Involvement of nuclear factor I-A1 in the regulation of regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells.
Nuclear factor I-A1 (NF1-A1) can bind to the TTGGC motif in the rat regucalcin gene promoter region. This study was undertaken to determine whether NF1-A1 is involved in the enhancement of the rat regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct with the deletion of -523/-435 including the TTGGC motif (mutant) in cloned normal rat kidney proximal tubular epithelial NRK52E cells. Cells were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with the deletion of -523/-435. NRK52E cells (wild-type) or NRK52E cells transiently transfected with HA-NF1-A1/phCMV2 were cultured for 48 h in a medium containing either vehicle or BS (5%) in the presence or absence of various factors. HA-NF1-A1 was localized in the nucleus of wild-type cells. Luciferase activity was significantly increased as compared to that of wild-type cells. This increase was significantly enhanced in the presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M). Such an enhancement was not seen by culture with Bay K 8644 (10(-6) M) or dibutyryl cyclic AMP (10(-4) M). The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1 was not observed in the presence of dibucaine (10(-6) M), staurosporine (10(-9) M), or PD 98059 (10(-8) M), which is an inhibitor of various protein kinases. Such an inhibition was also seen in the presence of vanadate (10(-6) M) or okadaic acid (10(-6) M), an inhibitor of protein phosphatase. The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1/ phCMV2 was not seen in the mutant with deletion of -523/-435. The increase in luciferase activity in HA-NF1-A1/ phCMV2-transfected NRK52E cells was not significantly enhanced in the cells transiently co-transfected with HA-RGPR-p117/phCMV2, which could increase the regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type). This study demonstrates that NF1-A1 enhances the regucalcin promoter activity which is related to the TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation in NRK52E cells.
Sawada
Natsumi
N
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Yamaguchi
Masayoshi
M
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases; D002460:Cell Line; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D051839:NFI Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D051839:NFI Transcription Factors; D011993:Recombinant Fusion Proteins; C486711:Sec16b protein, rat; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D008156:Luciferases; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D019311:Staurosporine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one