Department of Obstetrics and Fetal-Maternal Medicine (S.H., G.M., P.V., G.R.O., J.P., M.K.), Reproductive Biology Unit, Medical University of Vienna, A-1090 Vienna, Austria; Division of Biomedical Sciences (G.W.), St Georges's University of London, Londo SW17 0RE, United Kingdom; and Gynmed Clinic (C.F.), A-1150 Vienna, Austria.
Endocrinology. 2014 Jan;155(1):263-74. doi: 10.1210/en.2013-1455. Epub 2013 Dec 20.
Failures in human extravillous trophoblast (EVT) development could be involved in the pathogenesis of pregnancy diseases. However, the underlying mechanisms have been poorly characterized. Here, we provide evidence that Notch signaling could represent a key regulatory pathway controlling trophoblast proliferation, motility, and differentiation. Immunofluorescence of first-trimester placental tissues revealed expression of Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand [DLL] 1 and -4 and Jagged [JAG] 1 and -2) in villous cytotrophoblasts (vCTBs), cell column trophoblasts (CCTs), and EVTs. Notch4 and Notch1 were exclusively expressed in vCTBs and in CCTs, respectively. Both proteins decreased in Western blot analyses of first-trimester, primary cytotrophoblasts (CTBs) differentiating on fibronectin. Luciferase reporter analyses suggested basal, canonical Notch activity in SGHPL-5 cells and primary cells that was increased upon seeding on DLL4-coated dishes and diminished in the presence of the Notch/γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or L-685,458. Bromodeoxyuridine labeling, cyclin D1 mRNA expression, and cell counting indicated that chemical inhibition of Notch signaling elevated proliferation in the different primary trophoblast model systems. Notch inhibition also increased motility of SGHPL-5 cells through uncoated and fibronectin-coated Transwells, motility of primary CTBs, as well as migration in villous explant cultures on collagen I. Accordingly, small interfering RNA-mediated gene silencing of Notch1 also elevated SGHPL-5 cell migration. In contrast, motility of primary cultures and SGHPL-5 cells was diminished in the presence of DLL4. Moreover, DAPT increased markers of differentiated EVT, ie, human leukocyte antigen G1, integrin α5, and T-cell factor 4, whereas DLL4 provoked the opposite. In summary, the data suggest that canonical Notch signaling impairs motility and differentiation of first-trimester CTBs.
人类绒毛外滋养细胞 (EVT) 发育失败可能与妊娠疾病的发病机制有关。然而,其潜在机制尚未得到充分描述。在这里,我们提供的证据表明,Notch 信号可能代表控制滋养细胞增殖、运动和分化的关键调节途径。对早孕期胎盘组织的免疫荧光显示,Notch 受体(Notch2 和 Notch3)和膜锚定配体(Delta-like 配体 [DLL]1 和-4 以及 Jagged [JAG]1 和-2)在绒毛细胞滋养细胞(vCTB)、细胞柱滋养细胞(CCT)和 EVT 中表达。Notch4 和 Notch1 分别仅在 vCTB 和 CCT 中表达。在纤维连接蛋白上分化的早孕期原代细胞滋养细胞 (CTB) 的 Western blot 分析中,两种蛋白的表达均减少。荧光素酶报告分析表明,SGHPL-5 细胞和原代细胞中存在基础的、经典的 Notch 活性,在接种 DLL4 包被的培养皿后增加,而在存在 Notch/γ-分泌酶抑制剂 N-[N-(3,5-二氟苯乙酰基)-L-苯丙氨酸]-S-苯甘氨酸叔丁酯(DAPT)或 L-685,458 时减少。溴脱氧尿苷标记、cyclin D1 mRNA 表达和细胞计数表明,Notch 信号的化学抑制可增加不同原代滋养细胞模型系统中的增殖。Notch 抑制还通过未包被和纤维连接蛋白包被的 Transwell 增加 SGHPL-5 细胞的运动性、原代 CTB 的运动性以及在胶原 I 上的绒毛外植体培养中的迁移。因此,Notch1 的小干扰 RNA 介导的基因沉默也增加了 SGHPL-5 细胞的迁移。相反,在 DLL4 存在下,原代培养物和 SGHPL-5 细胞的运动性降低。此外,DAPT 增加了分化的 EVT 标志物,即人类白细胞抗原 G1、整合素 α5 和 T 细胞因子 4,而 DLL4 则引起相反的作用。总之,数据表明,经典的 Notch 信号会损害早孕期 CTB 的运动性和分化。