Overexpression of RGPR-p117 suppresses apoptotic cell death and its related gene expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells

Abstract

The novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. The role of RGPR-p117 in cell function has not been fully clarified. This study was undertaken to determine whether over-expression of RGPR-p117 regulates various types of signaling factor-induced apoptotic cell death in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's modified Eagle's medium containing 5% bovine serum (BS). NRK52E cells with subconfluent monolayers were cultured for 24-72 h in a medium without BS. The presence of tumor necrosis factor-α (TNF-α; 1.0 or 10 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 μg/ml), Bay K 8644 (10-6 or 10-5 M), or thapsigargin (10-8 or 10-7 M) caused a significant decrease in the number of NRK52E wild-type cells or phCMV2-transfected (mock-type) cells. The effect of TNF-α, LPS, Bay K 8644, or thapsigargin in decreasing cell number was significantly suppressed in the presence of the caspase-3 inhibitor (10 -8 M) in wild-type cells cultured for 48 h. The effect of TNF-α, LPS, or Bay K 8644 in decreasing cell number was significantly inhibited in the transfectants, while the effect of thapsigargin on cell death was not inhibited in the transfectants. Culture with TNF-α or LPS caused DNA fragmentation in wild-type cells. These effects were significantly suppressed in the transfectants. The result of reverse transcription-polymerase chain reaction analysis using specific primers for the genes of apoptotic cell death-related proteins showed that IAP-1, FADD, caspase-8, caspase-9, and caspase-3 mRNA levels were significantly decreased in the transfectants, while Akt-1, Bid, Apaf-1, and glyceroaldehyde-3-phosphate dehydrogenase mRNA levels were not significantly altered in the transfectants. Culture with TNF-α, LPS, Bay K 8644, or thapsigargin caused a significant increase in Apaf-1 or caspase-3 mRNA levels. Such an effect was not seen in the transfectants. This study demonstrates that overexpression of RGPR-p117 has a suppressive effect on cell death and apoptosis induced by TNF-α, LPS, or Bay K 8644 whose actions are mediated through intracellular signaling pathways. This study also demonstrates that RGPR-p117 regulates the gene expression of apoptosis-related proteins.