Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells -: Identification and characterization of a novel PDE4D5 promoter

被引:82
作者
Le Jeune, IR
Shepherd, M
Van Heeke, G
Houslay, MD
Hall, IP
机构
[1] Univ Nottingham Hosp, Div Therapeut, Nottingham NG7 2UH, England
[2] Univ Nottingham Hosp, Inst Cell Signalling, Nottingham NG7 2UH, England
[3] Gartnavel Royal Hosp, Dept Resp Med, Glasgow G12, Lanark, Scotland
[4] Univ Glasgow, Mol Pharmacol Grp, Div Biochem & Mol Biol, Inst Biomed & Life Sci, Glasgow G12 8QQ, Lanark, Scotland
[5] Novartis Horsham Res Ctr, Horsham RH12 5AB, W Sussex, England
关键词
D O I
10.1074/jbc.M204832200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phosphodiesterase 41) (PDE4D), part of the complex cAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation by catalyzing the hydolysis of cAMP. Its gene on chromosome 5q12 encodes 5 splice variants, which show tissue-dependent expression and regulation. The genomic arrangement of PDE4D was determined using in silico methods, and a putative promoter of one of the protein kinase A-activated, long isoforms, PDE4D5 was identified. Promoter-luciferase constructs, transiently transfected into a beta(2) adrenoreceptor-expressing CHO-K1 cell line, were used to demonstrate that the PDE4D5 promoter up-regulated reporter gene expression in response to increased cell cAMP. Site-directed mutagenesis of the cAMP-response element (CRE) at position -201 identified this as the principal component of the mechanism underlying this cAMP responsiveness. In the second part of this study, cAMP-dependent induction of PDE4D5 transcript in primary cultured human airway smooth muscle cells (hASMs) was demonstrated using both qualitative reverse-transcriptase PCR and quantitative real-time PCR. Isolated PDE4D5 isoenzyme activity, measured after selective immunoprecipitation from hASMs, confirmed that this increase in expression led to an up-regulation of functional activity. We present evidence for cAMP-driven PDE4D5 up-regulation in hASMs and suggest a CRE-containing, isoform-specific promoter as the primary mechanism.
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页码:35980 / 35989
页数:10
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