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

我们的小组研究了小鼠模型中胎盘发育的遗传和细胞基础。我们发现，TMED 2，一种跨膜蛋白，参与蛋白质在ER和高尔基体之间的顺行运输，表达和正常胎盘发育所需。在Tmed 2纯合突变体（Tmed 2-/-）胚胎中，功能性迷路层未能形成。此外，在Tmed 2-/-胚胎中没有发现迷路层的功能和分化所需的合胞体滋养层细胞。TMED 2在人类中是保守的，并且在细胞滋养层细胞和合体滋养层中表达。为了表征TMED 2在合体滋养层分化中的作用，我们转向体内人滋养层细胞培养模型。我们发现，TMED 2 mRNA和蛋白质水平较高的融合感受态BeWo细胞系相比，非融合Jeg-3细胞系。我们产生了具有TMED 2敲低的稳定BeWo细胞和具有TMED 2的可诱导过表达的稳定JEG-3细胞。我们发现TMED 2敲低的BeWo细胞中合体滋养层形成显著减少，而表达TMED 2的JEG-3细胞中合体滋养层形成显著减少。我们假设TMED 2是一个关键因素，运输一个或多个蛋白质所需的合体滋养层形成。这项建议的目的是： 1.为了确定合胞体滋养层分化中Tmed 2的组织和细胞类型特异性需求-TMED 2在绒毛膜和尿囊中表达，其附着形成迷路层。我们将产生在Tmed 2中具有条件突变的小鼠，以便将其在尿囊或绒毛膜中扩增。组织特异性基因敲除中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胞体滋养层形成的TMED 2货物-候选方法和无偏蛋白质组学筛选将用于鉴定具有TMED 2诱导表达的JEG 3细胞、具有TMED 2敲低的BeWo细胞和Tmed 2突变组织中的TMED 2货物。将通过蛋白质印迹分析、免疫荧光和免疫共沉淀来检查TMED 2货物的表达和定位。ER和高尔基体组分的蛋白质组学将用于鉴定异常转运蛋白。 3.为了鉴定调节合胞体滋养层形成的TMED 2货物蛋白-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守的经验证的新型TMED 2货物蛋白。shRNA将用于敲低滋养层细胞中的特定货物。将通过透射电子显微镜和组织学分析具有TMED 2货物蛋白突变的胚胎胎盘的迷路层异常。 拟议的研究将提供新的见解TMED 2在合体滋养层形成的作用。

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.