Comparative methodologic study of NF kappa B activation in cultured endothelial cells

The transcriptional regulatory protein nuclear factor ICE (NF kappaB) parti cipates in the control of gene expression of many modulators of the inflamm atory and immune responses. Various activators trigger NF kappaB release an d nuclear translocation after phosphorylation and proteolytic degradation o f I kappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shi ft assay (EMSA), and Western blotting to detect NF kappaB activation in end othelial cells (ECs) and to investigate the role of homocysteine (Hcy) in N F kappaB activation. ECs were treated with interleukin-1 beta (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NF kappaB activators. Hcy, a thiol-con taining amino acid, has been shown to directly damage ECs in vitro. Experim ental evidence suggests that the atherogenic propensity associated with hyp erhomocysteinemia results from EC dysfunction. When ECs were pretreated wit h an inhibitor (pyrrolidine dithiocarbamate, 100 mu mol/L) or with staurosp orine (5 muL/mL), no NF kappaB activation was observed. NF kappaB activatio n in ECs could be detected with all five techniques, clearly showing NF kap paB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference m ethod, has the disadvantages of being radioactive, expensive, and time cons uming. Western blot analysis detected the NF kappaB p50 subunit implicated in NF kappaB activation. The techniques usually used to detect NF kappaB ac tivation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.