Identification of an E-box motif as a transcriptional repressor element in the proximal promoter region of the GCLC gene in rat lung epithelial L2 cells.
Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5'-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5'-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from -403 to -111 and from -705 to -613 are involved in positive regulation and the region from -745 to -705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region -745 to -705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at -729/-724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.
Cheng
Lin-Ling
LL
Guangzhou Institute of Respiratory Disease, Guangzhou Medical College, Guangzhou, Guangdong 510182, People's Republic of China.
Li
Bing
B
Luo
Jian-Dong
JD
Tu
Hong-Bin
HB
Liu
Qi-Cai
QC
Ran
Pixin
P
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D015536:Down-Regulation; D024721:E-Box Elements / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005721:Glutamate-Cysteine Ligase / Q000235:genetics* / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D051381:Rats; D051720:Upstream Stimulatory Factors / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D005721:Glutamate-Cysteine Ligase