KGF and Pancreatic Duct Figure 9. Inactivation of PI3K/AKT pathway by wortmannin abolishes KGF-induced duct-to-beta cell differentiation but does not alter the effect of KGF on proliferation. (A) Double staining for insulin (red cytosol) and PDX1 (brown nuclei) were performed on pancreatic sections of STZ, STZ/KGF, STZ/wortmannin and STZ/wortmannin/KGF rats (wortmannin was administrated at the dose of 0.75 mg/kg body weight). Differentiated beta cells express both insulin and PDX1 (blue arrow). A subset of ductal cells express PDX1 (red arrow) while the majority of ductal cells are PDX1 negative (green arrow) (actual magnification 6500). d = duct. (B) PDX1-positive / insulin-negative ductal cells were quantified after insulin/PDX1 staining on pancreatic sections of the 2-day-old STZ, STZ/KGF, STZ/wortmannin and STZ/wortmannin/KGF rats. Results are expressed as the percentage of PDX1-positive/insulin-negative cells over total ductal cells. Four rats were analyzed in each experimental group. * p,0.05; ** p,0.01. (C) Ductal cell proliferation was assessed by BrdU staining of pancreatic sections of the 2-day-old STZ, STZ/KGF, STZ/wortmannin and STZ/ wortmannin/KGF rats and the results were expressed as the percentage of BrdU-positive ductal cells. Four rats were analyzed in each experimental group. * p,0.05; ** p,0.01. doi:10.1371/journal.pone.0004734.g009 protein PDX1 is a prominent transcription factor for pancreas morphogenesis during development [20,51,52] and a transcrip- tional regulator of insulin synthesis and secretion in mature beta cells [53–55]. It has been strongly suggested that during the postnatal life, PDX1 may also be essential for neogenesis of endocrine cells by differentiation from precursor cells that reside among the cells lining the pancreatic ducts [56–58]. The examination of pancreatic sections revealed that the number of PDX1-positive/insulin-negative cells was significantly higher among ductal cells of KGF treated STZ rats. PDX1-positive/ insulin-negative cells can be considered as cells committed toward a beta cell-like phenotype without having achieved terminal differentiation yet. This novel finding designates for the first time KGF as a differentiation inducing agent through the stimulation of PDX1 expression in ductal cells. In a study in primary islets and in INS1 beta cells, ERK1/2 activation has been shown to be required for the regulation of important transcription factors such as Beta2, PDX-1, and E12/47 [59]. Therefore we assessed the implication of ERK1/2 pathway in the induction of PDX1 expression in ductal cells induced by KGF. We found that the PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4734