McCahill Angela, Campbell Lachlan, McSorley Theresa, Sood Arvind, Lynch Martin J, Li Xiang, Yan Chen, Baillie George S, Houslay Miles D
Neuroscience and Molecular Pharmacology, Wolfson Link and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK.
Cell Signal. 2008 Nov;20(11):2071-83. doi: 10.1016/j.cellsig.2008.07.017. Epub 2008 Aug 5.
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.
磷酸二酯酶4A10（PDE4A10）环磷酸腺苷（cAMP）磷酸二酯酶同工型的转录本存在于包括心脏在内的多种大鼠组织中。对假定的人类和小鼠启动子进行序列比较，发现了许多保守区域，包括一个Sp1结合位点和一个CREB结合位点。从小鼠基因组DNA中扩增出假定的小鼠PDE4A10启动子，并亚克隆到荧光素酶报告载体中，以研究其在新生心肌细胞中的活性。用该构建体转染后，在新生心肌细胞中鉴定出高水平的荧光素酶表达。令人惊讶的是，通过涉及蛋白激酶A（PKA）而非交换蛋白直接激活cAMP（EPAC）信号传导的过程，细胞内cAMP升高会下调这种活性。啮齿动物PDE4A10启动子活性对cAMP水平升高的这种抑制作用与迄今为止描述的PDE4启动子形成对比。定点诱变显示，启动子位置-348至-336处的Sp1结合位点负责小鼠PDE4A10的基础组成型表达。位置-370至-363处保守的CREB结合基序也有助于基础启动子活性，但本身并不会赋予PDE4A10启动子cAMP抑制作用。电泳迁移率变动分析（EMSA）证实了CREB和Sp1结合位点的真实性。转录起始位点被确定为小鼠PDE4A10启动子中-55位的腺嘌呤残基。我们提供的证据表明，PDE4A10的这种新型下调是由转录因子诱导型cAMP早期抑制因子（ICER）以PKA依赖的方式介导的。下调PDE4A10的心肌细胞中的cAMP池受β-肾上腺素能受体偶联的腺苷酸环化酶活性调节，并通过水解作用主要由磷酸二酯酶4（PDE4，cAMP磷酸二酯酶-4）而非磷酸二酯酶3（PDE3，cAMP磷酸二酯酶-3）的作用决定。我们认为，cAMP增加可能不仅通过增加特定PDE4 cAMP磷酸二酯酶的表达，还通过下调特定同工型来重塑cAMP介导的信号事件，如此处显示的心肌细胞中的PDE4A10。
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