Atf3 Role In Sublyticc5b-9 Compound Induces Apoptosis Of Gmcs

Part I Construction and identification of ATF3 expression vector and eukaryotic vectors expressing shRNA targeting rat ATF3 geneAIMS: (1) To construct rat activating transcription factor 3 (ATF3) expression vector and ATF3 small hairpin RNA (shRNA) for exploring the role of ATF3 in the glomerular mesangial cells (GMCs) apoptosis induced by sublytic C5b-9 complexes.METHODS: (1) The coding sequence of the ATF3 gene was obtained by reverse transcription-PCR (RT-PCR) with the designed primers and inserted into the Sfi I site of pcDNA3.1 vector to construct pcDNA3.1/ATF3 plasmid. Five reverse repeated motifs targeting rat ATF3 gene were synthesized and cloned into eukaryotic expression plasmid pGCsi-U6/Neo/GFP. (2)After being screened and sequencing confirmed,the recombinant plasmids were transfected into rat GMCs respectively, with or without sublytic C5b-9 attack, then the levels of ATF3 protein were measured using Western blot. RESULTS: (1) The eukaryotic expression plasmid pcDNA3.1/ATF3 was successfully constructed. RT-PCR and Western blot confirmed pcDNA3.1/ATF3 can successfully express ATF3 protein in GMCs. (2) Five eukaryotic vectors expressing shRNA targeting rat ATF3 gene were successfully constructed. Western blot results showed that the optimal shRNA plasmid which could most effectively silence the target gene was shRNA-1. CONCLUSIONS: The expression plasmid pcDNA3.1/ATF3 was successfully constructed. The eukaryotic vectors expressing shRNA targeting rat ATF3 gene were successfully constructed and identified. These vectors built foundation for exploring the role of ATF3 in sublytic C5b-9-induced GMCs apoptosis. Part II The role of activating transcription factor 3 (ATF3) in glomerular mesangial cells (GMCs) apoptosis induced by sublytic C5b-9 complexesAIMS: To explore the role of activating transcription factor 3 (ATF3) in glomerular mesangial cells (GMCs) apoptosis induced by sublytic C5b-9 complexes. METHODS: (1) The expression of ATF3 in the cultured GMCs following sublytic C5b-9 attack were measured by reverse transcription PCR (RT-PCR), Real-time PCR, Western blot and Immunohistochemistry. The apoptosis of GMCs following sublytic C5b-9 attack was evaluated by Flow cytometry after Annexin V(AV)-propidium iodide (PI) staining. (2)The cultured rat GMCs were transfected with pcDNA3.1/ATF3 and ATF3 shRNA respectively, with or without sublytic C5b-9 attack, then the cells were divided into different groups. The level of ATF3 expression was assessed by fluorescent microscope and Western blot. The nuclear morphology of GMCs was observed with Hoechst 33342 staining. The apoptosis of GMCs was evaluated by Flow cytometry after AV-PI staining. RESULTS: (1) The mRNA transcripts of ATF3 from GMCs with sublytic C5b-9 attack were found to be significantly up-regulated at 3 h compared to control groups by RT-PCR and Real-time PCR, which was similar to changes in previous microarray experiments. Meanwhile, the ATF3 protein from GMCs with sublytic C5b-9 treatment was markedly increased at 3 h and the distribution of ATF3 protein was largely in the nuclei of GMCs. The relative apoptosis number of GMCs following sublytic C5b-9 attack was markedly increased compared with that of other groups. (2) In sublytic C5b-9-treated GMCs and pcDNA3.1/ATF3-transfected GMCs, the typical apoptotic chromatin condensation and fragmentation were seen and the ratio of apoptotic cells was increased. This was more obvious in pcDNA3.1/ATF3-transfected GMCs plus sublytic C5b-9 attack. In constract, the numbers of apoptotic GMCs, in response to sublytic C5b-9 attack were subdued mostly by ATF3 shRNA through knock-down of ATF3 gene. CONCLUSIONS: ATF3 was upregulated in the cultured GMCs following sublytic C5b-9 attack. Upregulation of ATF3 increased GMCs apoptosis which induced by sublytic C5b-9 attack.