Wild A C, Gipp J J, Mulcahy T
Department of Human Oncology, University of Wisconsin Medical School, 600 Highland Avenue, K4/316 CSC, Madison, WI 53792, USA.
Biochem J. 1998 Jun 1;332 ( Pt 2)(Pt 2):373-81. doi: 10.1042/bj3320373.
gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and beta-naphthoflavone (beta-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCAGCG-3') containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and beta-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and beta-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or beta-NF-induced expression of the GCSh gene.
γ-谷氨酰半胱氨酸合成酶（GCS）是谷胱甘肽（GSH）从头合成中的限速酶，是一种异二聚体，由一个催化亚基（GCSh）和一个调节亚基（GCSl）组成。我们先前证明，GCSh基因的组成型表达以及β-萘黄酮（β-NF）诱导的表达是由一个位于转录起始位点上游3.1 kb处的远端抗氧化反应元件（ARE）即ARE4介导的[Mulcahy、Wartman、Bailey和Gipp（1997年）《生物化学杂志》272，7445 - 7454]。ARE4由一个包含嵌入的佛波酯反应元件（TRE，下划线标注）的共有ARE序列（5'-GTGACTCAGCG-3'）组成。通过突变分析确定了这两个重叠反应元件对GCSh基因组成型表达和β-NF诱导表达的相对重要性。内部激活蛋白-1（AP-1）结合序列介导启动子/报告基因转基因的组成型表达，但对于β-NF反应性并非必需。在凝胶迁移实验中，TRE对于从未经处理 的HepG2细胞制备的核提取物中的蛋白质结合是必需的。相反，β-NF诱导依赖于完整的ARE序列，特别是ARE4的末端GC框。ARE4的GC框对于报告基因构建体的基础表达和β-NF诱导的表达均至关重要。该元件还影响核蛋白与ARE4的结合，特别是在从β-NF处理的HepG2细胞中分离的提取物中。因为先前的研究表明ARE4可能与一个单独的假定ARE协同作用，所以还评估了位于ARE4下游34个碱基处的相邻序列（ARE3）的作用。在GCSh启动子/报告基因内对该元件进行突变并没有改变转基因的基础表达或β-NF诱导的表达，表明ARE3不影响GCSh基因的组成型表达或β-NF诱导的表达。
