KGF and Pancreatic Duct Results I- in vitro studies Stimulation of duct cell proliferation by KGF. Panc-1 cells have been rendered quiescent by 72 h serum deprivation (control group) and were then treated with KGF or 10% FBS for 24 h. The exposure to KGF potently stimulated the mitotic activity of the cells (Figure 1A) as evidence by the 200% increase of the rate of cell proliferation in those cells as compared to that of the control group. The stimulation of proliferation induced by KGF was similar to that measured in cells treated with 10% FBS (Figure 1A). Activation of ERK1/2 pathway by KGF. The time-course analysis of ERK1/2 phosphorylation in cells treated with KGF showed a prompt activation of ERK1/2 which peaked at 5 min after stimulation. The activation of ERK1/2 was transient and the basal levels of ERK1/2 phosphorylation were recovered 1 h after stimulation (Figure 1B). At the time-point 20 min after stimulation, KGF-induced activation of ERK1/2 resembled that induced by 10% FBS. Inactivation of ERK1/2 pathway by MEK1 inhibitors and its effects on KGF-induced cell proliferation. To establish the functional link between ERK1/2 activation and the stimulation of cell proliferation induced by KGF, we proceeded to the inactivation of the only known upstream activator of ERK1/2 proteins. MEK1 was first inactivated by its pharmacological inhibitor PD98059. KGF-induced proliferation of Panc-1 cells assessed by BrdU staining (Figure 2B) was completely abolished by blockade of ERK1/2 phosphorylation caused by PD98059 (Figures 2A and 2C). In contrast, MEK1 inactivation and the consequent inhibition of ERK1/2 pathway, caused only a slight and non significant decrease in the rate of cell proliferation induced by 10% FBS (Figure 2C). Our results from experiments with pharmacological MEK1 inhibitor clearly designate MEK1-ERK1/2 pathway as the main pathway involved in the transduction of KGF biological actions on Figure 2. Inactivation of ERK1/2 pathway by pharmacological inhibitor abolishes KGF-induced Panc-1 cell proliferation. (A) Panc-1 cells were pre-incubated for 1 h with 20 mM, 10 mM and 1 mM of PD98059, an inhibitor of MEK1. Cells were then treated for 5 minutes with 30 ng/ml KGF. Cell lysates were analyzed by western blot using specific antibodies against P-ERK1/2 and total-ERK1/2. (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 24 h. Cell proliferation was assessed by BrdU staining (brown nuclei). Red arrows show BrdU-positive Panc-1 cells and blue arrows show BrdU-negative Panc-1 cells (actual magnification 6250). (C) Panc-1 cells were treated or not with 20 mM PD98059, 1 h prior to the 24 h incubation with KGF or 10% FBS. BrdU labelling index of Panc-1 cells was evaluated and expressed as the percentage of BrdU-positive Panc-1 cells. Results are presented as mean values 6SEM of three independent experiments. ** p,0.01. doi:10.1371/journal.pone.0004734.g002 PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4734