Holmberg M, Leonardsson G, Ny T
Department of Medical Biochemistry and Biophysics, Umeå Unviersity, Sweden.
Eur J Biochem. 1995 Jul 15;231(2):466-74. doi: 10.1111/j.1432-1033.1995.tb20720.x.
In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.
在大鼠卵巢细胞中，促性腺激素通过cAMP依赖途径诱导组织型纤溶酶原激活剂（tPA），且这种诱导与体内卵泡破裂时间相关。然而，在小鼠中，促性腺激素诱导的是相关但不同的蛋白酶——尿激酶型纤溶酶原激活剂（uPA）。对大鼠、小鼠和人类tPA基因的比较显示，启动子存在物种特异性差异，这可以解释这些物种之间tPA基因调控的差异。在大鼠启动子含有共有cAMP反应元件（CRE）的位置，小鼠和人类的对应序列含有一个单核苷酸取代的CRE变体。大鼠神经胶质细胞和颗粒细胞的瞬时转染实验表明，由大鼠tPA启动子而非小鼠或人类tPA启动子驱动的报告基因构建体可被cAMP诱导剂福斯可林或促卵泡激素有效诱导。将小鼠和人类类似CRE的序列转化为大鼠共有CRE后，这些启动子变得对cAMP有反应。相反，将大鼠共有CRE转化为相应的小鼠和人类类似CRE序列后，大鼠启动子失去了对cAMP有效反应的能力。利用脱氧核糖核酸酶I足迹分析和电泳迁移率变动分析来检测核因子与共有CRE和变体CRE的相互作用。与大鼠CRE相比，小鼠和人类类似CRE的序列对被鉴定为cAMP反应元件结合蛋白的核因子的结合亲和力大幅降低。因此，小鼠和人类tPA启动子不能有效响应福斯可林和促卵泡激素，似乎是由于这些类似CRE的序列不能有效结合转录因子CRE结合蛋白。
