ABSTRACT: Under stress conditions mammalian cells activate compensatory mechanisms to survive and maintain cellular function. During catabolic conditions, such as low nutrients, systemic inflammation, cancer or infections, protein breakdown is enhanced and aminoacids are released from muscles to sustain liver gluconeogenesis and tissues protein synthesis. Proteolysis in muscle is orchestrated by a set of genes named atrophy-related genes. A system that is activated both in short and prolonged stress conditions is the family of Forkhead Box (Fox) O transcription factors. Here, we report that muscle-specific deletion of FoxO members resulted in protection from muscle loss because FoxO family is required for induction of autophagy-lysosome and ubiquitin-proteasome systems. Importantly, FoxOs are required for Akt activity but not for mTOR signalling underlining the concept that FoxOs are upstream mTOR for the control of protein breakdown when nutrients are lacking. Moreover, FoxO family controls the induction of critical genes belonging to several fundamental stress response pathways such as unfolded protein response, ROS detoxification and translational regulation. Finally, we identify a set of novel FoxO-dependent ubiquitin ligases including the recent discovered MUSA11 and a new one, which we named Specific of Muscle Atrophy and Regulated by Transcription (SMART). Our findings identify the critical role of FoxO in regulating a variety of genes belonging to pathways important for stress-response under catabolic conditions. Gene expression in muscles of muscle-specific FoxO 1,3,4 knock-out mice that were fed normally or starved We generated knocked-out FoxO 1,3,4 specifically in muscle by crossing FoxO1-3-4-floxed mice (FoxO1,3,4 f/f) with a transgenic line expressing Cre recombinase under the control of MLC1f promoter to generate muscle-specific FoxO1,3,4 triple knockout mice. These mice were either fed ad libitum or starved. Subsequently gene expression of the gastrocnemius muscles was analyzed. For each of the 4 conditions (FOXO1,3,4 f/f fed or starved, FOXO1,3,4-/- fed or starved), we isolated the gastrocnemius muscles of 3 mice thus yielding 6 muscles per condition. RNA was prepared from these muscles using the TRIzol method (Life Technologies) followed by cleanup with the RNeasy kit (QIAGEN). RNA concentration was determined by spectrophotometry and quality of the RNA was monitored using the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA of the 6 muscles per condition was pooled equimolarly and used for further microarray analysis. cRNA was prepared, labelled and hybridised to Affymetrix Mouse Genome 430 2.0 Arrays using Affymetrix-supplied kits and according to standard Affymetrix protocols. Expression values were summarized using the Mas 5.0 algorithm. Genes that were up- or downregulated upon starvation compared to the fed condition were determined using Excel software. A threshold of 1.5 was used for the fold up-or downregulation consistent with the fold change that can be reliably detected with these type of arrays.
