Hong Zhang

Targeted inhibition of gene expression of pancreatitis‐associated proteins exacerbates the severity of acute pancreatitis in rats

Background: Pancreatitis‐associated protein (PAP) is a secretory protein not normally expressed in healthy pancreas but highly induced during acute pancreatitis. While PAP has been shown to be anti‐bacterial and anti‐apoptotic in vitro, its definitive biological function in vivo is not clear. Methods: To elucidate the function of PAP, antisense oligodeoxyribonucleotides (AS‐PAP) targeting all three isoforms of PAP were administered via intrapancreatic injections (5 mg kg −1 day −1 , 2 days) to rats prior to induction of pancreatitis. Results: Severity of pancreatitis and cytokine gene expression in peripheral blood mononuclear cells (PBMC) were evaluated. Administration of AS‐PAP, but not the scrambled oligodeoxyribonucleotide (SC‐PAP) control, reduced pancreatitis‐induced PAP expression by 55.2 ± 6.4%, 44.0 ± 8.9%, and 38.9 ± 10.7% for PAP isoforms I, II, and III, respectively, compared to saline‐treated controls (P < 0.05 for all). Inhibition of PAP expression significantly worsened pancreatitis: serum amylase activity, pancreas wet weight (reflecting edema), and serum C‐reactive protein levels all increased in AS‐PAP‐treated animals compared to SC‐PAP‐treated controls (by 3.5‐, 1.7‐, and 1.7‐fold, respectively; P < 0.05 for all). Histopathologic evaluation of pancreas revealed worsened edema, elevated leukocyte infiltration, and fat necrosis after AS‐PAP treatment. Gene expressions of IL‐1 μm and IL‐4 were significantly higher in PBMC isolated from AS‐PAP‐treated rats compared to SC‐PAP controls. Conclusion: This is the first in vivo evidence indicating that PAP mediates significant protection against pancreatic injury. Our data suggest that PAP may exert its protective function by suppressing local pancreatic as well as systemic inflammation during acute pancreatitis.

Pancreatic Reg I Binds MKP-1 and Regulates Cyclin D in Pancreatic-Derived Cells

BACKGROUND The pancreatic regenerating (reg I) gene and its protein product are derived from acinar cells and are mitogenic to beta- and ductal cells. We studied the mechanism of this mitogenic response. MATERIALS AND METHODS ARIP (rat ductal) and RIN 1046-38 (rat beta-) cell lines were exposed to exogenous reg I in culture or transfected with a reg I expression vector. Mitogenesis was assessed by MTS assay (CellTiter 96; Promega, Inc., Madison, WI), and cellular mRNA was subjected to gene microarray analysis to determine signal transduction pathways. Yeast two-hybrid technology was then used to determine intracellular binding of reg I protein. RESULTS Cells exposed to exogenous reg I showed a mitogenic response; cells transfected with reg I expression vector showed inhibited growth. Microarray analysis of the former showed induction of cyclin pathways and mitogen-activated protein kinase phosphatase (MKP-1); cyclins were inhibited in the latter. Northern analysis confirmed gene induction of cyclin D1 and MKP-1; JNK was phosphorylated prior to expression of both. Yeast two-hybrid analysis confirmed a protein-protein interaction with MKP-1; this was confirmed by immunoprecipitation. CONCLUSIONS Pancreatic-derived cells exposed to reg I grow by activation of signal transduction pathways involving the mitogen-activated protein kinase phosphatases and cyclins, with concomitant induction of MKP-1. However, high intracellular levels of reg I lead to decreased growth, likely via a binding to and inactivation of MKP-1. Inhibition of cell growth, and possible induction of apoptosis, may lead to differentiation of these cells to other cell types.