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 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. RESULTS: 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. CONCLUSIONS: RKIP is important in beta-cell proliferation, and its downregulation may play a role in islet neoplasia.
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 humaninsulinomas 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. RESULTS: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 humaninsulinomas 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. CONCLUSIONS:RKIP is important in beta-cell proliferation, and its downregulation may play a role in islet neoplasia.
Authors: Jiyoung Lee; Jinho Lee; Kevin S Farquhar; Jieun Yun; Casey A Frankenberger; Elena Bevilacqua; Kam Yeung; Eun-Jin Kim; Gábor Balázsi; Marsha Rich Rosner Journal: Proc Natl Acad Sci U S A Date: 2014-01-06 Impact factor: 11.205
Authors: Olga Martinho; António Gouveia; Paula Silva; Amadeu Pimenta; Rui Manuel Reis; José Manuel Lopes Journal: Virchows Arch Date: 2009-08-25 Impact factor: 4.064