Figure 7. Activation of duct- to- beta cell differentiation by KGF. PDX1-positive/insulin-negative ductal cells were quantified after insulin/PDX1 double staining of pancreatic sections of the 2-day-old STZ/Std, STZ/Std/KGF and STZ/MEK1-AS/KGF rats. Results are expressed as the percentage of PDX1-positive/insulin-negative ductal cells over total ductal cells. Four rats were analyzed in each experimental group. * p,0.05. doi:10.1371/journal.pone.0004734.g007 tion was associated with complete inhibition of proliferation induced by KGF. From these data we clearly established that ERK1/2 activation is necessary and sufficient for the mediation of growth promoting effects of KGF in pancreatic ductal cells in vitro. The mediation of mitogenic effects of KGF by ERK1/2 signaling has also been reported in other cell types such as endometrial cells [42,43] while other effects of KGF such as regulation of cell migration were mediated by MAP Kinase P38 [43]. The cell cycle is regulated at various checkpoints, of which the G1–S phase represents the most important step. At this checkpoint, extra-and intracellular signals are integrated and transmitted into cells to determine whether they enter the cell division phase, apoptosis or the quiescence G0 phase [44]. Cyclins are major regulators of cell cycle transition and by their binding to cyclin-depedent kinases they act as molecular switches for the G0/ G1-S phase transition in many cell types. We first established that cyclin D1 was expressed in Panc-1 cells while cyclin D2 was not detectable in this cell type. KGF treatment of Panc-1 cells significantly increased the levels of cyclin D1 protein and the blockade of the ERK signaling by PD98059 prevented the induction of cyclin D1 expression by KGF. Kornmann et al. have previously showed that cyclin D1 is over-expressed in ductal cancer cells and treatment of cells with cyclin D1 antisense was associated with significant inhibition of in vitro duct cell proliferation [45]. However the induction of cyclin D1 by KGF in pancreatic ductal cells has to date not been addressed. Thus, our report in support of previous studies [45,46] suggests that cyclin D1 may represent a final common effector for mitogenic signaling via KGF receptor. Although previous in vitro studies have partly identified the signaling pathways of growth factor-mediated proliferation in pancreatic ductal cells in vitro [37,47], the intracellular mecha- nisms of KGF-induced duct cell proliferation in vivo are still poorly understood. Therefore, in this study we explored how the pathways induced by KGF in vitro, translate into their in vivo KGF and Pancreatic Duct Figure 8. Activation of duct- to- beta cell differentiation by KGF. Glut 2-positive/insulin-negative cells were quantified after insulin/ Glut 2 double staining of pancreatic sections of the 4-day-old STZ/saline and STZ/KGF groups. Results are expressed as the percentage of Glut 2- positive/insulin-negative ductal cells over total ductal cells. Four rats were analyzed in each experimental group. * p,0.05. doi:10.1371/journal.pone.0004734.g008 activation. In a previous work we have reported that in vivo administration of KGF to neonatal diabetic rats stimulated beta cell regeneration via the induction of duct cell proliferation and their subsequent differentiation into insulin producing cells [6]. Here we confirmed the in vivo growth promoting effect of ductal cells by KGF in the pancreas of STZ diabetic rats. To determine the relevance of our in vitro findings to the in vivo situation we assessed the implication of ERK1/2 pathway in the KGF-induced duct cell proliferation. We used a specific approach based on the administration of anti-MEK1 antisense oligonucleotides to the pups. We demonstrated for the first time that MEK1 can be successfully knocked down in the pancreas by the use of antisense- oligonucleotides in vivo. This approach, more specific than pharmacological inhibition and less harmful than adenovirus mediated gene inactivation was well tolerated by the pups and no general deleterious effects due to the downregulation MEK1 in other organs during the time course of our study were observed (not shown). This is the first demonstration of the in vivo use of morpholino-oligonucleotides as a safe and specific post-transcrip- tional gene silencing approach in newborn rats. The blockade of ERK1/2 in vivo totally abolished the KGF-induced proliferation of ductal cells indicating that, at least in the neonatal pancreas, ERK1/2 pathway is the exclusive signaling pathway responsible for duct cell proliferation induced by KGF. The mediation of growth promoting effects of KGF by ERK1/2 signaling has also been reported in culture of embryonic pancreatic rudiments [47]. ERK1/2 activation has been shown to occur in a model of adult pancreas regeneration after pancreatectomy [48]. Immunohisto- chemical analysis revealed that in this model, ERK phosphory- lation can be seen only in duct cells and not in acinar cells during pancreatic regeneration [49,50]. Ductal compartment of the pancreas is admitted to harbour precursor cells for islet neogenesis. Therefore, the activation of ERK proteins in the duct in regenerating pancreas may play an indirect role in endocrine cell neogenesis by increasing the number of so-called progenitor cells. Here we have further explored the possibility that, beyond the stimulation of cell proliferation in the ductal compartment, other steps of beta cell neogenesis from ducts could be directly modulated by KGF. In order to address this question, we examined the expression of beta cell specific markers in ductal cells in the pancreas of neonatal diabetic rats. The homeodomain PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4734