Raf kinase inhibitory protein inhibits beta-cell proliferation.

Abstract

Raf-1 kinase inhibitory protein (RKIP) was recently identified as a physiologic endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway. The expression and role of RKIP within the pancreas are unknown. RKIP expression in normal pancreas and human insulinomas was examined by using paraffin-embedded sections. Co-localization of RKIP within islet cell subtypes was performed by using double immunofluorescence staining with antibodies directed toward RKIP and endocrine markers. To examine the role of RKIP in beta-cell proliferation, stable expression of sense (ss) and antisense (as) RKIP was established in HIT-T15 beta cells. The effect of RKIP on the ERK-signaling pathway in beta cells was determined by Western blotting with the use of phospho-specific antibodies directed against mitogen-activated protein kinase kinase (MEK) and ERK. The role of RKIP in beta-cell proliferation was assessed by using MTS assay and FACS analysis. RKIP was expressed only within pancreatic islet cells. Immunofluorescent double staining revealed that RKIP was expressed in most beta cells and a subset of pancreatic polypeptide-expressing cells. Based on the known function of RKIP, we hypothesized that RKIP expression would be downregulated in insulinomas: 8 of 9 human insulinomas demonstrated no RKIP staining, with decreased expression in 1 of 9 insulinomas. Studies using asRKIP and ssRKIP demonstrated that RKIP blocked activation of MEK and ERK by Raf-1 in beta cells. We also showed that RKIP inhibited beta-cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis. RKIP is important in beta-cell proliferation, and its downregulation may play a role in islet neoplasia.