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 glucose metabolism in islet beta-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that beta-cell depolarization and Ca(2+) influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca(2+)/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca(2+)-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser(103)-phosphorylated SRF in nuclear extracts, indicated that the SRE.SRF complexes contribute to the Ca(2+)-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.
Bernal-Mizrachi
E
E
Washington University School of Medicine, Division of Endocrinology, Diabetes and Metabolism, St. Louis, MO 63110, USA. aplab1@imgate.wustl.edu
Wice
B
B
Inoue
H
H
Permutt
M A
MA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003981:Diazoxide / Q000494:pharmacology; D004232:Diuretics; D004305:Dose-Response Relationship, Drug; D051766:Early Growth Response Protein 1; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D017874:Immediate-Early Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D026362:Serum Response Factor; D015398:Signal Transduction; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014044:Tolbutamide / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002147:Calmodulin; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004232:Diuretics; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D007093:Imidazoles; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D026362:Serum Response Factor; D049993:Sodium Chloride Symporter Inhibitors; D014157:Transcription Factors; C031938:calmidazolium; D004533:Egtazic Acid; D011189:Potassium Chloride; D014044:Tolbutamide; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose; D003981:Diazoxide; D002118:Calcium