Regulation of human PDE5A2 intronic promoter by cAMP and cGMP: identification of a critical Sp1-binding site.
PDE5A gene encodes type 5 phosphodiesterase (PDE5), the principal cGMP-catalyzing enzyme in the penis and the primary target of sildenafil (Viagra). We have previously reported the isolation of three alternatively spliced PDE5A isoforms in humans. We also reported the identification of three corresponding alternative first exons and an intronic promoter in the human PDE5A gene. The intronic promoter is situated upstream from the PDE5A2-specific first exon but downstream from the PDE5A1- and A3-specific first exons. In the current study we showed that the intronic promoter could be upregulated by either cAMP or cGMP. In order to identify possible regulatory elements in the promoter, we created deletion and base-substitution mutants targeting one AP2- and four Sp1-binding sequences. Loss of function of these mutants to bind to the respective transcription factors was verified by DNase I footprint analysis, and changes in promoter function were analyzed with a luciferase reporter system. Mutation of the AP2-binding sequence and deletion of the 3'-most Sp1-binding site (within the exon) had little effects on the basal or the cyclic nucleotide-inducible promoter functions. Mutation of the 5'-most Sp1-binding site had much more severe effects on the basal and the cyclic nucleotide-inducible promoter functions. Mutation of a neighboring site that contains two overlapping Sp1-binding sequences completely nullified the basal and cyclic nucleotide-inducible promoter activities. Thus, the PDE5A2 intronic promoter depends on the overlapping Sp1-binding site for basal and cyclic nucleotide-inducible functions.
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Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-1695, USA.
Chow
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Lau
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000235:genetics*; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011687:Purines; D000068677:Sildenafil Citrate; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013450:Sulfones; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D016329:Sp1 Transcription Factor; D013450:Sulfones; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000068677:Sildenafil Citrate; D000242:Cyclic AMP; D008156:Luciferases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP