Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κβ in airway smooth muscle cells.
BACKGROUND
Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling.


OBJECTIVE
We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms.


METHODS
HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids.


RESULTS
IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4.


CONCLUSIONS
These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.
Matsukura
S
S
First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, Japan. smatsuku@med.showa-u.ac.jp
Odaka
M
M
Kurokawa
M
M
Kuga
H
H
Homma
T
T
Takeuchi
H
H
Notomi
K
K
Kokubu
F
F
Kawaguchi
M
M
Schleimer
R P
RP
Johnson
M W
MW
Adachi
M
M
D001249:Asthma / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D002460:Cell Line; D054413:Chemokine CCL11 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D018793:Interleukin-13 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000494:pharmacology; D018418:Th2 Cells / Q000276:immunology; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
D054413:Chemokine CCL11; D018793:Interleukin-13; D016328:NF-kappa B; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D015847:Interleukin-4