Dose-dependent biphasic induction and transcriptional activity of nuclear factor kappa B (NF-kappaB) in EA.hy.926 endothelial cells after low-dose X-irradiation.

PURPOSE: Low-dose radiotherapy is known to exert an anti-inflammatory effect, but the underlying radiobiological mechanisms are still elusive. It was recently reported that transforming growth factor (TGF) beta1 essentially contributes to the reduced adhesion of peripheral blood mononuclear cells to endothelial cells at low-dose X-irradiation. As the transcription factor nuclear factor kappa B (NF-kappaB) is crucially involved in mediating an inflammatory response by inducing the expression of cytokines and adhesion molecules, NF-kappaB DNA binding and transcriptional activity as well as its impact on the expression of TGF-beta1 in EA.hy.926 endothelial cells were analysed subsequently to low-dose radiotherapy.
MATERIALS AND METHODS: Human EA.hy.926 endothelial cells were grown to subconfluence. Twenty hours after X-irradiation with single doses ranging from 0.3 to 3 Gy, the cells were activated with tumour necrosis factor-alpha. Four hours later, the cells were harvested. NF-kappaB DNA-binding activity of nuclear extracts was analysed by electrophoretic mobility shift assay. The NF-kappaB subunits p50, p65/RelA, c-Rel and RelB of the NF-kappaB complexes were quantified by enzyme-linked immunoabsorbant assay. The transcriptional activity of NF-kappaB was measured using luciferase reporter gene assays in EA.hy.926 endothelial cells transiently transfected with the plasmid pB2xLuc. To correlate transcriptional activity to TGF-beta1 expression, NF-kappaB decoy oligonucleotides were used to inhibit NF-kappaB activity and TGF-beta1 secretion.
RESULTS: After low-dose radiotherapy, an increased NF-kappaB DNA-binding activity was observed in stimulated EA.hy.926 endothelial cells with a relative maximum (threefold induction) at 0.5 Gy. The NF-kappaB activation then decreased after X-irradiation at 0.6-0.8 Gy and subsequently increased again at doses of 1 and 3 Gy. This biphasic induction profile of NF-kappaB was confirmed by the analysis of the NF-kappaB-specific transcriptional activity. The latter showed a relative maximum at 0.5 Gy, a relative minimum between 0.5 and 1.0 Gy, and an increase at 3 Gy. Transfection of EA.hy.926 endothelial cells with NF-kappaB decoy oligonucleotides before irradiation resulted in a 50% reduction of TGF-beta1 secretion at 0.5 Gy compared with control oligonucleotides or untreated cells.
CONCLUSIONS: Low-dose radiotherapy induces a biphasic activation of NF-kappaB with a relative maximum at 0.5 Gy. The induction by NF-kappaB of TGF-beta1 in endothelial cells might contribute to the anti-inflammatory properties of low-dose ionizing irradiation.