Ca2+ administration stimulates the binding of AP-1 factor to the 5'-flanking region of the rat gene for the Ca2+-binding protein regucalcin.
mRNA of the Ca2+-binding protein, regucalcin, is mainly expressed in the liver and only to a small extent in the kidney, and the expression of hepatic regucalcin mRNA is markedly stimulated by Ca2+ administration [Shimokawa and Yamaguchi (1992) FEBS Lett. 305, 151-154]. The existence of nuclear factors that bind to the 5'-flanking region of the rat regucalcin gene was investigated. When nuclear proteins obtained from various rat tissues were used in gel mobility-shift assays, tissue-specific formation of a protein-DNA complex was found in the liver and kidney. An additional novel protein-DNA complex was formed when liver nuclear extracts obtained from Ca2+-administered rats (10mg of Ca2+/100g body weight) were used. Competition gel mobility-shift experiments using consensus and mutant oligonucleotides for AP-1 factor showed that the additional novel complex was formed from binding of the AP-1 factor to the regucalcin gene. Ca2+-induced binding of the AP-1 factor to the regucalcin gene was completely inhibited by simultaneous administration of trifluoperazine, an antagonist of calmodulin, suggesting that the activation of nuclear AP-1 protein is partly mediated through a Ca2+/calmodulin-dependent pathway. Moreover, the 5'-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed the promoter activity in H4-II-E hepatoma cells. This promoter activity was enhanced by treatment with Bay K 8644, a Ca2+-channel agonist. The present study demonstrates that the Ca2+-response sequences are located within the 5'-flanking region of the rat regucalcin gene.
Murata
T
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan.
Yamaguchi
M
M
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D006528:Carcinoma, Hepatocellular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014268:Trifluoperazine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002120:Calcium Channel Agonists; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014268:Trifluoperazine; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D004247:DNA; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D002118:Calcium