blockade of this pathway by the administration of anti-MEK1 antisense-oligonucleotides did not affect the KGF-induced expres- sion of PDX1 in ducts, suggesting that the induction of PDX1 in those cells in not dependent upon ERK1/2 activation. Several papers by Evers group have reported that in the context of pancreas regeneration after pancreatectomy in adult rodents, the induction of PDX1 expression in duct cells is regulated by the PI3K pathway [49,50]. However whether KGF acts through this pathway to induce PDX1 expression in ductal cells had not been reported. We addressed this question in the present study and showed that the effect of KGF on the ductal expression of PDX1 is totally abolished in animals treated with wortmannin, providing evidence on the role of the PI3K/AKT pathway in the mediation of the KGF signal for PDX1 expression in ducts. As an additional hallmark of the progression of duct-to-beta cell differentiation process, we investigated the expression of Glut 2 in ductal cells. Glut 2, a transcriptional target of PDX1, is an essential effector of the glucose sensing machinery in terminally differentiated beta cells. However, Glut 2 has also been reported as a marker of precursor cells during the early stages of pancreas development [60] and in a subset of precursor cells during beta cell regeneration in experimental models of diabetes [18]. Our histological examination of the pancreatic sections revealed that the number of Glut 2-positive/insulin-negative cells among the ductal compartment is significantly increased in rats treated with KGF, suggesting that this growth factor induces duct-to-beta cell differentiation by the sequential activation of beta cell specific markers in ductal cells. Whether the expression of Glut 2 in ductal cells is induced by KGF directly or by transactivation effect of PDX1 is not clear but the fact that at an earlier stage (day 2 post natal) the number of Glut 2-positive ductal cells was not greater than that found in the age-matched untreated control group, suggests that the increased number of Glut 2-positive ductal cells observed at day 4 post natal might be due to the induction of Glut 2 by PDX1. Although PDX1 and Glut 2 are required for the initiation of the process of beta cell differentiation, the expression of many other factors including transcription factors are necessary for the full differentiation of beta cells [61]. However, only the presence of insulin can testify that the process of beta cell neogenesis has reached full completion. Therefore we evaluated the number of single or small clusters of insulin containing cells within or in the vicinity of the ductal epithelium. References 1. Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, et al. (2004) The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr Diabetes 5 Suppl 2: 16–22. 2. Werner S (1998) Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev 9: 153–65. 3. Finch PW, Rubin JS (2004) Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Adv Cancer Res 91: 69–136. 4. Finch PW, Rubin JS, Miki T, Ron D, Aaronson SA (1989) Human KGF is FGF- related with properties of a paracrine effector of epithelial cell growth. Science 245: 752–5. 5. Krakowski ML, Kritzik MR, Jones EM, Krahl T, Lee J, et al. (1999) Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells. Am J Pathol 154: 683– 91. 6. Movassat J, Portha B (2007) Early administration of keratinocyte growth factor improves {beta}-cell regeneration in rat with streptozotocin-induced diabetes. J Endocrinol 195: 333–40. 7. Movassat J, Beattie GM, Lopez AD, Portha B, Hayek A (2003) Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells. Diabetologia 46: 822–9. 8. Yi ES, Yin S, Harclerode DL, Bedoya A, Bikhazi NB, et al. (1994) Keratinocyte growth factor induces pancreatic ductal epithelial proliferation. Am J Pathol 145: 80–5. KGF and Pancreatic Duct The previous [6] and the present observation that the number of duct-associated insulin-positive cells was greater in KGF treated rats at the age of 4 and 7 days post natal compared to the age- matched untreated rats suggests that ductal cells expressing PDX1 and Glut 2 eventually differentiate into mature insulin producing beta cells. To the best of our knowledge, our study is the first in providing evidence of a direct differentiation promoting effect of KGF on ductal cells and for the identification of the signaling pathways involved in this process. We demonstrated that in vivo, in the context of beta cell deficiency, exogenous KGF acts on pancreatic ductal cells through two distinct mechanisms: 1) via the induction of their proliferation through an ERK1/2 dependent mechanism. The activation of cell proliferation in ductal compartment which includes that of progenitor cells would increase the number of the latter and indirectly promote the process of beta cell neogenesis. 2) via the induction of PDX1 expression through a PI3K dependent mechanism in a subpopulation of ductal cells, committing those cells towards differentiation into beta cell. Different duct cell subpopulations may differ in their precursor capacity. We have demonstrated that KGF acts in a unique cell type, via distinct signaling pathways to trigger the commitment of different subsets of ductal cells towards a beta cell-like phenotype. The transition from duct to beta cell phenotype requires a complex network of signaling pathways whose activation / inactivation in a finely coordinated manner ensures the proper differentiation process to take place [35]. The identification of agents with the potential to trigger this transition is of great interest. Our present work designates KGF as an effective agent for the in vivo induction of duct-to-beta cell differentiation and might have have important implications for the regenerative therapies of diabetes. Acknowledgments We thank AMGEN Inc. for providing KGF (palifermin) used in this study. We are grateful to D. Bailbe´ for expert assistance in animal breeding. Author Contributions Conceived and designed the experiments: JM. Performed the experiments: BU FF. Analyzed the data: BU JM. Wrote the paper: BU JM. Critical reading, helpful discussion and intellectual participation: BP. 9. Movassat J, Saulnier C, Serradas P, Portha B (1997) Impaired development of pancreatic beta-cell mass is a primary event during the progression to diabetes in the GK rat. Diabetologia 40: 916–25. 10. Bouwens L (1998) Cytokeratins and cell differentiation in the pancreas. J Pathol 184: 234–9. 11. Lardon J, Huyens N, Rooman I, Bouwens L (2004) Exocrine cell transdiffer- entiation in dexamethasone-treated rat pancreas. Virchows Arch 444: 61–5. 12. Fabian MA, Biggs WH 3rd, Treiber DK, Atteridge CE, Azimioara MD, et al. (2005) A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 23: 329–36. 13. Kumar N, Afeyan R, Kim HD, Lauffenburger DA (2008) Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2- overexpressing mammary epithelial cells. Mol Pharmacol 73: 1668–78. 14. Lupfer C, Stein DA, Mourich DV, Tepper SE, Iversen PL, et al. (2008) Inhibition of influenza A H3N8 virus infections in mice by morpholino oligomers. Arch Virol 153: 929–37. 15. Kurokawa M (2008) Effects of nestin-antisense treatment in mice on memory- related performance. Int J Neurosci 118: 693–704. 16. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H (1998) beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev 12: 1763–8. 17. Li Y, Cao X, Li LX, Brubaker PL, Edlund H, et al. (2005) beta-Cell Pdx1 expression is essential for the glucoregulatory, proliferative, and cytoprotective actions of glucagon-like peptide-1. Diabetes 54: 482–91. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4734