Pax-6 Activates Endogenous Proglucagon Gene Expression in the Rodent Gastrointestinal Epithelium

Pax-6 Activates Endogenous Proglucagon Gene Expression in the Rodent Gastrointestinal Epithelium Denny K.Y. Trinh 1 , Kai Zhang 1 , Moazzem Hossain 1 , Patricia L. Brubaker 2 and Daniel J. Drucker 1 1 Department of Medicine, Banting and Best Diabetes Center, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada 2 Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada Abstract The proglucagon gene encodes pancreatic glucagon and the glucagon-like peptides, which exert diverse effects on nutrient absorption and assimilation. The therapeutic potential of glucagon-like peptide-1 (GLP-1) has fostered interest in development of cellular engineering approaches to augment endogenous intestinal-derived GLP-1 for the treatment of type 2 diabetes. We have used adenovirus technology to examine the potential roles of the transcription factors Cdx-2/3 and Pax-6 as activators of endogenous proglucagon gene expression in enteroendocrine cell lines and in nontransformed rat intestinal cells. Adenoviral-expressed Cdx-2/3 and Pax-6 activated proglucagon promoter-luciferase activity in baby hamster kidney (BHK) fibroblasts, HEK 293 cells, and enteroendocrine cell lines. Pax-6, but not Cdx-2/3, induced expression of the endogenous proglucagon gene in enteroendocrine cell lines, but not in heterologous fibroblasts. Furthermore, transduction of primary rat intestinal cell cultures in vitro, or the rat colonic epithelium in vivo, with Ad-Pax-6 activated endogenous proglucagon gene expression. These data demonstrate that Pax-6, but not Cdx-2/3, is capable of activating the endogenous proglucagon gene in both immortalized enteroendocrine cells and the nontransformed intestinal epithelium in vivo. Footnotes Address correspondence and reprint requests to Dr. Denny K. Y. Trinh, Toronto General Hospital, MBRC-4R402, Research-2, 200 Elizabeth St., Toronto, Ontario M5G 2C4. E-mail: d.trinh{at}utoronto.ca . Received for publication 4 June 2002 and accepted in revised form 13 November 2002. DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility shift assay; GLP, glucagon-like peptide; MOI, multiplicity of infection; PFU, plaque-forming unit; PGDP, proglucagon-derived peptide; RT+, presence of reverse transcriptase; RT−, absence of reverse transcriptase; SI, sucrase isomaltase. DIABETES