Ca2+ administration stimulates the binding of AP-1 factor to the 5'-flanking region of the rat gene for the Ca2+-binding protein regucalcin

• Published in: Biochemical journal 1998-01, Vol.329 ( Pt 1) (1), p.157-163
• Main Authors: Murata, T, Yamaguchi, M
• Format: Article
• Language: English
• Subjects: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester - pharmacology; Animals; Calcium - administration & dosage; Calcium - pharmacology; Calcium Channel Agonists - pharmacology; Calcium-Binding Proteins - genetics; Calmodulin - antagonists & inhibitors; Calmodulin - metabolism; Carcinoma, Hepatocellular; DNA - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Genes, Reporter - genetics; Intracellular Signaling Peptides and Proteins; Liver - metabolism; Male; Nuclear Proteins - metabolism; Promoter Regions, Genetic - genetics; Rats; Rats, Wistar; Sulfotransferases; Transcription Factor AP-1 - genetics; Transcription Factor AP-1 - metabolism; Transcription, Genetic - genetics; Trifluoperazine - pharmacology; Tumor Cells, Cultured
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3290157

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.