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.2007 Jul 1;405(1):131-7.
doi: 10.1042/BJ20061754.

PPARalpha and AP-2alpha regulate bombesin receptor subtype 3 expression in ozone-stressed bronchial epithelial cells

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PPARalpha and AP-2alpha regulate bombesin receptor subtype 3 expression in ozone-stressed bronchial epithelial cells

Yu-rong Tan et al. Biochem J..

Abstract

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.

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Figures

Figure 1
Figure 1. Screen of transcription factors binding to the humanBRS-3 promoter region
(A) Depicted is the 781 bp DNA sequence of the humanBRS-3 5′-FR. The translation start codon ATG is in bold and underlined. Probes 1–10 used for EMSA are indicated by underlined sequences. Transverse lines represent possible nuclear-factor-binding sites in corresponding probes. The shaded residues represent the mutated sites in different mutated probes, which are listed alphabetically. (B) Specific DNA–protein complex binding to the humanBRS-3 promoter was demonstrated using probes 1, 4, 8 and 10 as competition with unlabelled primer significantly reduced binding intensity. (C) Transcription factors MTF-1, PPARα, AP-2α and HSF-1 were identified as binding proteins to these four specific DNA sequences with EMSAs and supershift assays.
Figure 2
Figure 2. ChIP of four nuclear factors bound to the humanBRS-3 gene
Cross-linked chromatin prepared from resting BECs or ozone-stressed BECs was immunoprecipitated with four kinds of specific antibodies or non-immune IgG as indicated.
Figure 3
Figure 3. Inhibition of transcription factors and humanBRS-3 expression subsequent to ASO treatment
(A) The protein expression levels of MTF-1, PPARα, AP-2α and HSF-1 in ozone-stressed BECs were assessed by Western blotting (left-hand panel) and the DNA-binding activity by EMSA (right-hand panel) with and without ASO treatment. NSO, nonsense oligonucleotide. (B) HumanBRS-3 mRNA expression was significantly induced by ozone exposure. The inducible expression was substantially reduced after ASO treatment specifically targeting PPARα and AP-2α. Results are means±S.D. for four experiments. **P<0.01 compared with resting BEC group;##P<0.01 compared with ozone-stressed BEC group without ASO treatment.
Figure 4
Figure 4. Transcriptional activation of the humanBRS-3 promoter–luciferase reporter in BECs (A) and HLFs (B)
Results are means±S.D. relative luciferase activities (normalized to β-gal) from at least five independent transient transfection experiments each performed in triplicate. Mutations BA, BP and BP+BA, but not BM and BH, of the pGL3/FR/luc reporter resulted in reduction of ozone-inducedBRS-3 promoter activation (**P<0.01 compared with pGL3/FR/luc under resting conditions;##P<0.01 compared with wild-type pGL3/FR/luc under ozone-stressed conditions).
Figure 5
Figure 5. Time course of humanBRS-3 mRNA expression and DNA–protein binding activities of AP-2α and PPARα
(A) The time course of AP-2α and PPARα DNA binding toBRS-3 promoter sequence under ozone stress was determined by EMSA. (B) Expression of humanBRS-3 mRNA was assayed by real-time PCR. Results are means±S.D. for four independent experiments. (C) Nuclear translocation of PPARα after ozone exposure in BECs was demonstrated by immunofluorescence. Magnification × 400.
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