Chronic pancreatitis (CP) is a major cause of morbidity and mortality. CP is also a major risk factor for the development of pancreatic cancer. In both diseases, fibrosis within the matrix alters function. In CP, fibrosis results in pain, pancreatic insufficiency and increases cancer risk. The desmoplastic response in pancreatic cancer increases the likelihood of metastatic disease. In both, matrix turnover leads to the loss of proteins such as pigment epithelium-derived factor (PEDF) that act as endogenous barriers to cellular proliferation. Thus, targeting enzymes that are important for matrix turnover may represent a novel target for treatment. One potential target is the vacuolar-ATPase (v-ATPase). The v-ATPase is a highly regulated proton pump that functions to move protons into organelles and out into the extracellular space. It plays an important homeostatic function in terms of pH regulation and acidifies compartments containing enzymes that work optimally at low pH. This occurs in cells such as osteoclasts where genetic mutations in v-ATPase result in severe skeletal abnormalities and death in humans and in mouse models. Cancer cells with v-ATPase on plasma membranes were also recently reported to behave more aggressively than cancer cells without plasma membrane v-ATPase. This suggests that v-ATPase on plasma membranes contributes to matrix degradation and cellular invasion. Our preliminary work has found that v-ATPase is present on the plasma membranes of stellate cells and pancreatic cancer cells, and its activation modulates matrix metalloproteinase (MMP) activities. Because MMPs degrade PEDF, inhibiting v-ATPase activity can preserve PEDF levels. This proposal examines the hypothesis that proton flux mediated by the v-ATPase results in MMP activation and turnover of proteins such as PEDF that maintain cellular quiescence. Moreover, stimuli such as ethanol and others increase v-ATPase translocation to plasma membranes. Our preliminary studies support this hypothesis by showing that: 1) Absence of PEDF in mice results in PSC activation. 2) PEDF null mice display enhanced expression of multiple pro-fibrogenic/inflammatory cytokines and proteases involved in matrix turnover. 3) Cerulein-induced pancreatitis results in impaired weight gain and increased collagen deposition in PEDF null mice compared to WT animals. 4) Ethanol challenge results in the loss of PEDF that is v-ATPase-dependent. 5) Ethanol or leptin challenge results in translocation of soluble v-ATPase (V1E) from cytosol to plasma membranes in stellate cells. 6) Ethanol/leptin challenge results in V1E co-localization with specific v-ATPase isoforms. 7) Pancreatic cancer cells display MMP zymogen activation that is v-ATPase dependent. 8) Pancreatic cancer cell lines with shRNA-mediated knockdown of v-ATPase subunit V1E increases PEDF levels and demonstrate decreased MMP-2/9 activities. 9) In human pancreatic tissue, a polarized v-ATPase distribution in PanIN lesions changes to a diffuse pattern in cancerous cells and correlates with cellular invasive potential. To test the role of v-ATPase activation in pancreatic fibrosis, in vitro and in vivo experiments will be performed that inhibit v-ATPase function in multiple ways. We will selectively target v-ATPase subunits using molecular methods to determine whether this will inhibit pancreatic stellate cell and pancreatic cell growth in vivo.
Ethanol abuse is a major health problem affecting the VA population and the leading cause of chronic pancreatitis (CP), increased scar tissue within the pancreas. CP results in the loss of pancreatic function and increases the risk of pancreatic cancer. How pancreatic scar tissue develops is incompletely understood. We have identified the activation of an enzyme called vacuolar-ATPase (v-ATPase) in certain cells of the pancreas in response to ethanol. V-ATPase activation increases the activity of enzymes that digest the normal tissue architecture in diseases such as CP. These enzymes also digest pigment epithelium-derived factor (PEDF), a protein that keeps the pancreas in its normal condition. The goal of this proposal is to test whether blocking v-ATPase activity prevents PEDF loss and inhibits pancreatic scar formation.