Pancreatic duct cells are considered a potential source of β-cell regeneration, and transforming growth factor-β (TGF-β) has been suggested to perform an important role in these processes, but the underlying mechanism of the signal pathways, especially in humans, remains poorly understood. To evaluate the role of TGF-β1, pancreatic duct cells were isolated from three brain-dead organ donors. Pancreatic cell clusters harvested after islet isolation were dispersed to single cells and cultured in monolayers, then treated with TGF-β1. We analyzed the characteristics of the cultured cells, the TGF-β1 intracellular signaling pathway, the proliferation, and transdifferentiation rates of the duct cells. We also evaluated the genes and protein expression patterns after TGF-β1 treatment. After TGF-β1 treatment, typical morphologic changes representative of EMT were observed and Erk1/2, JNK, and AKT phosphorylation, Ras downstream effectors, were increased. β cell-specific transcription factors including PDX-1, Beta2/NeuroD, Ist-1, and NGN3 were markedly suppressed and the rate of transdifferentiation into β cells was also suppressed. Genomic and proteomic analyses suggested that TGF-β1 induces marked changes in a variety of structural genes and proteins associated with EMT. In conclusion, TGF-β1 induces EMT in cultured human pancreatic duct cells, but suppresses its proliferation and transdifferentiation into β cells. Our results are the first report of TGF-β1 effects for EMT and ductal cell transdifferentiation and proliferation at the protein level in human pancreatic duct cells.
Pancreatic duct cells are considered to be a major source for beta-cell regeneration or neogenesis. Although epidermal growth factor (EGF) is a well-known important growth factor for pancreas development, the control of pancreatic duct cell growth and differentiation by EGF is poorly understood. In this study, we focused on identifying the genes that were differentially up-regulated in response to EGF stimulation using monolayer cultured porcine neonatal pancreas cells. Cells were obtained from 1 to 3 day old pigs, dispersed and cultured for 8 days. Monolayer cultured porcine pancreas cells were comprised of duct cells and some endocrine and mesenchymal cells (75.2 +/- 15.1, 19.6 +/- 4.9, and 9.5 +/- 3.1%, respectively). After 16 h in serum free media, cells were treated with 100 microg/L EGF for 24 h. Differentially expressed genes were screened by subtractive hybridization. (3)H-thymidine uptake was significantly increased by EGF with time (untreated vs. 24 h treated, untreated vs. 48 h treated: 305.5 +/- 3.5 cpm vs. 380.3 +/- 17.3 cpm (P < 0.05), 309.2 +/- 4.51 vs. 929 +/- 9.19 cpm, (P < 0.005), respectively). Three hundred and fifty cDNA clones were obtained by subtractive hybridization and the inserts were confirmed in 161 colonies and then sequenced. Finally, we found increased mRNA expression of five unknown and five known genes, including cytochrome c oxidase subunit I (COI), cyclooxygenase-2 (COX-2), matrix metalloproteinase-13 (MMP-13), Wiskott-Aldrich syndrome protein interacting protein (WASPIP), and hyaluronan synthase-2 (HAS-2). We confirmed the up-regulation of these genes by Northern blot and semi-quantitative RT-PCR at various time points. The present findings opened new targets for the research on the mechanisms of pancreatic duct cell proliferation by EGF.
The purpose of this study was to determine the effects of duration and timing of glucocorticoid treatment on the expansion and differentiation of porcine neonatal pancreas cell clusters
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