Effect Of HIV-1 Nef On ICAM-1 Gene Overexpression Of Endothelial Cells Via ERK/MAPK Signaling Pathway

Objectives: HIV / AIDS has become a human disastrous disease in 21st century. Research on the pathogenesis of AIDS and related treatments have been urgent requirements. It has been documented that Nef plays an important role in HIV pathogenesis.Therefore, the effects of HIV-1 Nef gene related to ICAM-1 over expression on the endothelial cells were studied to analysis the reasons of ICAM-1 over expression of endothelial cells in HIV-1 infected patients and investigate molecule mechanism of HIV-1 Nef in AIDS pathogenesis.These will provide research basis for clinical therapy of HIV-1 infection.Methods:1) To construct eukaryotic expression vector pcDNA 3.1(+)-Nef, HIV-1 Nef gene was amplified from plasmid HIV-1 HXB2 by PCR and inserted into pcDNA 3.1(+). 2) Plasmids pcDNA 3.1(+) -Nef and pcDNA 3.1(+) were stably transfected into endothelial cell line ECV304 cells respectively. Nef protein was expressed and confirmed with RT-PCR, Western blot and cell immunofluorescence stain. 3) ICAM-1 expression of ECV304-Nef was measured by real-time PCR, Western blot, FCM and cellular adhesion assay.4) Kinase inhibitor PD98059 was used in the cells to analyse the signaling pathways .Results:1)To construct plasmid pcDNA3.1(+)-Nef, digestion and ligation of Nef gene and vector pcDNA3.1(+) were performed and then the ligated product was transformed into E.Coli. DH5α. The DNA fragments of 5400 bp and 621 bp were observed on the agarose gel by double enzyme digesting pcDNA3.1(+)-Nef .Sequenced Nef gene was coincident with the theoretical sequence.2) The expression of Nef in transfected cells was identified by following experiments. RT-PCR product of Nef (621bp) could be detected in cDNA from ECV304 cells transfected with pcDNA3.1(+)-Nef; the red fluorescence was seen mainly in the cytoplasm of Nef expressed ECV304 cells under fluorescence microscope; using Western blot a 27 kD strap was displayed in the Nef positive cell lysis.3) Compared with the control group, the ICAM -1 mRNA expression level was 4.3±0.2 fold in ECV304-Nef cells by RT-PCR and real-time PCR assays. Cellular adhesion assay indicated that adhesion of Jurkat cells to ECV304-Nef cells was significantly increased (P<0.05). FCM results showed that the percentage of ICAM-1 positive cells in ECV304-Nef and ECV304 pcDNA 3.1(+) cells was 35.3±2.2% and 12.5±0.8%respectively(P<0.01). ICAM-1 protein expression level increased in ECV304-Nef cells by western blot.4) To analyse signal transduction passway, p-ERK expression was detected and significantly increased in ECV304-Nef cells.The p-ERK inhibitor PD98059 almost completely blocked Nef-induced up-regulation of the p-ERK and ICAM-1. FCM results showed that when p-ERK inhibitor added, the percentage of ICAM-1 positive cells in ECV304-Nef and ECV304 pcDNA 3.1(+) cells was 11.4±1.1% and 10.4±1.5%respectively(P>0.05).Conclusions : 1) We successfully constructed eukaryotic expression plasmid pcDNA 3.1(+)-Nef. 2) Nef stable expression cells ECV304-Nef was performed and identified. 3) Results demonstrated that HIV-1 Nef can enhance ICAM-1 gene expression in vascular endothelial cells. 4) A discrepancy effect of inhibitor PD98059 suggested that ERK/MAPK signaling pathway may contribute to adhesion molecules ICAM-1 gene expression in HIV-1 Nef positive cells.