Double-stranded RNA activates RANTES gene transcription through co-operation of nuclear factor-kappaB and interferon regulatory factors in human airway epithelial cells.
BACKGROUND
Regulated on activation, normal T cells expressed and secreted (RANTES) is a member of the CC chemokine family and contributes to viral-induced airway inflammation including exacerbations of asthma. Double-stranded RNA (dsRNA) is known to be synthesized during replication of many viruses and a ligand of Toll-like receptor 3. We hypothesized that dsRNA may mimic viral infection and induce RANTES expression in airway epithelial cells.


OBJECTIVE
We first confirmed that dsRNA up-regulated RANTES mRNA and protein synthesis in the airway epithelial cells. We next focused our studies on the transcriptional regulation of RANTES.


METHODS
Airway epithelial cell line BEAS-2B and normal human bronchial epithelial cells were used in vitro study. Levels of RANTES mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by electrophoretic mobility shift assay and dual luciferase assay using RANTES promoter-luciferase reporter plasmids.


RESULTS
Activation of nuclear factor-kappaB (NF-kappaB) was confirmed by nuclear protein binding to a DNA probe derived from the RANTES promoter. Activity of the RANTES promoter was increased by dsRNA. The stimulation with dsRNA was partially inhibited in plasmids mutated at either of the binding sites for NF-kappaB or IFN regulatory factors (IRFs). When both sites were mutated, the activation was totally abrogated.


CONCLUSIONS
These results imply that dsRNA activates NF-kappaB and IRFs and these transcription factors activate transcription of the RANTES promoter and its protein expression in airway epithelial cells.
Ieki
K
K
First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan.
Matsukura
S
S
Kokubu
F
F
Kimura
T
T
Kuga
H
H
Kawaguchi
M
M
Odaka
M
M
Suzuki
S
S
Watanabe
S
S
Takeuchi
H
H
Schleimer
R P
RP
Adachi
M
M
D001980:Bronchi / Q000378:metabolism*; D002460:Cell Line; D018946:Chemokine CCL5 / Q000032:analysis / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007372:Interferons / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D014158:Transcription, Genetic
D018946:Chemokine CCL5; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D007372:Interferons