Regulation of autophagy in mammalian cells by stress activated signaling pathways

Autophagy is an intracellular bulk degradation process of cytoplasmic proteins and organelles aimed to preserve cellular homeostasis. It involves the formation of double-membrane structures, called autophagosomes that transport their cargo to the lysosomes where it becomes degraded. The molecular mechanisms underlying induction of autophagy, biogenesis, trafficking and turnover of autophagosomes have been extensively studied. However, the regulation of these steps by signaling pathways that adjust the autophagy flux to different cellular stress situations is still poorly understood. The aim of this work was to investigate the regulation of autophagy upon particular stress conditions in a time-dependent manner. We particularly focused on the control of autophagy by p38. For this purpose we applied a MCF-7/NeuT cell model of oncogene-induced senescence (OIS). In this model, doxycycline-inducible expression of an oncogenic variant of ERBB2 (NeuT) in the breast cancer cell line MCF-7 leads to premature senescence, a process that was shown to be accompanied by aberrant accumulation of autophagic vesicles. We found that autophagy becomes initially induced but that its maturation is later impaired upon oncogenic stress. In addition we identified p38/MAPK as the signaling pathway mediating the block of autophagic flux. This was evidenced by the fact that pharmacological inhibition of p38 by the small molecule compound SB203580 can restore the autophagic flux. This result was confirmed in time-dependent rescue experiments with SB203580. Furthermore, mechanisms by which p38 mediates the autophagic flux blockade were identified. These involved post-translational modifications of the microtubule-associated proteins MAP4 and OP18/STMN1, as well as down regulation of a particular dynein motor protein subunit DYNLL1, which all together provoke microtubule disruption and defective autophagosome transport to the lysosome. The biphasic behaviour of autophagy upon stress (early induction, late block) was confirmed in several cell lines exposed to a different stress-stimulus (anisomycin). Here we could show that also p38/MAPK is involved in these effects in a dual way, with an early induction of initial steps of autophagy and a later block of maturation. In addition, the p38-mediated block of the autophagic maturation was shown to occur via the same mechanism described above.In conclusion, this work revealed new aspects of the control of autophagy by p38 signaling, mainly that the duration and intensity of p38 activation are key determinants of the autophagic flux rate. Low or transitory stress conditions that moderately activate p38 stimulate the autophagic flux. In contrast, more permanent and intensive stress that causes a strong activation of p38 blocks the autophagic flux