Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet beta-cells

The results of the current studies define the major elements whereby glucos e metabolism in islet beta-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets, Glucose sti mulation (2-20 mM) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, a s did the depolarizing agents KCl and tolbutamide. This response was inhibi ted by diazoxide and EGTA, indicating that beta-cell depolarization and Ca2 + influx, respectively, are essential. Pharmacological inhibition of the Eg r-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-acti vated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 o r phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A a nd Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 pro moter revealed that the proximal -198 base pairs containing two serum respo nse elements (SREs) and one cAMP-response element retained the depolarizati on response. Depolarization resulted in phosphorylation of cAMP-response el ement-binding protein, yet partial inhibition by a dominant negative cAMP-r esponse element-binding protein, along with a robust response of a cAMP-res ponse element-mutated Egr-1 promoter suggested the presence of a second Ca2 +-responsive element, Depolarization activation of 5XSRE-LUC and serum resp onse factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co- transfection with constitutively active calmodulin kinase IV and protein ki nase A, and binding of Ser(103)-phosphorylated SRF in nuclear extracts, ind icated that the SRE.SRF complexes contribute to the Ca2+-mediated transcrip tional regulation of Egr-1, The results of the current experiments demonstr ate for the first time SRE-dependent transcription and the role of SRF, a t ranscription factor known to be a major component of growth responses, in g lucose-mediated transcriptional regulation in insulinoma cells.