KGF and Pancreatic Duct Figure 6. Inactivation of ERK1/2 pathway by MEK1-AS abolishes KGF-induced duct cell proliferation. (A) Rats from STZ/Std and STZ/ MEK1-AS groups were sacrificed at day 2 post natal. Pancreases were removed and pancreatic protein extracts were analyzed by western blot using a specific MEK1 antibody. Representative blots and MEK1 protein quantification are shown in Figure 6A. Results are expressed as mean values 6SEM. Four rats were analyzed in each experimental group. * p,0.05. (B) BrdU (brown nuclei) and CK20 staining (red cytosol) of pancreatic sections from 2- day-old STZ/Std/KGF and STZ/MEK1-AS/KGF newborns (actual magnification 6500). (C) Ductal cell proliferation was assessed by BrdU staining of pancreatic sections of STZ/Std, STZ/MEK1-AS, STZ/Std/KGF and STZ/MEK1-AS/KGF groups. BrdU labelling index of ductal cells in all experimental groups was evaluated in 2-day-old pups and the results were expressed as the percentage of BrdU-positive ductal cells. Three to five rats were analyzed in each experimental group. ** p,0.01. doi:10.1371/journal.pone.0004734.g006 stimulated serum deprived Panc-1 cell proliferation as potently as did 10% FBS. The induction of cell proliferation by KGF in cultures of primary human ducts has been previously reported [34,37]. However in these studies a thorough analysis of growth promoting potential of KGF and the signaling pathways involved, had not been made. The expression of specific KGF receptor (FGFR2IIIb) having been described in this cell line [38,39], we sought to investigate the post-receptor signaling molecules which mediate the effects of KGF on cell proliferation. One of the most common signaling pathways linked to cell response to growth factors is the mitogen-activated protein kinases (MAPK) pathway. Among the three main MAPK cascades (ERK1/2, JNK and P38), ERK1/2 are predominantly activated by growth factors whereas P38 and JNK transduce stress-related stimuli such as heat shock and inflammatory cytokines [40,41]. We analyzed the implication of ERK1/2 pathway in the mediation of KGF proliferative effects on ductal cells. The only known upstream activators of ERK1/2 are MEK proteins. We first inactivated MEK1 by its pharmaco- logical inhibitor PD98059. We further confirmed the specificity of MEK inactivation by the use of antisense morpholino-oligonucle- otides (MEK1-AS) specifically designed to inhibit MEK1 transla- tion. Strikingly, in both cases blockade of ERK1/2 phosphoryla- PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4734