(1/768) The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration.
Islet duodenal homeobox 1 (IDX-1/PF-1/STF-1/PDX-1), a homeodomain protein that transactivates the insulin promoter, has been shown by targeted gene ablation to be required for pancreatic development. After 90% pancreatectomy (Px), the adult pancreas regenerates in a process recapitulating embryonic development, starting with a burst of proliferation in the epithelium of the common pancreatic duct. In this model, IDX-1 mRNA was detected by semiquantitative reverse transcription-polymerase chain reaction in total RNA from isolated common pancreatic ducts at levels 10% of those of isolated islets. The IDX-1 mRNA levels were not significantly different for common pancreatic ducts of Px, sham Px, and unoperated rats and did not change with time after surgery. By immunoblot analysis, IDX-1 protein was only faintly detected in these ducts 1 and 7 days after Px or sham Px but was easily detected at 2 and 3 days after Px. Similarly, IDX-1 immunostaining was barely detectable in sham or unoperated ducts but was strong in ducts at 2-3 days after Px. The increase of IDX-1 immunostaining followed that of BrdU incorporation (proliferation). These results indicate a posttranscriptional regulation of the IDX-1 expression in ducts. In addition, islets isolated 3-7 d after Px showed higher IDX-1 protein expression than control islets. Thus, in pancreatic regeneration IDX-1 is upregulated in newly divided ductal cells as well as in islets. The timing of enhanced expression of IDX-1 implies that IDX-1 is not important in the initiation of regeneration but may be involved in the differentiation of ductal cells to beta-cells. (+info)
(2/768) Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells.
Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF. (+info)
(3/768) The FHIT gene is expressed in pancreatic ductular cells and is altered in pancreatic cancers.
We examined 2 normal pancreata, 21 primary pancreatic ductal cancers, and 19 pancreatic cancer cell lines for Fhit expression and FHIT gene status. The normal pancreas expressed Fhit protein in the cytoplasm of ductular cells, whereas interlobular and larger ducts, acini, and insulae of Langerhans were negative. Fhit protein was detected by immunoblot assay in 11 pancreatic cancer cell lines; of the 8 cell lines lacking Fhit protein, 7 lacked FHIT mRNA and 1 showed an abnormally sized transcript. DNA from five of these eight cell lines showed homozygous loss of FHIT exon 5. In 8 of the 21 primary cancers, Fhit expression was detected by immunohistochemistry. Reverse transcription-PCR analysis of 6 of the 13 cases lacking Fhit showed normal-sized FHIT product in 3 cases and a mixture of normal and abnormal products in the other 3. Sequencing showed that abnormal bands were missing variable numbers of exons. Loss of microsatellite DNA markers internal to the FHIT gene was observed in 10 of 13 primary cancers lacking Fhit protein (homozygous in two cases) and in only 1 of the 8 cancers expressing Fhit protein. In nine primary cancers, four expressing and five lacking Fhit protein, it was possible to obtain pure cancer DNA by microdissection. Three of the five microdissected cases lacking Fhit protein exhibited homozygous deletion of FHIT exon 5. In conclusion, the lack of Fhit protein in pancreatic cancers correlated with absence or alteration of FHIT mRNA and was often associated with FHIT gene anomalies. (+info)
(4/768) Modulation of Ca2+-dependent anion secretion by protein kinase C in normal and cystic fibrosis pancreatic duct cells.
The study investigated the role of protein kinase C (PKC) in the modulation of agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. The short-circuit current (ISC) technique was used to examine the effect of PKC activation and inhibition on subsequent ATP, angiotensin II and ionomycin-activated anion secretion by normal (CAPAN-1) and cystic fibrosis (CFPAC-1) pancreatic duct cells. The ISC responses induced by the Ca2+-mobilizing agents, which had been previously shown to be attributed to anion secretion, were enhanced in both CAPAN-1 and CFPAC-1 cells by PKC inhibitors, staurosporine, calphostin C or chelerythrine. On the contrary, a PKC activator, phorbol 12-myristate 13-acetate (PMA), was found to suppress the agonist-induced ISC in CFPAC-1 cells and the ionomycin-induced ISC in CAPAN-1 cells. An inactive form of PMA, 4alphad-phorbol 12, 13-didecanote (4alphaD), was found to exert insignificant effect on the agonist-induced ISC, indicating a specific effect of PMA. Our data suggest a role of PKC in modulating agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. Therapeutic strategy to augment Ca2+-activated anion secretion by cystic fibrosis pancreatic duct cells may be achieved by inhibition or down-regulation of PKC. (+info)
(5/768) Angiotensin II-mediated signal transduction events in cystic fibrosis pancreatic duct cells.
Different signal transduction pathways, i.e. Ca2+- and cAMP-dependent, involved in mediating the effects of angiotensin II (AII) were investigated separately using the short-circuit current (Isc) technique and radioimmunoassay (RIA) in a cystic fibrosis pancreatic cell line (CFPAC-1) which exhibits defective cAMP-dependent but intact Ca2+-dependent anion secretion. The AII-induced Isc could be inhibited by the specific antagonist for AT1, losartan (1 microM), but not the antagonist for AT2, PD123177 (up to 10 microM). The AII-induced Isc was also reduced by the treatment of the cells with a Ca2+ chelator, BAPTA-AM (100 microM), indicating a dependence of the AII-induced anion secretion on the intracellular Ca2+. Treatment of the cells with pertussis toxin (0.1 microg/ml) or a phospholipase C (PLC) inhibitor, U73122 (5 microM), resulted in a substantial reduction in the AII-induced Isc indicating involvement of Gi and PLC in the Ca2+-dependent anion secretion. RIA measurements showed that AII stimulated an increase in cAMP production which could be reduced by losartan, pertussis toxin and U73122 but not BAPTA-AM. In addition, inhibitors of cyclooxygenase, indomethacin (10 microM) and piroxicam (10 microM), did not have any effect on the AII-induced cAMP production, excluding the involvement of prostaglandins. Our results suggest that both AII-stimulated cAMP and Ca2+-dependent responses are mediated by the AT1 receptor and Gi-coupled PLC pathway. However, the AII-stimulated cAMP production in CFPAC-1 cells is not dependent on Ca2+ or the formation of prostaglandins. (+info)
(6/768) Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts.
We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis. (+info)
(7/768) Sustained proliferation of PDX-1+ cells derived from human islets.
Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined. (+info)
(8/768) Pancreatic islet cell survival following islet isolation: the role of cellular interactions in the pancreas.
The purpose of this study was to characterize the trophic effect of pancreatic duct cells on the islets of Langerhans. Ductal epithelium and islets were isolated from hamster pancreata. In addition, duct-conditioned medium (DCM) was prepared from primary duct cultures that had been passaged twice to remove other cellular elements. Three experimental groups were then established: Group 1, 100 islets alone; Group 2, 100 islets+80 duct fragments; and Group 3, 100 islets in 25% DCM. All tissues were embedded in rat tail collagen for up to 12 days and the influence of pancreatic ductal epithelium on islet cell survival was examined. By day 12, 20.6+/-3. 0% (S.E.M.) of the islets cultured alone developed central necrosis, compared with 6.7+/-2.0% of the islets co-cultured with ducts and 5.6+/-1.5% of the islets cultured in DCM (P<0.05). The presence of apoptotic cell death was determined by a TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and by a specific cell death ELISA. DNA fragmentation in islets cultured alone was significantly increased compared with islets cultured either in the presence of duct epithelium or in DCM (P<0.05). More than 80% of TUNEL-positive cells were situated in the inner 80% of the islet area, suggesting that most were beta-cells. DCM was analysed for known growth factors. The presence of a large amount of IGF-II (34 ng/ml) and a much smaller quantity of nerve growth factor (4 ng/ml) was identified. When the apoptosis studies were repeated to compare islets alone, islets+DCM and islets+IGF-II, the cell death ELISA indicated that IGF-II produced the same beneficial result as DCM when compared with islets cultured alone. We conclude that pancreatic ductal epithelium promotes islet cell survival. This effect appears to be mediated in a paracrine manner by the release of IGF-II from cells in the ductal epithelium. (+info)