Peptide YY reverses TNF-alpha induced binding of interferon regulatory factor-1 and P53 in pancreatic acinar cells

Abstract

Background: Cytokine activation in acute pancreatitis (AP) promotes local and systemic cellular damage. The transcription factors interferon regulatory factor-1 (IRF-1) and the tumor suppressor gene p53 share a common pathway in cell cycle regulation during disease progression. TNF-α induces the binding of many transcription factors, including NF-κB. Our laboratory has previously shown that GI inhibitory peptide, Peptide YY (PYY), improves acute pancreatitis both in vitro and in vivo. We hypothesized that TNF-α would induce IRF-1 and p53 protein binding in pancreatic acinar cells and that PYY would attenuate this effect. Methods: Rat pancreatic acinar cells were treated with rat recombinant TNF-α (200ng/ml). To verify that our TNF-α model was inducing pancreatitis, amylase activity was measured in cell culture supernatant by fluorescence spectroscopy. PYY 3-36 was added at 500pM, 30 minutes post-TNF-α treatment and cells were harvested at 2 hours for nuclear protein extraction. Transcription factor binding of IRF-1 and p53 were determined by protein/DNA array analysis using chemiluminescence detection, and relative spot densities were determined by densitometry. A two-fold increase or decrease in spot density was considered significant. Results: Amylase enzyme activity was significantly (p<0.05) elevated in the TNF-α treated cells by 2 hours and restored to baseline by PYY treatment. Protein /DNA array analysis revealed significant up-regulation of both IRF-1 and p53 in nuclear extracts. Induction by TNF-α increased IRF-1 binding 3.5 fold, while binding levels of p53 protein increased 6-fold. The addition of PYY to TNF-α treated cells reduced IRF-1 and p53 binding to control levels. Conclusions: We have shown for the first time that short-term exposure to TNF-α induces the binding activity of IRF-1 and p53 in pancreatic acinar cells, and that the addition of PYY normalizes their binding. Regulation of transcription factor activity by PYY may explain its salutary effects in AP and provide a therapeutic target in altering disease progression.