Sustained elevation of NF-kappaB DNA binding activity in radiation-induced lung damage in rats.
OBJECTIVE
To characterize the cellular distribution and DNA binding activity of the nuclear factor kappaB (NF-KappaB) in a model of radiation-induced lung damage in the rat.


METHODS
The right lung of Fischer rats was irradiated with a single dose of 20 Gy. The cellular distributions of NF-KappaB proteins and mRNA were detected with immunohistochemistry and in-situ hybridization respectively. The DNA binding activity of NF-KappaB, nuclear and cytoplasmic levels of NF-KappaB proteins, and kinase activity towards IkappaBalpha (IKappaBAlpha) were determined using electrophoretic mobility shift assays (EMSA), Western blots and kinase assays, respectively. The mRNA level of interleukin 6 (IL-6) was determined using quantitative room temperature polymerase chain reaction.


RESULTS
There was a continuous elevation of NF-KappaB DNA binding activity in the rat lung after ionizing irradiation over 6 months. The irradiated lung tissue exhibited an increased kinase activity towards IKappaBAlpha and a selective loss of nuclear IKappaBAlpha. The NF-KappaB-DNA binding complex switched from p50-p65 heterodimers in normal lung tissue to p50 homodimers in irradiated lung tissue. The increased level of IL-6 mRNA suggests transcriptional activation of NF-KappaB-dependent genes in the irradiated rat lung.


CONCLUSIONS
The DNA binding activity of NF-KappaB is continuously activated after irradiation of the rat lung by loss of nuclear IKappaBAlpha. This might play a role in sustaining chronic inflammation and hyperproliferation of mesenchymal cells after irradiation.
Haase
M G
MG
Department of Pathology, Carl Gustav Caruis Medical Faculty, Dresden University of Technology, Germany. michael.haase@mailbox.tu-dresden.de
Klawitter
A
A
Geyer
P
P
Alheit
H
H
Baumann
M
M
Kriegel
T M
TM
Kasper
M
M
Baretton
G B
GB
D000818:Animals; D015153:Blotting, Western; D001980:Bronchi / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D005260:Female; D005355:Fibrosis; D005822:Genetic Vectors; D020672:I-kappa B Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D015850:Interleukin-6 / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000528:radiation effects* / Q000648:ultrastructure; D055370:Lung Injury; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000096:biosynthesis / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding; D011650:Pulmonary Alveoli / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D051996:Transcription Factor RelA
D020672:I-kappa B Proteins; D015850:Interleukin-6; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606206:Nfkbia protein, rat; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA