Molecular mechanism of placental development: The role of TMED2, a vesicular transport protein during syncytiotrophoblast formation

本课题组在小鼠模型中研究胎盘发育的遗传和细胞基础。我们发现TMED2是一种参与内质网和高尔基体之间蛋白质顺行转运的跨膜蛋白，它是胎盘正常发育所必需的。在Tmed2纯合突变体（Tmed2-/-）胚胎中，功能迷路层未能形成。此外，在Tmed2-/-胚胎中没有发现迷宫层功能和分化所需的合胞滋养细胞。TMED2在人体内是保守的，在细胞滋养层细胞和合胞滋养层细胞中均有表达。为了表征TMED2在合胞滋养细胞分化中的作用，我们转向人体内滋养细胞培养模型。我们发现，与未融合的Jeg-3细胞系相比，具有融合能力的BeWo细胞系中TMED2 mRNA和蛋白水平更高。我们产生了稳定的BeWo细胞，TMED2被敲低，以及稳定的JEG-3细胞，诱导TMED2过表达。我们发现，在表达TMED2的BeWo细胞中，合体滋养细胞的形成明显减少，而在表达TMED2的JEG-3细胞中，合体滋养细胞的形成明显减少。我们假设TMED2是运输一种或多种合胞滋养细胞形成所需蛋白质的关键因子。本提案的目的是：******1。为了确定Tmed2在合胞滋养细胞分化中对组织和细胞类型的特异性需求- Tmed2在绒毛膜和尿囊中均有表达，它们附着形成迷宫层。我们将产生Tmed2条件突变的小鼠，以使其在尿囊或绒毛膜中失活。组织特异性敲除的胎盘异常将通过组织学，免疫组织化学和原位杂交来表征。******2。为了鉴定和表征参与合体营养细胞形成的TMED2货物-候选方法和无偏倚的蛋白质组学筛选将用于鉴定诱导表达TMED2的JEG 3细胞、敲低TMED2的BeWo细胞和TMED2突变组织中的TMED2货物。TMED2基因的表达和定位将通过免疫印迹分析、免疫荧光和共免疫沉淀进行检测。内质网和高尔基体部分的蛋白质组学将用于鉴定异常转运的蛋白质。******为了鉴定调节合胞滋养层形成的TMED2货运蛋白，将在滋养层细胞和小鼠突变胚胎中研究在人类和小鼠中保存的经过验证的新型TMED2货运蛋白。shRNA将被用来敲除滋养细胞中的特定货物。TMED2转运蛋白突变胚胎的胎盘将通过透射电镜和组织学分析迷宫层的异常。***拟议的研究将为TMED2在合胞滋养细胞形成中的作用提供新的见解

Our group investigates the genetic and cellular basis of placental development in the mouse model. We discovered that TMED2, a transmembrane protein involved in anterograde transport of proteins between the ER and Golgi, was expressed and required for normal placental development. In Tmed2 homozygous mutant (Tmed2-/-) embryos the functional labyrinth layer fails to form. Furthermore, syncytiotrophoblast cells required for function and differentiation of the labyrinth layer are not found in Tmed2-/- embryos. TMED2 is conserved in human and is expressed in both cytotrophoblast cells and in syncytiotrophoblast. To characterize the role of TMED2 in syncytiotrophoblast differentiation, we turned to in vivo human trophoblast cell culture models. We found that TMED2 mRNA and protein levels were higher in the fusion competent BeWo cell line when compared to the non-fusing Jeg-3 cell line. We generated stable BeWo cells with knockdown of TMED2, and stable JEG-3 cells with inducible over-expression of TMED2. We found significantly reduced syncytiotrophoblast formation in BeWo cells with knockdown of TMED2, and syncytiotrophoblast formation in JEG-3 cells expressing TMED2. We hypothesise that TMED2 is a key factor that transports one or more proteins required for syncytiotrophoblast formation. The Aims of this proposal are:******1. To determine the tissue and cell type-specific requirement for Tmed2 in syncytiotrophoblast differentiation - TMED2 is expressed in both the chorion and allantois, which attach to form the labyrinth layer. We will generate mice with conditional mutation in Tmed2 in order to inactivate it in the allantois or chorion. Placental abnormalities in tissue-specific knockouts will be characterized by histology, immunohistochemistry and in situ hybridization.******2. To identify and characterize the TMED2 cargo(es) involved in syncytiotrophoblast formation - Candidate approaches and unbiased proteomic screens will be used to identify TMED2 cargoes in JEG 3 cells with induced expression of TMED2, in BeWo cells with knockdown of TMED2, and in Tmed2 mutant tissues. Expression and localization of TMED2 cargoes will be examined by western blot analysis, immunofluorescence, and co-immunoprecipitation. Proteomics of ER and Golgi fractions will be used to identify abnormally transported proteins.******3. To identify TMED2 cargo proteins that regulate syncytiotrophoblast formation -Validated novel TMED2-cargo proteins conserved in human and mouse will be studied in trophoblast cells and mouse mutant embryos. shRNA will be use to knockdown the specific cargo in trophoblast cells. Placentas of embryos with mutation in TMED2 cargo proteins will be analyzed by transmission electron microscopy and histology for abnormalities in the labyrinth layer. ***The proposed studies will provide novel insights into the role of TMED2 in syncytiotrophoblast formation.**