Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are, differentially expressed and regulated in rat tissue
Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are, differentially expressed and regulated in rat tissue
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DOI：
10.1042/bj20050030
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发表时间：
2005-06-15
影响因子：
4.1
通讯作者：
Conti, M
中科院分区：
文献类型：
--
作者：
Richter, W;Jin, SLC;Conti, M
环核苷酸PDE4(磷酸二酯酶4)抑制剂正被开发为用于慢性肺部疾病的有效抗炎药物，但PDE4家族的复杂性阻碍了这一过程。PDE4家族的四个基因PDE4A、PDE4B、PDE4C和PDE4D均以多种剪接变异体的形式表达。鉴定内源性表达的PDE4变异体最常用的标准是它们在SDS/PAGE上的迁移。然而，当PDE4D3选择性抗体用于免疫沉淀时，获得的表达模式并不证实SDS/PAGE预测的表达。这一观察结果，加上最近发现的其他PDE4D转录本，促使我们重新评估这些变体的表达模式。克隆了9个大鼠PDE4D剪接变异体PDE4D1至PDE4D9，比较了它们的电泳性，并测定了它们在体内的mRNA和蛋白质水平。使用这种方法，我们发现PDE4D剪接变异体的分布模式比以前报道的更复杂。多个变异体在单个免疫反应条带中共同迁移，而变异体选择性抗体是区分剪接变异体所必需的。被认为只表达PDE4D3的组织表达三种密切相关的蛋白，其中PDE4D8和PDE4D9是主要的表达形式。此外，cAMP信号的激活会导致除PDE4D3以外的其他变异体的磷酸化和激活，而PDE4D mRNA的表达并不总是与蛋白质表达模式相关。由于PDE4抑制剂对不同的PDE4D剪接变体有不同的亲和力，我们的结果表明，需要更好地定义PDE4的表达模式才能进行靶标验证。
Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.