Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter.
Phosphodiesterase 4D (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.
Division of Therapeutics and Institute of Cell Signalling, University Hospital, Nottingham NG7 2UH, United Kingdom.
Ivan R
IR
Le Jeune
Malcolm
M
Shepherd
Gino
G
Van Heeke
Miles D
MD
Houslay
Ian P
IP
Hall
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002895:Chromosomes, Human, Pair 5; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015536:Down-Regulation; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014132:Trachea / Q000166:cytology*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D016895:Culture Media, Serum-Free; D020033:Protein Isoforms; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D008156:Luciferases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human