Protein Kinase C Delta (PKCd) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of Cip1/WAF1 the Cell Cycle Inhibitor p21 Felicia Ranta1, Johannes Leveringhaus1, Dorothea Theilig1, Gabriele Schulz-Raffelt1, Anita M. Hennige1, Dominic G. Hildebrand 2 , Rene´ Handrick3, Verena Jendrossek4, Fatima Bosch5, Klaus Schulze-Osthoff2, Hans-Ulrich Ha¨ ring1, Susanne Ullrich1* 1 Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, Department of Internal Medicine, University of Tu¨ bingen, Tu¨ bingen, Germany, 2 Interfaculty Institute for Biochemistry (IFIB), University of Tu¨ bingen, Tu¨ bingen, Germany, 3 Institute for Pharmaceutical Biotechnology, Biberach University of Applied Sciences, Biberach, Germany, 4 Institute of Cell Biology, University of Essen, Essen, Germany, 5 Center of Animal Biotechnology and Gene Therapy, Universita Auto` noma Barcelona, Bellaterra and CIBER de Diabetes y Enfermedades Metabo´ licas Asociadas (CIBERDEM), Barcelona, Spain Abstract Background: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCd) in b-cells. To understand the role of PKCd in more detail the impact of changes in PKCd activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. Methodology and Principal Findings: Using genetic and pharmacological approaches, the effect of reduced and increased PKCd activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCd (PKCdWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21Cip1/WAF1. This nuclear extrusion was mediated by PKCd-dependent phosphorylation of p21Cip1/WAF1 at Ser146. In kinase dead PKCd (PKCdKN) overexpressing cells and after inhibition of endogenous PKCd activity by rottlerin or RNA interference phosphorylation of p21Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCdWT transgenic mice the inhibitor resides cytosolic. Conclusions and Significance: These observations disclose PKCd as negative regulator of p21Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCd into its known pro-apoptotic role. Citation: Ranta F, Leveringhaus J, Theilig D, Schulz-Raffelt G, Hennige AM, et al. (2011) Protein Kinase C Delta (PKCd) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1. PLoS ONE 6(12): e28828. doi:10.1371/journal.pone.0028828 Editor: Kathrin Maedler, University of Bremen, Germany Received August 16, 2011; Accepted November 15, 2011; Published December 27, 2011 Copyright: 2011 Ranta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported for consumables/material expenses by a grant from the German Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD e.V.) and by financial supports of DFG grants (GRK1302/1, UL140/7-1 and UL140/7-2) for consumables/ material expenses and staff appropriations. Human islets were provided through JDRF Award 31-2008-413 (ECIT Islet for Basic Research Program). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: susanne.ullrich@med.uni-tuebingen.de Introduction Sufficient b-cell mass is required for adequate insulin secretion. Consequently, an elevated demand of insulin is controlled by increased proliferation of pancreatic endocrine cells while insufficient insulin secretion and the development of type-2 diabetes have been associated with b-cell death [1]. A variety of molecular changes are involved in b-cell failure including reduced insulin/IGF-1 receptor signaling, endoplasmic reticulum stress and mitochondrial dysfunction [2–10]. These changes are triggered by obesity-linked factors, such as oxidative stress, saturated free fatty acids, cytokines and interleukins. Previous observations from our and other groups suggested that protein kinase C delta (PKCd) plays a decisive role in b-cell failure induced by cytokines and free fatty acids [11–15]. Thus, mice with targeted overexpression of a kinase-negative PKCd (PKCdKN) mutant in b-cells are protected against high fat diet-induced glucose intolerance and show increased survival of islet b-cells [14]. Conversely, we have previously shown that exposure of b-cells to high concentrations of palmitate promotes PKCd-mediated nuclear accumulation of FOXO1, a pro-apoptotic transcription factor activated under stress conditions [14]. Furthermore, PKCd has been found to mediate iNOS mRNA stabilization induced by IL-1b, whereas ablation of PKCd protected mice against streptozotozin-induced hyperglycemia [11,12]. Thus, under cer- tain stress conditions, PKCd promotes signaling pathways leading to apoptotic b-cell death. Very few studies have investigated the role of PKCd for normal b-cell function, in particular under stress-free conditions. Surpris- ingly, mice with increased transgenic expression of PKCd in b-cells PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28828