showed that the PKD-family redundantly relieves HDAC-mediated inhibition of TFEB and TFE3. Altogether I propose a mechanistic basis for the control of muscle atrophy via the PKD/HDAC/TFEB-TFE3 axis. Besides that, TFEB is known to be a master regulator of lysosomal biogenesis and autophagy. However, if this function has physiological implications for skeletal muscle is uncertain. To address this question, I performed muscle-specific gain- and loss-of-function experiments both in vitro and in vivo. We generated a muscle specific Tfeb knockout mice; importantly, we did not observe a difference in autophagy-mediated protein degradation in muscle. In the in vitro part, I successfully established a retrovirus-based strategy to stably overexpress Tfeb in C2C12 muscle cells. My results suggest that TFEB does not induce ALP mediated protein degradation in C2C12 cells. Although the vast majority of studies support TFEB as master regulator of ALP, I propose that the effects of TFEB on ALP-mediated protein degradation are different between muscle and non-muscle cells. Interestingly, I found in my study that overexpression of TFEB in C2C12 myoblasts attenuates myoblast differentiation and keeps these cells in at an undifferentiated blast stage. Using a proteomics-based approach we found that TFEB-overexpression resulted in a reduction of proteins in charge of cell-cell fusion and cytoplasmic architecture such as α-actin, actinin, muscle specific cadherin and most of tropomyosin isoforms. Additionally, we found an increased amount of the proliferation marker Ki67 indicative for the incompetence of C2C12 cells to exit the cell cycle, to differentiate and to generate muscle fibers upon TFEB overexpression. Accordingly, late differentiation markers such as myosin heavy chain proteins were greatly reduced in TFEB treated C2C12 cells. In summary, my thesis sheds new light on the transcriptional regulation of the MiTF-family of transcription factors in myocytes and implicates that TFEB is involved in myogenic differentiation. Although TFEB is known as a master regulator of ALP-mediated protein degradation in non-myocytes, my data argue for alternative functions of TFEB in myocytes.
