In this study we investigated whether the nuclear localization of regucalcin in cloned normal rat kidney tubular epithelial NRK52E cells is regulated after culture with hormonal signaling factors. Stable regucalcin/pCXN2 transfectants with subconfluent monolayers were further cultured for 24 or 48 h in a serum-free medium containing either vehicle, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), parathyroid hormone (PTH), phorbol 12-myristate 13-acetate (PMA), or other factors. Culture with TNF-α (1.0 ng/ml of medium) or TGF-β1 (5.0 ng/ml) for 48 h caused a significant decrease in regucalcin mRNA levels in NRK52E cells (wild-type), while regucalcin mRNA levels were markedly increased in the presence of PMA (10−6 M), an activator of protein kinase C, in wild-type cells. Immunocytochemical observation showed that HA-regucalcin was markedly localized in the nucleus of HA-regucalcin/ phCMV2-transfected cells. The nuclear localization was enhanced in culture with BS (5%), PTH (10−7 M), Bay K 8644 (2.5x10−6 M), or PMA (10−6 M) for 24 or 48 h. Culture with staurosporine, an inhibitor of protein kinase C, caused a remarkable decrease in the localization of HA-regucalcin in the nucleus of HA-RGPR-p117/phCMV2-transfected cells with PMA. Culture with PMA (10−6 M) for 24 or 48 h caused a remarkable increase in nuclear regucalcin protein levels. The effect of PMA in increasing nuclear regucalcin levels was completely absent in culture with staurosporine (10−8 M). The nuclear localization of regucalcin in the stable regucalcin/pCXN2-transfected cells (transfectant) increased markedly as compared with that of wild-type cells, whereas the increase was less evident in the transfectants cultured with staurosporine. This study demonstrated that regucalcin localizes in the nucleus of cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that its nuclear localization is enhanced through an intracellular signaling process which involves protein kinase C.

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