Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene

1 Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul; 2 Department of Food Science and Nutrition, The Catholic University of Korea, Puchon; and 3 Department of Family Medicine, Uijeongbu St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea Submitted 14 April 2006 ; accepted in final form 14 August 2006 Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 ( cyclin D1 ) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic -cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between –73 and –46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at –69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at –139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser 133 to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser 133 or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1 . Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser 133 is necessary for induction of the EGR-1 transcription by exendin-4. early growth response factor-1; cyclic adenosine monophosphate response element-binding protein; protein kinase A Address for reprint requests and other correspondence: Y.-H. Jo, Dept. of Physiology, The Catholic University of Korea, Seoul 137-701, Korea (e-mail: yhjo{at}catholic.ac.kr )