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Objective To examine the effect of angiotensin Ⅱ (Ang Ⅱ) on nuclear factor-kappa B (N F-κB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang Ⅱ activat es NF-κB. Methods ECV304 cells were transiently cotransfected with an NF-κB/ luciferase reporter gene and inactive NF-κB-inducing kinase (NIK), IκB kinase α (IKK_α), I κB kinase β (IKK_β) mutants or vectors, respectively. The effect on NF- κB was detected by using an electrophoretic mobility shift assay (EMSA) and overex pression of the mutants enabled blocking of reporter gene activation induced by Ang Ⅱ. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, d efinite cytoplasmic-to-nuclear translocations of NF-κB activation were detec ted using subunits p50 and p65 induced by Ang Ⅱ. Results The translocation of p50 in nuclei was highly remarkable 2 hours after Ang Ⅱ st imulation, and the activity was somewhat reduced 6 hours after stimulation to th e 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang Ⅱ for 18 hours. Conclusion Ang Ⅱ is effective in stimulating NF-κB activation through a pathway dependen t on NIK, IKK_α and IKK_β, and induces PDGF-B transcription in the endothel ial cell line, ECV304.
Objective To examine the effect of angiotensin Ⅱ (Ang Ⅱ) on nuclear factor-kappa B (NF-κB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang Ⅱ activat es NF-κB. Methods ECV304 cells were transiently cotransfected with an NF-κB / luciferase reporter gene and inactive NF-κB-inducing kinase (NIK), IκB kinase α (IKK_α), I κB kinase β (IKK_β) mutants or vectors, respectively. by using an electrophoretic mobility shift assay (EMSA) and overex pression of the mutants enabled blocking of reporter gene activation induced by Ang II. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, d efinite cytoplasmic- to-nuclear translocations of NF-κB activation were detec ted using subunits p50 and p65 induced by Ang Ⅱ. Results The translocation of p50 in nuclei was highly remarkable 2 hours after Ang Ⅱ st imulati on, and the activity was somewhat reduced 6 hours after stimulation to e 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang Ⅱ for 18 hours. Conclusion Ang Ⅱ is effective in stimulating NF-κB activation through a pathway dependen t on NIK, IKK_α and IKK_β, and induces PDGF-B transcription in the endothelium ial cell line, ECV304.