Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: Change in apoptosis‐related gene expression

Abstract

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.

Increased regucalcin gene expression extends survival in breast cancer patients: Overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro

“…Regucalcin has been shown to suppress transcriptional activity in the nucleus of MDA-MB-231 cells (15). Thus molecular mechanism showed similarity to the action of regucalcin in cloned normal rat kidney proximal epithelial cells and cloned rat hepatoma H4-II-E cells in vitro (28,29). Suppressive effects of regucalcin on the proliferation were independent on the death in MDA-MB-231 cells, since regucalcin prevents cell death induced by various stimulatory factors.…”

“…MDA-MB-231 wild-type cells (1x10 5 /ml/well) and MDA-MB-231 cells (1x10 5 /ml/well) transfected with regucalcin cDNAs of either full length, deleted exon 4 or deleted exons 4 and 5 were cultured using a 24-well plate in DMEM containing 10% FBS and 1% penicillin and streptomycin for 1, 2, 3 or 7 days in a watersaturated atmosphere containing 5% CO 2 and 95% air at 37˚C (28,29). In separate experiments, MDA-MB-231 wild-type cells (1x10 5 /ml/well) or full length regucalcin transfectants were cultured in DMEM containing 10% FBS and 1% penicillin and streptomycin in the presence of sodium butyrate (10 and 100 µM), roscovitine (10 and 100 nM), sulphoraphan (1 and 10 nM), dibucain (0.1 or 1 µM), Bay K 8644 (1 or 10 µM), PD98059 (1 or 10 µM), wortmannin (0.1 or 1 µM), DRB (0.1 or 1 µM), or gemcitabine (50 or 100 nM) for 3 days.…”

“…Subconfluent cells were cultured for additional 3 days in the presence or absence of LPS (0.1 or 1 µg/ml), TNF-α (0.1 or 1 ng/ml) (30). In separate experiments, wild-type MDA-MB-231 cells (1x10 5 /ml/well) or transfectants were cultured for 5 days to confluent, and then for an additional 24 h in the presence or absence of LPS (1 ng/ml) or Bay K 8644 (10 µM) with or without caspase-3 inhibitor (10 µM) (29). After culture, cells were detached with trypsin from each culture dish.…”

“…After trypsinization of each culture dish using 0.2% trypsin plus 0.02% EDTA in Ca 2+ /Mg 2+ -free PBS for 2 min at 37˚C, the detached cells from the dish were collected by centrifugation (28)(29)(30). Cells were resuspended on PBS solution and stained with eosin.…”

Overexpression of regucalcin suppresses gene expression of insulin signaling-related proteins in cloned rat hepatoma H4-II-E cells: Involvement of insulin resistance

“…In addition, regucalcin has an activatory effect on Ca 2+ -ATPase (calcium pump enzymes), proteolysis (protease), and superoxide dismutase in cells (14)(15)(16). Regucalcin has been shown to have suppressive effects on cell proliferation (17,18) and apoptotic cell death (19,20). Thus, regucalcin plays a pivotal role in maintaining cell homeostasis and function (14).…”