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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Published on: June 25, 2014
Pancreatic reg I binds MKP-1 and regulates cyclin D in pancreatic-derived cells
Cathy M Mueller1, Hong Zhang, Michael E Zenilman
1Department of Surgery, SUNY Downstate Medical Center, Brooklyn, New York 11203, USA.
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.
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