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.