Functional Characterization Of The Transcription Factor P8 Involved In Autophagy Regulation

Autophagy is an evolutionarily conserved process that allows cells to maintain homoeostasis in numerous physiological situations, by which cytoplasmic components, including macromolecules and organelles, are degraded by the lysosome. This process also functions as an essential protective response to ER stress, which promotes the removal and degradation of unfolded proteins; however, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. Increasing evidence suggests that phosphorylated AMP-activated protein kinase (p-AMPK) and target of rapamycin (TOR) play key roles in the regulation of autophagy. However, the regulation of autophagy in quiescent cells remains unclear, despite the fact that autophagy is known to be critical for normal development, regeneration and degenerative diseases.Here, we used encysted Artemia embryos as a model system because they produce and release encysted embryos that enter a state of obligate dormancy in cell quiescence to withstand various environmental threats. We observed that autophagy was increased before diapause embryo formation but dropped to extremely low levels in the diapause stage in Artemia. Western blotting analysis and RNA interference indicated that autophagy regulation is AMPK/TOR independent during diapause embryo formation. Importantly, the level of p8, a stress-inducible transcription cofactor (Ar-p8), was elevated at the stage just before diapause and was absent in encysted embryos, indicating that Ar-p8 may regulate autophagy. The results of Ar-p8 knockdown revealed that Ar-p8 regulated autophagy during diapause formation in Artemia. Moreover, we observed that activating transcription factors 4 and 6 (ATF4 and ATF6) respond to Ar-p8-regulated autophagy, indicating that autophagy targets the endoplasmic reticulum during diapause formation in Artemia. Additionally, AMPK/mTOR-independent autophagy was validated in human gastric cancer MKN45 cells overexpressing Ar-p8 and Hu-p8. Meanwhile, different types of autophagy were identified in MKN45 cells in response to the stress induced by nutrient starvation and by lipotoxicity, in which the regulation of these pathways is AMPK/mTOR independent and Hu-p8 dependent, respectively. Western blotting analysis showed that autophagy was regulated by Hu-p8 and AMPK/mTOR in response to the protein kinase-like endoplasmic reticulum kinase (PERK)/activating ATF6-mediated ER stress of lipotoxicity and the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that the autophagy induced by lipotoxicity is AMPK/mTOR independent, but this autophagy pathway was regulated by p8 via p53- and PKCa-mediated signaling in MKN45 cells. The findings presented here may provide insights into the role of p8 in regulating autophagy in quiescent cells and fat accumulated lipotoxic cells.