The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned normal rat kidney proximal tubular epithelial NRK52E cells over-expressing regucalcin. NRK52E cells were transfected with regucalcin (RC) pCXN2 vector and the multiple neomycin-resistant clones that stably overexpress regucalcin were selected. The regucalcin content of RC/pCXN2-transfected cells used in this study was about 21-fold as compared with that of the parental wild-type NRK52E cells. Wild-type NRK52E cells, pCXN2 vector-transfected cells (mock-type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of bovine serum (5%). The cell numbers of wild- and mock-type were significantly increased with the time course of the culture. Cell numbers of transfectants were significantly suppressed as compared with that of wild- and mock-type. The decrease in cell number of wild-type cultured for 72 h in the presence of butyrate (8.3 x 10(-6) or 8.3 x 10(-5) M), rescovitine (10(-8) or 10(-7) M), or sulforaphane (10(-9) M), which is an inhibitor of the cell cycle, was not observed in transfectants. The effect of PD98059 (10(-8) M), staurosporine (10(-10) M) or dibucaine (10(-8)-10(-6) M), which is an inhibitor of protein kinases, in decreasing cell number of wild-type was not seen in transfectants. Moreover, the culture with wortmannin (10(-8) or 10(-7) M), an inhibitor of phosphatidylinositol 3 (PI3)-kinase, or Bay K 8644 (10(-8) or 10(-7) M), an agonist of calcium entry in cells, caused a significant decrease in cell number of the wild-type. This decrease was not observed in transfectants. The result of reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers showed that c-jun and chk2 mRNA levels were significantly decreased in transfectant. p53 mRNA level was significantly increased in transfectants. The expression of c-myc, c-fos, cdc2, p21, and G3PDH mRNAs in transfectants was not significantly changed. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation, which is mediated through various signaling pathways, in the cloned normal rat kidney proximal tubular epithelial NRK52E cells.