REGULATION OF AUTOPHAGY AND THE UBIQUITIN- PROTEASOME SYSTEM BY THE FoxO TRANSCRIPTIONAL NETWORK DURING MUSCLE ATROPHY
Year: 2016
Type: dissertation
Abstract: Skeletal muscle can adapt its mass in response to physical activity, metabolism and hormones. The control of muscle size depends on the coordinated balance between protein synthesis and protein degradation. Mechanical overload or anabolic hormonal stimulation shifts the balance towards protein synthesis leading to an increase in fiber size, a process called hypertrophy. Conversely, in catabolic conditions protein degradation exceeds protein synthesis resulting into muscle weakness and muscle atrophy. Muscle loss and weakness occurs in several pathological conditions like disuse, denervation, immobilization, sepsis, burn injury, cancer, AIDS, diabetes, heart and renal failure and in aging. Muscle atrophy is an active process which requires the activation of specific signalling pathways and transcriptional programs. Gene expression studies revealed a set of genes, named atrogenes (atrophy related genes), which are commonly up- and down-regulated in different catabolic conditions (Bodine et al., 2001; Gomes et al., 2001; Lecker et al., 2004; Sacheck et al., 2007; Sandri et al., 2004). These genes encode for enzymes that catalyze important steps in autophagy-lysosome pathway, ubiquitin-proteasome system, unfolded protein response, ROS detoxification, DNA repair, mitochondrial function and energy balance pathways. Skeletal muscle growth is regulated by the IGF1-AKT-mTOR-FoxO signalling pathway (Sandri, 2013). FoxO family of transcription factors activity is suppressed during growth by AKT. On the other hand, in the absence of growth factors, like IGF1 or insulin, AKT is not activated and does not block FoxOs which can translocate into the nucleus and interact with promoters of their target genes (Sandri et al., 2004). Activation of FoxOs promotes the expression of the essential atrophy-related ubiquitin ligases atrogin-1 and MuRF-1 and leads to a dramatic loss of muscle mass (Kamei et al., 2004; Sandri et al., 2004). In addition, autophagy and expression of autophagy-related genes is also under FoxO3 control (Mammucari et al., 2007; Zhao et al., 2007). Thus FoxO coordinates the two major proteolytic system of the cell, the autophagy-lysosome and the ubiquitin-proteasome. However, the activation of the few atrogenes reported to be regulated by FoxO cannot sustain all the protein breakdown during atrophy. Nevertheless several other genes among the atrogenes are of potential interest but their particular role and regulation in muscle wasting is still unknown. Moreover, other unknown players are certainly important for muscle protein breakdown. For this reason, in this PhD project we propose, by a loss of function approach, to unravel the role of FoxO family in gene regulation and muscle adaptation during catabolic conditions. The FoxO family in skeletal muscle is comprised of four isoforms: FoxO1, FoxO3, FoxO4 and FoxO6. Therefore, to simultaneously delete the three major expressed FoxO genes in skeletal muscle, we generated muscle-specific conditional FoxO1,3,4 knockout mice. These animals resulted to be fully viable, phenotypically normal and indistinguishable in appearance from aged-matched control mice. To characterize the role of FoxO1,3,4 in skeletal muscle, we analyzed the phenotype of knockout mice under conditions of muscle wasting. Initially we used starvation as a model of muscle loss. because it is an established condition that induces nuclear translocation of FoxO members and their binding to target promoters (Mammucari et al., 2007; Sandri et al., 2004). Interestingly, FoxO1,3,4 knock-out mice were completely protected from muscle loss and muscle weakness after fasting. Moreover, deletion of FoxO resulted in suppression of autophagy during fasting. To further determine if the role of FoxO1,3,4 is critical in different catabolic conditions, we then used denervation as another model of muscle atrophy. Quantification of fibre size revealed that FoxO-deficient muscles were partially protected from atrophy. Thus, FoxO family members are necessary for muscle loss but their involvement in the atrophy programme depends on the catabolic condition. Since muscle atrophy is characterized by a transcriptional-dependent regulation of atrogenes we decided to perform gene expression profiling in fed and fasting conditions in FoxO1,3,4 -/- and controls to identify genes under FoxO regulation. Then, we compared the list of FoxO-dependent genes with the list of atrogenes and we founded that 29 of 63 atrophy related-genes were not induced in FoxO null muscles during fasting. Interestingly, the lists of FoxO-dependent genes during denervation and fasting do not completely overlap confirming that FoxO trascription factors are more sensitive in conditions of nutrient deprivation. Finally we identified new FoxO-dependent ubiquitin ligases including MUSA1 recently identified in our lab and another one that we name SMART (Specific of Muscle Atrophy and Regulated by Transcription). We confirmed by IP experiments that SMART forms an SCF complex with Skp1, Cullin1 and Roc1 and therefore belongs to the SCF family of E3 ligases. To validate the hypothesis that SMART had a role in promoting atrophy during denervation, we knocked down SMART in tibialis anterior in vivo in innervated and denervated muscles. Importantly, SMART inhibition significantly protected denervated muscles from atrophy. Therefore, SMART is an additional critical gene whose upregulation is required for atrophy. In conclusion, this PhD project identified the FoxO signature in protein breakdown. This information would be valuable in order to block muscle wasting in many systemic disease and to promote pharmacological strategies against muscle loss.
Author: PESCATORE, FRANCESCA
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6996267773