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β-naphthoflavone affects rat glutamyl cysteine synthetase catalytic subunit gene expression

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Author:
No author available
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
3
DOI:
10.3760/cma.j.issn.1674-1927.2010.03.001
Key Word:
β萘黄酮;谷氨酸-半胱氨酸连接酶;谷氨酰半胱氨酸合成酶催化亚单位;谷胱甘肽;细胞特异性;Beta- naphthoflavone;Glutamate-cysteine ligase;Glutamyl cysteine synthetase catalytic subunit;Glutathione;Cells specificity

Abstract: Objective To elucidate the gene transcription regulation mechanism of rat γ-glutamyl cysteine synthetase (γ- GCS) by investigating the effect of β- naphthoflavone (β- NF) , an exogenous stimulating factor, on rat glutamyl cysteine synthetase catalytic subunit (GCLC) gene transcription.methods RTE and H4 Ⅱ E cells were tranfected with GCLC 5' -upstream regulatory sequence driven GCLC-PGL3-enhancer- Luciferase reporter vector (GCLC-Luc) and assigned to DMSO control group and several groups treated by different levels of stimulating factors. Possible stimulating factors of rat GCLC gene regulation were screened by comparing luciferase activity among the groups. The effect of β-NF on expression of GCLC - Luc in RTE and in H4ⅡE cells was also compared. The level ofγ- GCS mRNA was measured by SYBR green fluorescent quantitative PCR in β - NF experimental group ( 10 μmol/L ) and compared with that in DMSO control group of RTE or H4ⅡE cells. The target site and DNA binding elements that related with β - NF effect were verified by transfecting r series sequential deletion reporter vectors and GCLC-Luc,point-mutation reporter vectors of AP- 1 and NF-κB into cells. The levels of luciferase activity in β-NF experimental groups were compared with the DMSO control group. Results β-NF at levels of 1, 10 and 100 μmol/L was shown to strongly inhibit GCLC gene expression in RTE cells, resulting in significantly lowered luciferase activity compared with that in the DMSO control group (16 135±1456, 2752±218, 1579±294 vs 25 971±1662, all P<0.01). Β-NF at levels of 1, 10 and 100 μmol/L was shown to stimulate GCLC gene expression in H4ⅡE cells, resulting in significantly higher luciferase activity compared with that in the DMSO control group (5686±441, 13 601±746, 13 978± 164 vs 3645±367, all P<0.01). Fluorescent quantitative PCR examination showed that the level of endogenous γ-GCS mRNA expression in 10 μmol/L β-NF experimental group of RTE cells was 0.73 fold, as was 1.98 fold in 10 μmol/L β-NF experimental group of H4ⅡE cells, compared with the DMSO control group. After transfection with GCLC-Luc or r series deletion reporter vectors, luciferase activities were lowered in all RTE cells (P<0.01) but elevated in all H4ⅡE cells (P<0.05) treated by β-NF compared with the DMSO control group. The active region of β-NF action was localized between nucleotides -390 bp upstream and +2 bp. While point-mutated AP-1 and NF-κB reporter vectors were transfected into cells, the luciferase activity in β-NF treated RTE cells further transfected with GCLC-Luc vector was (91.50±0.32)% lower than that in the DMSO control group, whereas no reduction was seen in those transfected with AP-1 and NF-κB vectors (P>0.05). In H4 Ⅱ E cells, the extent of β-NF increased luciferase activity of AP-1 point-mutation reporter vector was lower than that of GCLC-Luc vector [ (2.08±0.19) fold vs (3.81 ±0.19) fold, P<0.05]. But there was no difference in rising amplitude of luciferase activity in cells transfected with NF-κB point-mutation reporter vector and GCLC- Luc vector.Conclusion Exposure of RTE cells and H4 Ⅱ E cells to β-NF results in cells specific response. Β-NF may regulate the expression of GCLC gene by partially activating antioxidant response element (ARE)-like AP-1 element in rat H4 Ⅱ E cells.

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