Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: involvement of PDX-1 and HNF3beta transcription factors.
Exendin-4 (EX-4), a long acting agonist of GLP-1, induces an endocrine phenotype in Capan-1 cells. Under culture conditions which include serum, approximately 10% of the cells contain insulin and glucagon. When exposed to EX-4 (0.1 nM, up to 5 days), the number of cells containing insulin and glucagon increased to approximately 40%. Western blot analysis detected a progressive increase in protein levels of glucokinase and GLUT2 over 3 days of EX-4 treatment. We explored the sequence of activation of certain transcription factors known to be essential for the beta cell phenotype: PDX-1, Beta2/NeuroD, and hepatocyte nuclear factor 3beta (HNF3beta). Double immunostaining showed that PDX-1 coexisted with insulin and glucagon in EX-4-treated cells. Treatment caused an increase in PDX-1 protein levels by 24 h and induced its nuclear translocation. Beta2/NeuroD protein levels also increased progressively over 24 h. HNF3beta protein level increased twofold as early as 6 h after EX-4 treatment. EMSA results indicated that EX-4 caused a 12-fold increase in HNF3beta binding to PDX-1 promoter area II. Beta2/NeuroD protein levels progressively increased after 24 h treatment. Differentiation to insulin-producing cells was also seen when Capan-1 cells were transfected with pdx-1, with 80% of these cells expressing insulin 3 days after transfection. PDX-1 antisense totally inhibited such conversion. During the differentiation of duct cells to endocrine cells, cAMP levels (EX-4 is a ligand for the GLP-1, G-protein coupled receptor) and MAP kinase activity increased. Our results indicate that EX-4 activates adenylyl cyclase and MAP kinase which, in turn, may lead to activation of transcription factors necessary for an endocrine phenotype.
Zhou
Jie
J
Diabetes Section, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA.
Pineyro
Marco A
MA
Wang
Xiaolin
X
Doyle
Maire E
ME
Egan
Josephine M
JM
D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000077270:Exenatide; D005934:Glucagon / Q000819:agonists / Q000378:metabolism; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D051542:Hepatocyte Nuclear Factor 3-beta; D018398:Homeodomain Proteins; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010183:Pancreatic Ducts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000819:agonists; D010455:Peptides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000819:agonists; D018027:Receptors, Glucagon / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014688:Venoms
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D018398:Homeodomain Proteins; D007328:Insulin; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D018027:Receptors, Glucagon; D015534:Trans-Activators; D014157:Transcription Factors; D014688:Venoms; C084010:pancreatic and duodenal homeobox 1 protein; D051542:Hepatocyte Nuclear Factor 3-beta; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000077270:Exenatide; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases