p62/SQSTM1, a Key Autophagy Mediator, Is Regulated in Pancreatitis Through mTOR-Mediated Pathways: 32

Introduction: p62/SQSTM1 is a multifunctional signaling protein that plays a key role in autophagy. p62 mediates autophagic clearance of protein aggregates; is specifically degraded via autophagy; and was recently shown to mediate ER and oxidative stress in a genetic pancreatitis model caused by IKKa deficiency. However, the pathways regulating p62 in pancreas are largely unknown. Here, we investigate the role and mechanisms of p62 degradation and synthesis in regulating pancreatic acinar cell p62 level, both basal and in experimental alcoholic and non-alcoholic pancreatitis. Methods: p62 levels and autophagy markers/mediators were measured in mouse models of pancreatitis: alcoholic, induced by 6-week feeding of Lieber-DeCarli diet followed by low-dose (5 μg/kg) cerulein; non-alcoholic (by L-arginine or high-dose cerulein); and ex-vivo model of CCK-hyperstimulated acinar cells. To examine the role of autophagy in regulating p62, we used lysosomal inhibitors and mice with genetic ablation of Atg5 in acinar cells (Spink-A5) We used pharmacologic inhibitors and genetically modified mice to study p62 regulation by mTOR, NF-κB, STAT3, CHOP, and by PI3K and p38 protein kinases. Results: p62 was greatly up-regulated in mouse models of alcoholic and non-alcoholic pancreatitis. Autophagy blockade with lysosomal inhibitors (E64d plus pepstatin) or in Spink-A5 mice increased p62 in acinar cells, indicating that its basal level is regulated by autophagic degradation. However, cerulein pancreatitis caused an additional p62 increase in Spink-A5 mice; and cycloheximide markedly decreased p62 in acinar cells. Thus, p62 accumulation in pancreatitis is mediated by both decreased degradation and increased synthesis. We found that mTOR was activated in pancreatitis and its' inhibition with Torin or PP242 largely prevented p62 rise. Torin and PP242 also prevented p62 increase in conditions in which autophagy is blocked, indicating that mTOR mediates p62 synthesis. The results further implicate the key transcription factor STAT3, but not NF-κB, in mTOR's effect on p62 synthesis. Conclusion: p62 is up-regulated in experimental pancreatitis (as well as in human disease).mTOR is a critical regulator of acinar cell p62 in pancreatitis: mTOR both inhibits p62 autophagic degradation and stimulates its synthesis. A generally novel effect is that mTOR stimulates p62 synthesis via STAT3. The results suggest mTOR-STAT3 inhibition as an approach to alleviate ER and oxidative stress in pancreatitis.