In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10).
Transcripts for the PDE4A10 cyclic AMP phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was amplified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position -348 to -336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position -370 to -363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position -55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated by beta-adrenoceptor coupled adenylyl cyclase activity and via hydrolysis determined predominantly by the action of PDE4 (cAMP phosphodiesterase-4) and not PDE3 (cAMP phosphodiesterase-3). We suggest that increased cAMP may remodel cAMP-mediated signalling events by not only increasing the expression of specific PDE4 cAMP phosphodiesterases but also by down-regulating specific isoforms, such as is shown here for PDE4A10 in cardiac myocytes.
McCahill
Angela
A
Neuroscience and Molecular Pharmacology, Wolfson Link and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK.
Campbell
Lachlan
L
McSorley
Theresa
T
Sood
Arvind
A
Lynch
Martin J
MJ
Li
Xiang
X
Yan
Chen
C
Baillie
George S
GS
Houslay
Miles D
MD
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D005576:Colforsin / Q000494:pharmacology; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D051786:Cyclic AMP Response Element Modulator / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000235:genetics*; D004252:DNA Mutational Analysis; D015536:Down-Regulation / Q000187:drug effects*; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects
C497412:Crem protein, rat; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D051786:Cyclic AMP Response Element Modulator; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4