day-old animals treated with KGF 20 minutes before the sacrifice (Figure 5). Inactivation of ERK1/2 pathway by MEK1 inhibitors and its effects on KGF-induced duct cell proliferation. In order to investigate the signaling pathways involved in the transduction of the mitogenic effects of KGF on neonatal pancreatic duct cells showed previously [6], we silenced MEK1 protein expression by the use of anti-MEK1 morpholino-oligonucleotides, administrated in vivo to the diabetic pups. During the treatments, all pups tolerated standard morpholinos and MEK1-AS well. There were no adverse effects involving general appearance, body weight, and glycemia (data not show) and no pups died in relation with the administrated treatment. Western blot analysis of whole pancreatic extracts from 2-day-old rats injected with MEK1-AS showed that the amount of MEK1 protein was significantly reduced (by 50%) in the pancreas, confirming the efficiency of morpholino-oligonucleotides to inhibit Figure 4. Induction of D-type cyclins by KGF. Cell lysates were analyzed by western blot for cyclin D1 and cyclin D2 expression. (A) Cells were cultured in medium with 10% FBS. Basal levels of cyclin D2 were not detectable in Panc-1 cells as compared to its expression in insulinoma cell line, INS-1 used as control for the specificity of antibody. (B) Panc-1 cells were cultured in serum-free medium. Cells were then exposed or not to KGF or PD98059 alone, or to the combination of the two agents for 8 h. Cell lysates were analyzed by western blot for cyclin D1 expression. Results are expressed as mean values 6SEM of three independent experiments. * p,0.05. doi:10.1371/journal.pone.0004734.g004 KGF and Pancreatic Duct MEK1 translation in vivo (figure 6A). STZ/Std group exhibited no difference in pancreas morphology compared to STZ/saline group. Moreover duct cell proliferation assessed by BrdU incorporation method in STZ/Std group was similar to that of STZ/saline group (8.160.8% vs 7.660.3% respectively). Therefore for duct cell proliferation and differentiation studies, STZ/Std group has been taken as control for comparison with all other groups. We showed that the inhibition of MEK1/ERK pathway dramatically reduced the proliferation of ductal cells induced by KGF in 2-day-old STZ rats as compared to STZ/Std group. These data indicate that the KGF-stimulated proliferation of neonatal duct cells is exclusively dependent on ERK1/2 activation (Figure 6B). We also found a decrease in the rate of proliferation of acinar cells (Figure 6B). Activation of duct-to-beta cell differentiation by KGF. In order to assess the mechanisms of ductal cell differentiation into beta cells in STZ rats, we analyzed the ductal expression of PDX1 and Glut 2, as intermediate steps in the process of beta cell neogenesis. We found that KGF significantly increased the number of PDX1-positive/insulin-negative ductal cells in the pancreas of 2-day-old pups (Figure 7). At the age of 4 days post natal, a slight but not significant increase in the number of PDX1- positive/insulin-negative ductal cells persisted in the KGF treated rats (not shown). PDX1 regulates the expression of a number of beta cell specific genes including Glut 2 [16,17]. We sought to determine whether the early induction of PDX1 expression by KGF is further followed by the activation of downstream effectors involved in the acquisition of mature beta cell phenotype. To address this point, we examined the expression of Glut 2, an important effector for glucose sensing in terminally differentiated beta cells, also reported as a marker of precursor cells with potential to differentiate into beta cells [18]. At the age of 2 days post natal (8 hours after KGF administration) the percentage of of Glut 2-positive/insulin negative ductal cells was not greater in the KGF treated group than that found in the age-matched untreated STZ group (not shown). At the age of 4 days post natal, the number of Glut 2-positive/insulin-negative ductal cells was significantly increased in the pancreases of rats treated with KGF compared to that found in the STZ control group (Figure 8). Beta cell neogenesis can be indirectly estimated by the evaluation of the number of single or small clusters of insulin expressing cells within or in the vicinity of the epithelial wall of pancreatic ducts. At the age of 4 (0.6160.17% of total ductal cells in the STZ group vs 1.6260.07% of total ductal cells in the STZ/ KGF group) and 7 days post natal (1.0760.09% of total ductal cells in the STZ group vs 2.2560.01% of total ductal cells in the STZ/KGF group), the number of single beta cells or clusters of beta cells budding from ducts was significantly higher (p,0.05) in the KGF treated groups compared to the age-matched untreated control groups. Inactivation of ERK1/2 pathway and its impact on KGF- induced expression of PDX1 in ductal cells. Rats treated with MEK1-AS exhibited a slight but not significant decrease in the number of PDX1-positive ductal cells in response to 8 h KGF treatment as compared to STZ/Std rats treated with KGF (Figure 7) suggesting that ERK1/2 activation is not required for the expression of PDX1 in ductal cells. Inactivation of PI3K/AKT pathway and its impact on KGF-induced expression of PDX1 in ductal cells. The implication of the PI3K/AKT pathway in the induction of PDX1 expression in exocrine cells had been reported in an adult model of pancreatic regeneration [19]. In order to assess whether this signaling pathway is involved in the transduction of KGF signal in ductal cells in in the neonatal regenerating pancreas, we PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4734