Overexpression of regucalcin suppresses cell proliferation of cloned normal rat kidney proximal tubular epithelial NRK52E cells.
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.
Nakagawa
Taeko
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
Sawada
Natumi
N
Yamaguchi
Masayoshi
M
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D002265:Carboxylic Ester Hydrolases; D002452:Cell Count; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000077546:Roscovitine; D015238:Sulfotransferases; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D002087:Butyrates; D002120:Calcium Channel Agonists; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D026902:Potassium Channel Blockers; D011687:Purines; D012333:RNA, Messenger; D013454:Sulfoxides; D013861:Thiocyanates; D000077546:Roscovitine; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D010741:Phospholipases A; C016766:sulforaphane; D000077191:Wortmannin