MAPK/P38/NF-kB signaling pathway for radiation increased IL-6 expression of endothelial cells with anti-apoptosis effect through Mcl-1 Free

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Several studies indicate that irradiation results in increased interleukin-6 (IL-6) in endothelial cells. The mechanism and functional role of IL-6 in endothelial cells are unclear. This study is to elucidate these effects by using human umbilical vascular endothelial cell (HUVEC). The HUVECs were irradiated with different doses. The concentration of IL-6 in cell supernatant was determined by EIA. The transcriptional regulation of IL-6 was investigated by RT-PCR, IL-6 promoter drived luciferase reporter assay, and transcription factor decoy oligonucleotides. The signal transduction pathway involved in radiation increased IL-6 expression was clarified by specific chemical inhibitors and confirmed by western blot. Finally, the anti-apoptosis effect of IL-6 was determined by Annexin-V expression using flow cytometry. On the other hand, the role of Mcl-1 was confirmed by anti-sense oligonucleotides. With different doses of radiation (0, 2, 4, 6, 8, 10 Gy), 4 Gy most significantly increased the IL-6 concentrations in cell supernatant at 48 hours. With this dose, the time-dependent IL-6 levels increased rapidly at 24 hours and remained steady until 72 hours. The RT-PCR results demonstrated that radiation increased IL-6 mRNA at 4 hours, and saturated the expression at 8 hours. The IL-6 promoter drived luciferase activity assay showed that radiation transcriptionally regulated IL-6 expression. With specific chemical inhibitor SB203580, the radiation increased IL-6 mRNA, IL-6 concentration in supernatant, and IL-6 promoter drived luciferase activity were all inhibited. It implies that the radiation activated IL-6 expression was mainly through MAPK/P38 signaling pathway. By using decoy oligonucleotides, it revealed that NF-kB but not AP-1 or NF-IL6 critically involved in radiation induced IL-6 expression. Furthermore, the NF-kB activated by radiation involving IL-6 expression was regulated by MAPK/P38 signal transduction pathway. IL-6 has been shown ineffective on HUVEC in vitro, given that gp80 domain (IL-6α chain receptor), in terms of soluble IL-6 receptor (sIL-6-Rα), is lacking. The functional role of radiation induced IL-6 in HUVEC was further investigated by adding sIL-6-Rα. In the presence of sIL-6-Rα radiation induced apoptosis of HUVECs was significantly reduced. The western blot demonstrated that radiation increased IL-6 was through sIL-6-Rα induced expression of anti-apoptosis protein Mcl-1. Besides, the protective effect of IL-6 on HUVEC was inhibited by Mcl-1 anti-sense oligonucleotides. In conclusion, radiation increased IL-6 expression was mainly through MAPK/P38/NF-kB signaling pathway. Functionally the anti-apoptosis effect of radiation on HUVEC from IL-6 was induced by Mcl-1 expression. The radiation increased IL-6 expression may play an important role in protecting endothelial cells by the autocrine effect.