Nuclear factor kappaB dominant negative genetic constructs inhibit X-ray induction of cell adhesion molecules in the vascular endothelium.
X-ray-induced expression of inflammatory mediators has been proposed to contribute to radiation injury in normal tissues. Radiation-inducible inflammatory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1. Nuclear factor (NF)kappaB is activated by X-rays and may participate in the transcriptional regulation of each of these inflammatory mediators. To determine whether NFkappaB inhibition abrogates X-ray induction of inflammatory mediators, we used two experimental approaches including NFkappaB inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells were treated with the NFkappaB inhibitors ALLN, PDTC, NAC, and MG132. After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and ICAM-1 expression was measured by use of immunofluorescence and fluorescence-activated cell-sorting analysis. E-selectin expression increased 7-fold, and ICAM-1 expression increased 4-fold after irradiation. All of the inhibitors attenuated E-selectin expression after irradiation. ALLN and MG132 attenuated radiation-induced ICAM expression. However, PDTC and NAC induced increased expression of ICAM-1 in HUVECs. Inhibition of X-ray induction of ICAM by these agents could not be demonstrated. In separate experiments, the NFkappaB dominant negative genetic construct was cotransfected with the promoter-reporter constructs by means of Lipofectin reagent. The ICAM promoter-reporter construct consists of the 1.2-kb segment of the human ICAM promoter upstream of the transcriptional start site linked to the luciferase reporter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-selectin promoter linked to the human growth hormone reporter gene (pE525-GH). Endothelial cells transfected with the ICAM-1 promoter-reporter construct showed a 3-fold induction after irradiation. Likewise, cells transfected with pE525-GH showed a 7-fold induction after irradiation. When cotransfected with the CAM reporter-promoter constructs, the NFkappaB dominant negative genetic construct abolished X-ray-induced transcriptional activation of the E-selectin and ICAM-1 promoters. NFkappaB inhibition is, therefore, a means of abrogating radiation-induced expression of CAMs.
Hallahan
D E
DE
Department of Radiation Oncology, Vanderbilt University, Nashville, Tennessee 37232-5671, USA. dennis.hallahan@mcmail.vanderbilt.edu
Virudachalam
S
S
Kuchibhotla
J
J
D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D019040:E-Selectin / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000196:embryology* / Q000528:radiation effects*; D005799:Genes, Dominant; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D012680:Sensitivity and Specificity; D015533:Transcriptional Activation / Q000528:radiation effects; D014965:X-Rays
D019040:E-Selectin; D016328:NF-kappa B; D018799:Intercellular Adhesion Molecule-1