Knöfler Martin, Saleh Leila, Bauer Sandra, Galos Barbara, Rotheneder Hans, Husslein Peter, Helmer Hanns
Department of Obstetrics and Gynecology, University of Vienna, Austria.
Endocrinology. 2004 Apr;145(4):1685-94. doi: 10.1210/en.2003-0954. Epub 2004 Jan 8.
Expression of the trophoblast-specific subunit of human chorionic gonadotropin, CGbeta, is associated with fusion of cytotrophoblasts into a multinuclear syncytium. Here, we studied regulation of the CGbeta5 gene in trophoblasts undergoing in vitro syncytialization. Transfection of luciferase reporters harboring different lengths of the CGbeta5 upstream sequence revealed that the proximal promoter region (-345 to +114) is sufficient to govern differentiation-dependent induction. Mutational analyses suggested that two selective promoter factor (Sp) and three activating protein 2 (AP-2) recognition sequences are necessary for full activity of the promoter. During syncytialization these elements interacted with increasing amounts of the transcription factors Sp1, Sp3, and AP-2alpha in electrophoretic mobility shift assay, but only AP-2alpha binding rose upon elevation of cAMP levels with forskolin. Increasing expression of different isoforms of Sp1 and Sp3 could also be detected by Western blot analyses. Sp1/Sp3 localized to syncytial nuclei both in differentiated cultures and in term placental tissue, suggesting assembly of functional transcriptional complexes. Costaining of the transcription factors with E-cadherin on term placental sections revealed that 47 and 33% of cytotrophoblast nuclei were negative for Sp1 and Sp3, respectively. In contrast, immunohistochemistry of early tissue demonstrated expression of Sp1 in the majority of cyto- and syncytiotrophoblasts, whereas Sp3 was absent from the syncytium. Sp1 and Sp3 induced wild-type/mutant promoter constructs upon transfection in Sp-deficient SL-2 cells, indicating that the Sp elements function as activating sequences. The data suggest that increasing concentrations of Sp1, Sp3, and AP-2alpha enhance transcription of CGbeta in differentiating term trophoblasts, whereas a different combination of factors may control expression in early placentas.
人绒毛膜促性腺激素滋养层特异性亚基CGβ的表达与细胞滋养层融合形成多核合体滋养层有关。在此，我们研究了体外合体化的滋养层中CGβ5基因的调控。转染携带不同长度CGβ5上游序列的荧光素酶报告基因表明，近端启动子区域（-345至+114）足以调控分化依赖性诱导。突变分析表明，两个选择性启动子因子（Sp）和三个激活蛋白2（AP-2）识别序列是启动子充分活性所必需的。在合体化过程中，这些元件在电泳迁移率变动分析中与转录因子Sp1、Sp3和AP-2α的结合量增加，但只有AP-2α的结合在福斯可林升高cAMP水平时增加。通过蛋白质免疫印迹分析也可检测到Sp1和Sp3不同异构体表达的增加。Sp1/Sp3在分化培养物和足月胎盘组织的合体细胞核中均有定位，提示功能性转录复合物的组装。足月胎盘切片上转录因子与E-钙黏蛋白的共染色显示，分别有47%和33%的细胞滋养层细胞核Sp1和Sp3呈阴性。相反，早期组织的免疫组织化学显示，大多数细胞滋养层细胞和合体滋养层细胞中均有Sp1表达，而合体滋养层中不存在Sp3。Sp1和Sp3转染Sp缺陷型SL-2细胞后可诱导野生型/突变型启动子构建体，表明Sp元件作为激活序列发挥作用。数据表明，Sp1、Sp3和AP-2α浓度的增加可增强足月分化滋养层中CGβ的转录，而不同的因子组合可能控制早期胎盘的表达。
