Abstract 361: Inhibition of Ras Homologue Enrich in Brain (Rheb) Enhances Cell Survival against Amino Acid Depletion through Stimulation of Autophagy
Rheb (Ras homologue enrich in brain) integrates signals from nutrients and growth factors to regulate cellular functions, including ribosomal biogenesis and metabolism. Rheb is negatively regulated by tubular sclerosis complex 2 (TSC2), whose activity is subjected to positive and negative regulation by AMPK and Akt, respectively. Rheb positively regulates mTOR, a master regulator of protein translation and autophagy, an intracellular bulk protein degradation process. We have recently shown that autophagy plays a protective role against nutrient starvation in cardiac myocytes. We examined the role of Rheb in regulating cell survival/death in nutrient deprived conditions and the involvement of autophagy in this process. Amino acid deprivation (AD) for 96h significantly induced cytoplasmic accumulation of mono- and oligo-nucleosomes, a hallmark of apoptosis, in cardiac myocytes. AD significantly reduced phosphorylation of Thr389 of p70S6 kinase (24.8±0.1% vs control, p<0.05), suggesting that the Rheb-mTOR pathway is suppressed. Adenovirus harboring Rheb S20N, a dominant negative Rheb, (Ad-S20N) reduced phosphorylation of p70S6 kinase in the presence of complete medium (CM, 42.2±2.5% vs control) and under AD (10.5±0.2%). Transduction of Ad-S20N 48h before AD significantly attenuated AD-induced apoptosis (48.8±0.5% vs AD, p<0.05), without affecting baseline apoptosis in CM. AD significantly increased LC3-II/LC3-I ratio(CM 5.2±0.1%, AD32.1±2.1%, p<0.05), an established marker for autophagy. Ad-S20N, but not control adenovirus, significantly enhanced LC3-II/LC3-I in the presence of AD (LacZ 35.2±3.2%, RhebS20N 50.4%±6.4%, p<0.05), suggesting that inhibition of Rheb increases autophagy. In contrast, wild type Rheb attenuated AD-induced increases in LC3-II/LC3-I ratio(20.1±4.1%) and enhanced AD-induced apoptosis (1.47±0.06 fold vs AD, p<0.05). These results suggest that AD suppresses Rheb, which in turn plays a protective role against AD through induction of autophagy. In conclusion, Rheb plays an important role in controlling cell survival in part through regulation of autophagy during nutrient starvation in cardiac myocytes.