cell regeneration. It induces proliferation of the pancreatic ductal cells via the activation of the MEK-ERK1/2 pathway and it triggers duct-to-beta cell differentiation directly by the induction of PDX1 expression in the neonatal ducts via the activation of PI3K/ AKT pathway. The duality of the effects of KGF in a different subsets of a unique cell type is an original finding and could have important implication in the potential use of this factor as differentiation promoting agent for regenerative therapies of diabetes. Materials and Methods Reagents The following chemicals and reagents were used in this study. DMEM was purchased from Promochem (Molsheim, France), penicillin–streptomycin, and trypsin–EDTA solutions from Invitrogen (Cergy Pontoise, France). Fetal bovine serum (FBS) was purchased from Abcys (Paris, France). Keratinocyte growth factor, palifermin (KGF) was generously provided by AMGEN (Thousand Oaks, CA, USA). The pharmacological MEK1 inhibitor, PD98059 and total-ERK1/2 antibody were purchased from Promega (Charbonnie`res, France). The phospho-ERK1/2 (Thr202/ Tyr204) and MEK1 antibodies were purchased from Ozyme (St Quentin en Yvelines, France), cyclin D1 antibody was from Microm (Francheville, France), cyclin D2 antibody and the protein assay reagents were purchased from Interchim (Montluc¸on, France), actin antibody and wortmannin from Sigma (L’Isle-d’Abeau Chesnes, France), PDX1 antibody from Millipore (St Quentin en Yvelines, France), insulin antibody from MP Biomedicals (Aurora, Ohio, USA), CK 20 antibody from Abcam (Paris, France) and Glut 2 antibody from AbD Serotec (Du¨sseldorf, Germany). Antisense morpholino-oligonucleotides to knock down endogenous MEK1 (MEK1-AS) expression were designed and produced by Gene Tools LLC (Philomath, Oregon, USA). Animals Wistar rats were fed ad libitum with pelleted chow (diet 113, SAVE, Villemoisson-sur-Orge, France). Females were caged with males for one night and pregnancy was detected by abdominal palpation after 14 days. Natural birth occurred 22 days after mating. All animal experiments were conducted with the approval of French Center for Scientific Research. Eight experimental groups were studied STZ/saline group. At the day of birth, rats of this group received an injection of 100 mg/g body weight of streptozotocin (STZ) (Sigma) freshly dissolved in citrate buffer (0.05 M, pH 4.5). From day 2 to day 6, rats received an i.p. injection of saline solution. Rats of this group were sacrificed at day 2, 4 or 7 after birth. STZ/KGF group. At the day of birth, rats of this group received a single daily i.p. injection of 100 mg/g body weight of STZ freshly dissolved in citrate buffer (0.05 M, pH 4.5). From day 2 to day 6, rats received an s.c. injection of KGF (palifermin) provided by AMGEN (Thousand Oaks, CA, USA) at the dose of 3 mg/kg body weight. For the evaluation of ERK1/2 activation by KGF, 2-day-old pups received an i.p. injection of KGF 20 minutes before sacrifice. STZ/MEK1-AS group. As for the above groups, Wistar newborns received a single daily i.p injection of STZ. Eight hours after STZ administration rats received a subcutaneous injection of antisense morpholino-oligonucleotides targeted to the MEK1 mRNA translation site (MEK1-AS, 1 nmole/g). On day 2, 8 h before sacrifice, rats underwent another s.c. injection of MEK1-AS. KGF and Pancreatic Duct STZ/Std group. Eight hours after STZ administration, rats of STZ group received a subcutaneous injection of non specific standard morpholino-oligonucleotides (Std). On day 2, rats underwent another s.c. injection of Std 8 hours before sacrifice. STZ/MEK1-AS/KGF group. In this group, 2-day-old STZ/ MEK1-AS treated animals received an injection of KGF (3 mg/kg body weight) 8 hours before sacrifice. STZ/Std/KGF group. In this group, STZ/Std treated animals received an injection of KGF (3 mg/kg body weight; i.p) 8 hours before sacrifice. STZ/Wortmannin group. Rats of this group were given a subcutaneous injection of wortmannin (0.75 mg/kg body weight), on day 2, 24 hours after the administration of STZ. STZ/Wortmannin/KGF group. In this group, rats were pre-treated with wortmannin for one hour on day 2 and then received an s.c injection of KGF. They were sacrificed 8 hours after KGF administration. In each group, the pups were left with the dams and kept in an environment of constant temperature, humidity, and day–night cycle. The number of animals per litter was kept at eight. For each experimental group the pups belonged to at least three different litters. All the neonates were tested for blood glucose concentration (Glucotrend, Roche) 24 h after STZ administration. Animals were included in the study only if their glycemia was higher than 9.35 mmol/l. Animals were killed on day 2, 4 or 7 after birth. One hour before the sacrifice, animals were given an injection of 59-bromo- 29-deoxyuridine (BrdU) (Sigma) at the dose of 50 mg/kg, i.p. Pancreases were removed and half of the tails and heads were used for immunohistochemistry and the other halves were used for western blot analysis. Immunohistochemistry Pancreases were fixed in aqueous Bouin’s solution for 24 h and embedded in Paraplast. Each piece of pancreatic tissue was serially sectioned (5 mm) throughout its length. Sections at a fixed interval throughout the block (every 35th section) were immunostained for BrdU using the cell proliferation kit (GE Healthcare, Orsay, France) as described previously [9]. To identify ductal cells, pancreatic sections were stained for cytokeratin 20 (CK20), a specific marker of ductal cells in rat [10,11]. To estimate the ductal cell replication rate, ductal cells were counted in CK20/BrdU double- stained pancreatic sections. After BrdU staining, sections were stained for CK20 using an alkaline phosphatase-conjugated antibody as secondary antibody. Results were expressed as the percentage of BrdU-positive ductal cells. At least 800 ductal cells were counted per pancreas, and four different sections were analyzed for each pancreas. In order to assess ductal cell differentiation, we sought to identify ductal cells with intermediate phenotype. These consisted of cells that expressed specific beta cell markers that are not normally expressed in ductal cells. As such markers, we investigated the expression of PDX1 and the beta cell specific glucose transporter, Glut 2. Since both CK20 and PDX1 antibodies were raised in rabbit, double staining using both antibodies on the same section could not be carried out. Therefore, in order to localize the PDX1 expressing ductal cells in the pancreatic sections of 2-day-old rats , double staining for insulin and PDX1 were performed in sections adjacent to those that were stained for CK20. To estimate the number of PDX1- positive cells, ductal cells that were positive for PDX1, but negative for insulin were counted and the results were expressed as the percentage of PDX1-positive/insulin-negative ductal cells over total ductal cells. For all in vivo studies at least 4 animals were used PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4734