Expression Inhibition Of GCLC Gene By Proximal AHR/ARNT Element In Rat Brochial Epithelial Cells

Oxidative stress has a variety of significant impacts on human health in the development of common diseases. Chronic obstructive pulmonary disease (COPD) is one of such chronic diseases. Glutathione (GSH) is important in the antioxidant and anti-inflammatory substance, playing an important role in the protection of organs from oxidative damage.γ-glutamylcysteine synthetase (γ-GCS) is the rate-limiting enzyme in GSH synthesis, the catalytic subunit ofγ-GCS has full catalytic activity. The enzyme gene expression and regulation can contribute to the understanding of the mechanisms of GSH changes at the molecular level. Our preliminary study has found that the upstream of GCLC gene exists a negative part in regulation, two E-box element (-804~-799,-729~-724) can repress the expression of GCLC in a variety of sources of cells and have tissue specificity. Some articles report that the ability which E-box can repress or activate the expression of GCLC is related with neighboring elements or the transcriptional factors binding its. Therefore, we analyze the neighboring sequences of E-box in the upstream of GCLC gene; the AHR/ARNT element attracts our attention. The upstream of GCLC gene contains two AHR/ARNT elements, the core sequence of which are CACGGG, one of AHR/ARNT elements (-1090~-1085) locates in the neighboring of E-box element. ARNT is the dimerized partner of many bHLH-PAS proteins, such as AHR, HIF1α,HIF2αand SIM, participating in many important biological processes. As some articles report that ARNT can bind transcriptional factors-USF1/2 and formulate heterodimer, it is trustful that the hypothesis that the E-box element can bind ARNT in theory. Moreover, the transcriptional factors binding E-box element are related with hypoxia and detoxification, so if this binding is real, we can explain many problems such as stress and oxidant/antioxidant. For this reason, we mainly analyze whether this two AHR/ARNT elements can regulate the expression of GCLC gene or not.ObjectiveTo explore the transcriptional inhibition role of two AHR/ARNT elements (-1090~-1085 and -215~-210) at the upstream of transcription initiation site in RTE cells ofγ-glutamyl cysteine synthetase (γ-GCS) catalytic subunit (GCLC)Method1,Reporter vectors and expression vectors construction: (1) AHR/ARNT element deleted reportor vectors construction: Based on a previously constructed rat GCLC 1.8kb proximal regulatory sequence luciferase reporter vector, two AHR/ARNT elements (-1090~-1085 or -215~-210) deleted mutant reporters were constructed by PCR point-directed method. Two amplified fragments would be connected to the pGEM-T Easy vector with sequence submitted. The recombinant plasmids were digested by Sac I and Xho I enzyme and connected to pGL3-enhancer. GCLC-DdelAHR/ARNT-Luc was also constructed by PCR point-directed method with the model of GCLC-delAHR/ARNT(-1090)-Luc. (2) AHR/ARNT element core sequence with neighboring sequence reporter vector: we synthesized the AHR/ARNT(-1090), AHR/ARNT(-215) and their reverse complementary chains. We matched two complementary chains with gradual annealing and connected these fragments to the pGL3-promoter by the restriction enzyme sites SacI and XhoI. (3) Expression vector construction: we extracted total RNA of rat liver by Trizol (Invitrogen). The mRNA was reverse transcribed to cDNA, the fragments of AHR, ARNT, ARNT2, AHRR were amplified by PCR. The PCR products were identified by 1.2% agarose gel electrophoresis and connected to pGEM-T easy vector. The recombinant plasmids were digested by HindIII and NotI enzyme and connected to pRc/CMV2 vector.2,Transfection and luciferase activity analysis: (1) The gene transcriptional regulatory efficiency between wild-type and its AHR/ARNT deletion mutant reporters were compared by measuring luciferase activity of transfected rat bronchial epithelial cells (RTE).3,Transcriptional factors AHR, AHRR, ARNT, ARNT2 overexpression experiments: Co-transfection of rat expression vectors and reporter vectors were performed to prove the involvement of AHR, AHRR, ARNT and ARNT2 in the regulatory of GCLC transcription.4,Electrophoretic mobility shift analysis (EMSA) and supershift: To observe whether AHR/ARNT element can specially bind to its nuclear protein in the RTE cell, EMSA and antibody supershift assay were used to confirm the specific binding of the transcription factor AHR and ARNT.Result1,We have successfully constructed two AHR/ARNT elements mutant reporters (GCLC-delAHR/ARNT(-1090)-luc and GCLC-delAHR/ARNT(-215)-luc), a reporter deleted both elements(GCLC-DdelAHR/ARNT-Luc), reporters containing AHR/ARNT element core sequence with neighboring sequence (AHR/ARNT(-1090)-Promoter and AHR/ARNT(-215)-Promoter)and expression vectors (AHR-CMV2,AHRR-CMV2,ARNT-CMV2 and ARNT2-CMV2)。2,The gene transcriptional regulatory efficiency between wild-type and its AHR/ARNT deletion mutant reporters were compared by measuring luciferase activity of transfected rat bronchial epithelial cells (RTE). The result showed that deletion of one AHR/ARNT element (-1090~-1085) and both elements (-1090~-1085, -215~-210)significantly increased the luciferase activity in RTE cells compared with wild-type regulatory sequence(P<0.05); deletion of another AHR/ARNT element (-215~-210)also increased the reporter transcription but without statistical significance(P>0.05). The reporter driven by AHR/ARNT(-1090~-1085) core and neighboring sequence increased luciferase activity compared with control reporter ( P < 0.05 ) , while AHR/ARNT(-215~-210) had no such effect(P>0.05).The results of this experiment showed that the AHR/ARNT element (-1090~-1085) can repress the expression of GCLC.3,Transfection of CMV2-AHR vector can repress luciferase expression of wild type and deletion mutants(P<0.05), but transfection of other expression vectors had no effect on luciferase expression of wild type and deletion mutants. The results of this experiment showed that overexpression of transcriptional factors AHR can repress the expression of GCLC.4,EMSA showed that the probes with complete AHR/ARNT element could bind nuclear protein of RTE. Supershift assay showed that the nuclear protein binding to the probes contained the transcription factor AHR and ARNT.ConclusionThe transcription factor AHR and ARNT could bind two AHR/ARNT elements, AHR/ARNT element(-1090~-1085) was an important transcriptional suppressor element ofγ-GCS gene.