FOXO HYPERACETYLATION AS A MECHANISM OF SUPPRESSION OF ATROPHY GENE PROGRAM INDUCED BY BETA2-ADRENERGIC SIGNALING IN RODENT SKELETAL MUSCLE

Abstract

The development of therapeutic strategies capable of inhibiting protein breakdown and preserving muscle mass is an important issue for human health. In previous studies, we have showed that beta2-adrenoceptors (AR) activation mitigates muscle atrophy through the suppression of the activity of ubiquitin (Ub)-proteasome (UPS) and autophagy-lysosomal (ALS) systems, the major proteolytic systems involved in muscle loss. Foxo transcription factors are key players in regulating both UPS and ALS. The phosphorylation of Foxo by Akt, a kinase that lies downstream of IGF-1/insulin signaling, decreases its activity and the expression of its target genes related to muscle wasting. We have also demonstrated that pharmacological activation of beta2AR increases Foxo3a phosphorylation and down-regulates the atrophy-related Ub-ligases Atrogin-1 and MuRF1 and the autophagic gene Gabarapl1 in muscles from 2-day fasted mice. Besides phosphorylation, Foxo proteins are regulated through other post-translational modifications including acetylation. Recent studies have shown that histone acetyltransferase (HAT) p300 and histone deacetylases (HDACs) regulate the acetylation levels of Foxo. Hyperacetylated Foxo has cytosolic localization and lower stability and activity in different cell types. In our post-doc project, we observed that beta2-agonists suppress Foxo activity and increase the acetylation levels of Foxo3a in muscles from fasted mice. However, the precise molecular mechanism by which sympathomimetics induces Foxo hyperacetylation and suppresses the expression of atrophy-related genes is not yet clarified. Thus, this project aims to investigate the in vivo involvement of HAT p300 and HDACs in Foxo acetylation and downregulation of atrophy-related genes induced by beta2-adrenergic signaling in skeletal muscle. Our hypothesis is that beta2-adrenergic stimulation in muscle might elevate the expression and the activation status of Sik1, a kinase downstream of PKA/CREB and an inhibitor of HDACs, via CREB and Akt, respectively. Thus, class IIa HDACs might remain in cytosol and the acetylation levels of Foxo would increase in nucleus, inducing its cytosolic translocation and reduced transcriptional activity. Additionally, beta2-agonist activates Akt and PKA, which in turn could stimulate p300/CBP activity and Foxo hyperacetylation. To test this hypothesis in collaboration with Dr. Marco Sandri (Italy), in vivo techniques of molecular biology, such as gene transfer by electroporation, the generation of mutant proteins, the in vivo knockdown of the mediators of acetylation by short hairpin RNA and the analysis of transcriptional factor activity by luciferase reporter assay will be used.