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D E Hallahan1, S Virudachalam, J Kuchibhotla
1Department of Radiation Oncology, Vanderbilt University, Nashville, Tennessee 37232-5671, USA. dennis.hallahan@mcmail.vanderbilt.edu
Abstract:
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.
Insights
Inhibiting nuclear factor kappa B (NFκB) can prevent X-ray-induced expression of cell adhesion molecules (CAMs) like E-selectin and ICAM-1, potentially reducing radiation injury in normal tissues.
Area of Science:
- Radiation Biology
- Molecular Biology
- Immunology
Background:
- X-ray exposure can trigger inflammatory responses in normal tissues, contributing to radiation injury.
- Radiation-inducible inflammatory mediators, such as E-selectin and ICAM-1, are implicated in this process.
- Nuclear factor kappa B (NFκB) activation by X-rays suggests its role in regulating these inflammatory mediators.
Purpose of the Study:
- To investigate whether inhibiting NFκB can abrogate the X-ray-induced expression of inflammatory mediators, specifically E-selectin and ICAM-1.
- To evaluate the efficacy of different NFκB inhibitory drugs and a dominant-negative genetic construct in blocking radiation-induced CAM expression.
Main Methods:
- Human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells were treated with NFκB inhibitors (ALLN, PDTC, NAC, MG132) and then irradiated.
- E-selectin and ICAM-1 expression was measured using immunofluorescence and fluorescence-activated cell-sorting (FACS).
- NFκB's role in transcriptional regulation was assessed using promoter-reporter constructs for ICAM-1 and E-selectin, cotransfected with a dominant-negative NFκB genetic construct.
Main Results:
- Irradiation significantly increased E-selectin (7-fold) and ICAM-1 (4-fold) expression in endothelial cells.
- Most NFκB inhibitors attenuated radiation-induced E-selectin expression; ALLN and MG132 also reduced ICAM-1 expression.
- A dominant-negative NFκB construct abolished X-ray-induced transcriptional activation of both E-selectin and ICAM-1 promoters.
Conclusions:
- NFκB plays a critical role in the transcriptional regulation of X-ray-induced E-selectin and ICAM-1 expression.
- Inhibition of NFκB is an effective strategy to abrogate radiation-induced expression of cell adhesion molecules (CAMs).
- Targeting NFκB may offer a therapeutic approach to mitigate radiation injury in normal tissues.
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