Abstract
To validate that the E-box element in the -745-705 region of rat gamma-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element. Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745 - 705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein. EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P < 0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P < 0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells. The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of gamma-GCS gene.
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