Autopaghy mediated by VMP1 expression is a survival mechanism in caerulein-treated AR42J Pancreas Cells

SACCHETTI, M.L.; GRASSO, D.; LO RE, A.; PARDO, R.; IOVANNA, J. L.; ALEJANDRO JAVIER ROPOLO; VACCARO, M. I.

Chicago, Illinois, USA.

Congreso; Digestive Disease Week 2008; 2008

American Gastroenterological Association

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes including human and experimental acute pancreatitis. We have recently characterized the Vacuole Membrane Protein 1 (VMP1), which is highly activated in acute pancreatitis, as a new autophagy-related protein that localizes in the membrane of pancreatitis-induced vacuoles. We have also shown that VMP1 is a novel autophagy related membrane protein that triggers autophagosome formation in mammalian cells. Our aim was to study the role of VMP1-induced autophagy in pancreas acinar cells. AR42J cells were cultured in nutrient and grown factor-replete conditions and treated with increasing doses of caerulein (Cae) in a time course scheme. We found that Cae treatment induces VMP1 expression in AR42J cells by RT-PCR and Western Blot analysis. We also found that Cae treatment induces autophagy; it was demonstrated by pRFP-LC3 recruitment and by Western Blot of endogenous LC3. Apart from that, using trypan blue and acrydine orange strategies, it was shown that Cae treatment eventually leads to cell death. Finally, in order to know whether VMP1 expression is directly involved in Caeinduced autophagy, VMP1 expression was silenced using two different specific VMP1-siRNAs. As a result we found out that Cae treatment failed to induce autophagy in VMP1-silenced cells and the effect was rescued after transfecting cells with a VMP1-expression`plasmid. Moreover, silencing VMP1 expression decreased significantly AR42J cells survival under Cae treatment. Our results indicate that VMP1 is involved in caerulein-induced autophagy and suggest that VMP1-mediated autophagy is a survival mechanism in AR42J pancreas cells.