Mechanisms of KGF Mediated Signaling in Pancreatic Duct Cell Proliferation and Differentiation Benjamin Uzan, Florence Figeac, Bernard Portha, Jamileh Movassat* Laboratory of Pathophysiology of Nutrition, Paris Diderot- Paris 7 University, Department of Life Science, CNRS/UMR 7059, Paris, France Abstract Background: Keratinocyte growth factor (KGF; palifermin) is a growth factor with a high degree of specificity for epithelial cells. KGF is an important effector of epithelial growth and tissue homeostasis in various organs including the pancreas. Here we investigated the intracellular signaling pathways involved in the mediation of pancreatic ductal cell proliferation and differentiation induced by exogenous KGF during beta-cell regeneration in diabetic rat. Methodology and Results: In vitro and in vivo duct cell proliferation was measured by BrdU incorporation assay. The implication of MAPK-ERK1/2 in the mediation of KGF-induced cell proliferation was determined by inactivation of this pathway, using the pharmacological inhibitor or antisense morpholino-oligonucleotides against MEK1. In vivo KGF-induced duct cell differentiation was assessed by the immunolocalization of PDX1 and Glut2 in ductal cells and the implication of PI3K/AKT in this process was investigated. We showed that KGF exerted a potent mitogenic effect on ductal cells. Both in vitro and in vivo, its effect on cell proliferation was mediated through the activation of ERK1/2 as evidenced by the abolition of duct cell proliferation in the context of MEK/ERK inactivation. In vivo, KGF treatment triggered ductal cell differentiation as revealed by the expression of PDX1 and Glut2 in a subpopulation of ductal cells via a PI3K-dependent mechanism. Conclusion: Here we show that KGF promotes beta-cell regeneration by stimulating duct cell proliferation in vivo. Moreover, we demonstrated for the first time that KGF directly induces the expression of PDX1 in some ductal cells thus inducing beta-cell neogenesis. We further explored the molecular mechanisms involved in these processes and showed that the effects of KGF on duct cell proliferation are mediated by the MEK-ERK1/2 pathway, while the KGF-induced cell differentiation is mediated by the PI3K/AKT pathway. These findings might have important implications for the in vivo induction of duct-to-beta cell neogenesis in patients with beta-cell deficiency. Citation: Uzan B, Figeac F, Portha B, Movassat J (2009) Mechanisms of KGF Mediated Signaling in Pancreatic Duct Cell Proliferation and Differentiation. PLoS ONE 4(3): e4734. doi:10.1371/journal.pone.0004734 Editor: Kathrin Maedler, University of Bremen, Germany Received August 7, 2008; Accepted January 13, 2009; Published March 6, 2009 Copyright: 2009 Uzan 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: The authors are supported by grants from the Centre National de la Recherche Scientifique/CNRS. Florence Figeac is a recipient of a fellowship from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche. 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: movassat@paris7.jussieu.fr Introduction The shortage of islet for transplantation is a major obstacle for the wide application of beta cell replacement therapy in type 1 diabetic patients. To make such a therapy readily available, new sources of insulin-producing cells must be identified. Pancreatic duct cells represent such an alternative source for generation of new beta cells in vitro [1]. Duct compartment has also implications in type 2 diabetes (T2D), since T2D is associated with beta cell loss, and the in situ induction of beta cell neogenesis from ductal progenitor cells may help to restore the beta cell mass in the pancreas of type 2 diabetic patients. Therefore, characterization of stimuli with potential to induce the expansion of duct cells and/or to drive their differentiation into beta cells is of great importance. A variety of growth factors have been described to induce growth and differentiation of ductal cells. Fibroblast growth factors (FGFs) comprise a growing group of structurally related polypeptide mitogens with more than 20 members, most of which stimulate proliferation of a variety of cell types [2,3]. Keratinocyte growth factor, also called FGF7, is a member of the FGF family. KGF is a paracrine-acting mitogen produced by cells of mesenchymal origin. It acts exclusively through a subset of FGF receptor isoforms (FGFR2IIIb) expressed predominantly by epithelial cells and therefore is a specific mitogen for these cells [4]. Preclinical data from several animal models have demonstrated that recombinant human KGF could enhance the regenerative capacity of epithelial tissues and protect them from a variety of toxic exposures [3]. We and others have previously reported that KGF induces ductal cell proliferation in the pancreas [5–8] and promotes beta cell regeneration in neonatal streptozotocin diabetic rats [6]. However the molecular mechanisms underlying the stimulation of beta cell regeneration through the induction of ductal cell proliferation and beta cell neogenesis by KGF are not known. The present study was undertaken to investigate the intracel- lular signaling pathways which mediate the growth promoting effects of KGF (palifermin) in pancreatic ducts and to dissect the key steps of duct-to-beta cell differentiation induced by KGF in a model of neonatal diabetes in rat. In our study, we have established that KGF acts on ductal cells by the the activation of distinct signaling pathways to promote beta PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4734