Abstract 1963 Poster Session II, Sunday, 5/2 (poster 185)

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that acts as a transcriptional activator or repressor of a number of genes. We cloned the 5′-flanking promoter region of the rat taurine transporter gene (TauT), which contains a perfect half-site with reversed orientation (5′-CCTGTTCAGA-3′) and two imperfect consensus p53 binding half sites. To test the role of wild-type p53 in TauT expression, a temperature-sensitive mutant p53 allele (ts p53) was stably transfected into NRK-52E, a normal rat kidney cell line expressing TauT. Switching the cells (NRK-52E/p53) from 37°C to 32°C overnight did not affect expression of ts p53. Expression of TauT was decreased about 3-fold when NRK-52E/p53 cells were cultured at 32°C, as compared with 37°C. Temperature switching did not affect expression of TauT in NRK-52E cells and NRK 52E/neo (CMV-neo control vector) cells. Taurine uptake was also decreased approximately 3-fold when NRK-52E/p53 cells were cultured at 32°C, as compared with 37°C. Temperature switching did not affect taurine uptake by NRK-52E and NRK-52E/neo cells that did not contain ts p53. To confirm these observations, construct pGL 189 (containing basal TauT promoter sequences) was transiently transfected into NRK-52E cells with or without co-transfection of either wild-type p53 or a mutant p53 (p53-281). Wild-type p53 decreased TauT promoter activity approximately 5-fold, whereas mutant p53 had no effect on TauT promoter function. No reduction in promoter activity was evident when pGL-189 was co-transfected with the CMV vector, which does not express p53. To extend these findings, construct pGL-189 was transiently transfected into cells of different species or tissues, including MCF-7 (human breast cancer, estrogen receptor (ER) positive), MDA-MB-231 (human breast cancer, ER negative), LLC-PK1 (pig kidney), MDCK (canine kidney), H9c2 (rat heart), and NRK-52E cells, both with and without co-transfection of either wild-type p53 or mutant p53-281. Wild-type p53 enhanced TauT promoter activity by nearly 20-fold in MCF-7 cells and 5-fold in H9c2 cells, respectively, but had no effect on MDA-MB-231 cells. Interestingly, wild-type p53, but not mutant p53, decreased TauT promoter activity by 5-10 fold in all tested renal cell lines, including LLC-PK1, MDCK, and NRK-52E cells. Induction of TauT promoter activity by wild-type p53 was observed in cells (MCF-7 and H9c2) that mainly express ERa. In contrast, down-regulation of TauT promoter activity by wild-type p53 was found in renal cells, such as NRK-52E, which express both ERa and ERβs (β1, β2). Thus, the taurine transporter gene is differentially regulated by wild-type p53 through a mechanism that one can speculate may be mediated by estrogen receptors.