ABSTRACT
Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K1) and a series of bacterial menaquionones (MK-n; vitamin K2). Menadione (vitamin K3) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5' rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter.
Subject(s)
Dimethylallyltranstransferase/metabolism , Gene Expression Regulation/physiology , YY1 Transcription Factor/physiology , Base Sequence , Blotting, Western , Chromatin Immunoprecipitation , DNA, Complementary , Dimethylallyltranstransferase/genetics , Electrophoretic Mobility Shift Assay , HEK293 Cells , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Transcription, GeneticABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/physiology , Cell Proliferation , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Androstadienes/pharmacology , Animals , Butyrates/pharmacology , Calcium Channel Agonists/pharmacology , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/genetics , Carboxylic Ester Hydrolases , Cell Count , Cell Cycle Proteins/genetics , Cell Line , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression/genetics , Genetic Vectors/genetics , Immunoblotting , Intracellular Signaling Peptides and Proteins , Isothiocyanates , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Male , Phosphoinositide-3 Kinase Inhibitors , Phospholipases A/antagonists & inhibitors , Potassium Channel Blockers/pharmacology , Purines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Roscovitine , Sulfotransferases , Sulfoxides , Thiocyanates/pharmacology , Transfection , WortmanninABSTRACT
The expression of regucalcin in rat bone marrow cells was investigated. The expression of regucalcin mRNA in the bone marrow cells of normal (wild-type) rat was shown by using reverse transcription-polymerase chain reaction (RT-PCR) with a specific primer of regucalcin cDNA. Regucalcin protein was detected in the marrow cells of normal (wild-type) rats using Western blot analysis. Regucalcin levels were significantly increased in the marrow cells of regucalcin transgenic (TG) male and female rats with increasing age (5-36 weeks old). When the marrow cells obtained from normal or regucalcin TG rats (36-week-old) were cultured for 7 days, the number of tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts, positive multinucleated cells (MNCs) were significantly increased in the marrow culture of regucalcin TG rats. This increase was remarkable in female TG rats as compared with male TG rats. The effect of parathyroid hormone [human PTH (1-34); 10(-7) M] or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10(-7) M] in stimulating TRACP-positive MNCs formation was significantly enhanced in female regucalcin TG rats. Calcium content in the femoral-diaphyseal and -meta-physeal tissues was significantly decreased in regucalcin TG rats (10- or 36-week-old). This decrease was greater in female than in male. Femoral-metaphyseal deoxyribonucleic acid (DNA) content was significantly reduced in regucalcin TG male and female rats (36-week-old). Moreover, serum inorganic phosphorus, triglyceride, HDL-cholesterol, and albumin concentrations were significantly increased in regucalcin TG female rats (36-week-old), while serum calcium, zinc, and glucose concentrations were not significantly altered in TG male and female rats. In TG male rats, serum triglyceride and HDL-cholesterol concentrations were significantly raised. This study demonstrates that regucalcin is expressed in rat bone marrow cells, and that osteoclastic bone resorption is stimulated in regucalcin TG rats with increasing age. Also, regucalcin TG aged rats was found to induce serum metabolic disorder.
