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Constitutive and β-Naphthoflavone-induced Expression of the Human γ-Glutamylcysteine Synthetase Heavy Subunit Gene Is Regulated by a Distal Antioxidant Response Element/TRE Sequence
Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance. The rate-limiting enzyme in th...
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| Published in: | The Journal of biological chemistry 1997-03, Vol.272 (11), p.7445 |
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| Main Authors: | , , , |
| Format: | Article |
| Language: | English |
| Online Access: | Get full text |
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| Summary: | Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive
oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of
cellular redox balance. The rate-limiting enzyme in the de novo synthesis of glutathione is γ-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing
catalytic and regulatory functions, respectively. Exposure of HepG2 cells to β-naphthoflavone (β-NF) resulted in a time- and
dose-dependent increase in the steady-state mRNA levels for both subunits. In order to identify sequences mediating the constitutive
and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5â²-flanking region (â3802
to +465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells. Constitutive expression
was maximally directed by sequences between â202 and +22 as well as by elements between â3802 to â2752. The former sequence
contains a consensus TATA box. Increased luciferase expression following exposure to 10 μ M β-NF was only detected in cells transfected with a reporter vector containing the full-length â3802:+465 fragment. Hence,
elements directing constitutive and induced expression of the GCS heavy subunit are present in the distal portion of the 5â²-flanking
region, between positions â3802 and â2752. Sequence analysis revealed the presence of several putative consensus response
elements in this region, including two potential antioxidant response elements (ARE3 and ARE4), separated by 34 base pairs.
When cloned into the thymidine kinase-luciferase vector, pT81-luciferase, and transfected into HepG2 cells, both ARE3 and
ARE4 increased basal luciferase expression approximately 20-fold. When cloned in tandem in their native arrangement the increase
in luciferase activity was in excess of 100-fold, suggesting a strong interaction between the two sequences. Luciferase expression
was elevated in β-NF-treated cells transfected with the ARE4- tk -luciferase vector and all DNA fragments containing ARE4. In contrast, ARE3 did not direct increased luciferase expression
in response to β-NF nor did it significantly modify the magnitude of induction directed by ARE4. The influence of the ARE4
oligonucleotide on constitutive and induced expression was eliminated by introduction of a single base mutation, convert |
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| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.272.11.7445 |