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

• Published in: The Journal of biological chemistry Vol. 277; no. 39; pp. 35980 - 35989
• Main Authors: Le Jeune, Ivan R, Shepherd, Malcolm, Van Heeke, Gino, Houslay, Miles D, Hall, Ian P
• Format: Journal Article
• Language: English
• Published: United States 27.09.2002
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; 8-Bromo Cyclic Adenosine Monophosphate - metabolism; Alternative Splicing; Animals; Base Sequence; CHO Cells; Chromosomes, Human, Pair 5; Cricetinae; Culture Media, Serum-Free - pharmacology; Cyclic AMP - metabolism; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Down-Regulation; Genes, Reporter; Humans; Kinetics; Luciferases - metabolism; Models, Genetic; Molecular Sequence Data; Muscle, Smooth - cytology; Mutagenesis, Site-Directed; Phosphoric Diester Hydrolases - biosynthesis; Phosphoric Diester Hydrolases - chemistry; Precipitin Tests; Promoter Regions, Genetic; Protein Isoforms; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Trachea - cytology; Transcription, Genetic; Transfection; Up-Regulation
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M204832200

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.