PPARalpha and AP-2alpha regulate bombesin receptor subtype 3 expression in ozone-stressed bronchial epithelial cells.
Previously, we found that bombesin receptor subtype 3 (BRS-3) significantly increased in an ozone-stressed airway hyperresponsiveness animal model and resulted in induced wound repair and protection from acute lung injury. In the present study, we determined molecular mechanisms of BRS-3 regulation in human BECs (bronchial epithelial cells) in response to ozone stress. Ten oligonucleotide probes corresponding to various regions of the BRS-3 promoter were used in EMSA (electrophoretic mobilityshift assays). Four were found to have an enhanced mobility shift with extracts from ozone-stressed cells. On the basis of the assay of mutated probes binding with extracts and antibody supershift, they were verified as MTF-1 (metal-regulatory-element-binding transcription factor-1), PPARalpha (peroxisome-proliferator-activated receptor alpha), AP-2alpha (activator protein 2alpha) and HSF-1 (heat-shock factor 1). Next, ChIP (chromatin immunoprecipitation) assay, site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe these transcription factors associated with the BRS-3 promoter. Only AP-2alpha and PPARalpha increased ozone-inducible DNA binding on the BRS-3 promoter and BRS-3 expression. The time courses of AP-2alpha and PPARalpha activation, followed by BRS-3 expression, were also examined. It was shown that ozone-inducible BRS-3 expression and AP-2alpha- and PPARalpha-binding activity correlated over a 48 h period. The translocation of PPARalpha was observed by immunofluorescence assay, which showed that PPARalpha nuclear translocation increased after ozone exposure. Our data suggest that AP-2alpha and PPARalpha may be especially involved in this ozone-inducible up-regulation mechanism of BRS-3 expression.
Tan
Yu-rong
YR
Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan, China.
Qin
Xiao-qun
XQ
Xiang
Yang
Y
Yang
Tao
T
Qu
Fei
F
Wang
Yue
Y
Liu
Hui-jun
HJ
Weber
H Christian
HC
D000818:Animals; D001483:Base Sequence; D001980:Bronchi / Q000166:cytology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010083:Oxidants, Photochemical / Q000494:pharmacology*; D010126:Ozone / Q000494:pharmacology*; D047493:PPAR alpha / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018004:Receptors, Bombesin / Q000235:genetics / Q000378:metabolism*; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D010083:Oxidants, Photochemical; D047493:PPAR alpha; D018004:Receptors, Bombesin; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C107120:bombesin receptor subtype 3; D010126:Ozone