L Ikonomou1, E Geras-Raaka, B M Raaka, M C Gershengorn. 1. Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-8029, USA.
Abstract
OBJECTIVES: Previously, we characterized human islet-derived precursor cells (hIPCs) as mesenchymal stem cells that migrate out from islets in vitro and can differentiate into functional islet-like structures following proliferative expansion. Here, we investigate the role of beta-catenin signalling in derivation and proliferation of hIPCs. MATERIALS AND METHODS: Localization of beta-catenin was performed using confocal microscopy. Expression levels of beta-catenin target genes were measured by quantitative real-time polymerase chain reaction. Loss-of-function studies were performed using specific short interfering RNAs. RESULTS: Immunostaining of islet outgrowths revealed translocation of beta-catenin from plasma membranes in intact islets to the nucleus in cells migrating out. There were no nuclear beta-catenin-positive cells in intact islets whereas between 35% and 70% of cells in established hIPC cultures exhibited nuclear beta-catenin. Transcripts for beta-catenin target genes were increased in hIPCs compared to those in islets. Beta-catenin translocated to the cell membrane when hIPCs formed epithelial cell clusters. In proliferating hIPCs, there was a strong correlation between markers of proliferation and nuclear beta-catenin. Treatment of hIPCs with the glycogen synthase kinase-3beta inhibitor (2'Z,3'E)-6-Bromoindirubin-3'-oxime increased intracellular beta-catenin but reduced nuclear beta-catenin, and was associated with reduced cell proliferation. Finally, knockdown of beta-catenin decreased beta-catenin target gene expression and hIPC proliferation. CONCLUSIONS: These results support a functional role for beta-catenin during proliferation of hIPCs and suggest that activated beta-catenin signalling may also be important during hIPC derivation from islets.
OBJECTIVES: Previously, we characterized human islet-derived precursor cells (hIPCs) as mesenchymal stem cells that migrate out from islets in vitro and can differentiate into functional islet-like structures following proliferative expansion. Here, we investigate the role of beta-catenin signalling in derivation and proliferation of hIPCs. MATERIALS AND METHODS: Localization of beta-catenin was performed using confocal microscopy. Expression levels of beta-catenin target genes were measured by quantitative real-time polymerase chain reaction. Loss-of-function studies were performed using specific short interfering RNAs. RESULTS: Immunostaining of islet outgrowths revealed translocation of beta-catenin from plasma membranes in intact islets to the nucleus in cells migrating out. There were no nuclear beta-catenin-positive cells in intact islets whereas between 35% and 70% of cells in established hIPC cultures exhibited nuclear beta-catenin. Transcripts for beta-catenin target genes were increased in hIPCs compared to those in islets. Beta-catenin translocated to the cell membrane when hIPCs formed epithelial cell clusters. In proliferating hIPCs, there was a strong correlation between markers of proliferation and nuclear beta-catenin. Treatment of hIPCs with the glycogen synthase kinase-3beta inhibitor (2'Z,3'E)-6-Bromoindirubin-3'-oxime increased intracellular beta-catenin but reduced nuclear beta-catenin, and was associated with reduced cell proliferation. Finally, knockdown of beta-catenin decreased beta-catenin target gene expression and hIPC proliferation. CONCLUSIONS: These results support a functional role for beta-catenin during proliferation of hIPCs and suggest that activated beta-catenin signalling may also be important during hIPC derivation from islets.
Authors: José Manuel González-Sancho; Oscar Aguilera; José Miguel García; Natalia Pendás-Franco; Cristina Peña; Santiago Cal; Antonio García de Herreros; Félix Bonilla; Alberto Muñoz Journal: Oncogene Date: 2005-02-03 Impact factor: 9.867
Authors: Marvin C Gershengorn; Anandwardhan A Hardikar; Chiju Wei; Elizabeth Geras-Raaka; Bernice Marcus-Samuels; Bruce M Raaka Journal: Science Date: 2004-11-25 Impact factor: 47.728