The Expression of the Vacuole Membrane Protein 1 (VMP1) is Necessary and Sufficient to Trigger Autophagy in pancreatic cells.

VACCARO MI; GRASSO D; ROPOLO A; PARDO R; GONZALEZ CD; IOVANNA JL

CHICAGO, USA

Congreso; 36th Annual Meeting of the American Pancreatic Association 2005; 2005

AMERICAN PANCREATIC ASSOCIATION

Autophagy, the process by which a cell recycles cytoplasm and disposes excess or defective organelles, has been described as a non-apoptotic programmed cell death. By morphological studies, autophagy has been linked to disease process in acute pancreatitis. We have previously characterized a novel membrane protein, whose expression is induced in acinar cell early during acute pancreatitis, correlates with vacuole formation and precedes cell death.  In order to study the role of VMP1, we proposed the hypothesis that the expression of VMP1 triggers autophagy in pancreatic acinar cells. Autophagic processes were assayed using AR4-2J pancreatic acinar cells. Polyclonal rabbit antibody was developed using a VMP1-specific peptide antigen. VMP1/V5 and VMP1/EGFP expression plasmids and VMP-RNAi were constructed. Electron microscopy studies showed that VMP1-induced vacuoles have ultraestructural features of autophagy. Autophagosomes, a double membrane structure containing cytoplasmic material, as well as autolysosomes, single membrane structure containing cytoplasmic components at various stages of degradation, were observed in VMP1 expressing cells. VMP1-induced vacuoles were stained with monodansylcadaverine, a fluorescent tracer for autophagy. LC3, a specific marker for autophagic membranes, co-localized with VMP1 transfected cells. These results strongly demonstrate that VMP1 expression induces autophagy. In other series of experiments, AR4-2J cells were treated with rapamycin, a drug that induces autophagy, and VMP1 expression was demonstrated by RT-PCR and Western blot analysis. Finally, RNAi silencing of VMP1 expression prevented autophagy induced by rapamycin demonstrating that VMP1 expression is required for autophagy. Our results demonstrate that VMP1 expression is necessary and sufficient to trigger autophagy. VMP1 expression is an early molecular event that may promote autophagy and could be involved in the regulation of acinar cell death during acute pancreatitis pathogenesis.