Raf kinase inhibitory protein inhibits β-cell proliferation

Background 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. Methods 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 β-cell proliferation, stable expression of sense (ss) and antisense (as) RKIP was established in HIT-T15 β cells. The effect of RKIP on the ERK-signaling pathway in β 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 β-cell proliferation was assessed by using MTS assay and FACS analysis. Results RKIP was expressed only within pancreatic islet cells. Immunofluorescent double staining revealed that RKIP was expressed in most β 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 β cells. We also showed that RKIP inhibited β-cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis. Conclusions RKIP is important in β-cell proliferation, and its downregulation may play a role in islet neoplasia.