Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10738958 | Free Radical Biology and Medicine | 2005 | 11 Pages |
Abstract
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 5â²-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5â²-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from â403 to â111 and from â705 to â613 are involved in positive regulation and the region from â745 to â705 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 â745 to â705. 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 â729/â724 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.
Keywords
NF1EMSAbHLHZipAP-4MZF1GCLMGCLCARNTNF-κBAP-1GCLHRPPBSFBSDTTELFC/EBPSp1aryl hydrocarbon receptor nuclear translocatorsodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGEmyeloid zinc finger 1Chronic obstructive pulmonary diseaseCOPDelectrophoretic mobility-shift assayOxidative stressdithiothreitolbasic helix-loop-helix leucine zipperfetal bovine serumnuclear factor 1antioxidant-response elementUpstream stimulatory factornuclear factor κBglutamate-cysteine ligaseEpithelial lining fluidAREpolymerase chain reactionPCRHorseradish peroxidaseStimulating protein 1activator protein 1Glutathione
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Authors
Lin-Ling Cheng, Bing Li, Jian-Dong Luo, Hong-Bin Tu, Qi-Cai Liu, Pixin Ran,