Linton E A, Rodriguez-Linares B, Rashid-Doubell F, Ferguson D J P, Redman C W G
Nuffield Department of Obstetrics and Gynaecology, Women's Centre, University of Oxford, John Radcliffe Hospital, Headington, OX3 9DU, Oxford, UK.
Placenta. 2003 Aug;24(7):745-57. doi: 10.1016/s0143-4004(03)00106-1.
Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types, particularly endothelium. A major structural component is the membrane protein caveolin-1 which associates with numerous signalling molecules, including endothelial nitric oxide (eNOS). Caveolin-1, which co-immunoprecipitates with eNOS in preparations from endothelial cells, regulates eNOS activity, holding it inactive. Controversy now exists regarding the presence of caveolae and caveolin-1 in trophoblasts, hence this study was carried out to examine whether the high levels of eNOS expressed in human syncytiotrophoblast are associated with caveolin-1, and to find out if caveolae are present in villous cytotrophoblasts and syncytiotrophoblast. Immunohistochemistry of term placentae revealed only weak labelling for caveolin-1 in the syncytiotrophoblast although the endothelium of the terminal villus vessels stained strongly. By electron microscopy, numerous caveolae were identified in the villus capillary endothelium but were extremely rare in the syncytium. Caveolin-1 staining was extensive in purified, isolated term villous cytotrophoblasts, with the purity of these cytokeratin positive cells confirmed by cytospin analysis and flow cytometry. Caveolae were clearly demonstrated in ultrastructural sections of the purified cytotrophoblasts. The time course of expression of caveolin-1 and eNOS during differentiation of villous cytotrophoblast into syncytiotrophoblast in culture was studied. Western analysis showed that caveolin-1 expression evident in day 1 whole cell lysates decreased at day 3 when the cells had syncytialized and declined further by day 6, while the levels of actin (control) remained high. eNOS expression in the same samples followed a different pattern, with the low levels in day 1 cells increasing substantially by 3 days in culture, subsiding again by day 6. eNOS association with caveolin-1 in day 1 and day 3 trophoblast cultures was evidenced by the demonstration that eNOS co-immunoprecipitates with caveolin-1 and vice versa. We conclude that human villous cytotrophoblasts express caveolin-1, which assembles into caveolae. Differentiation into syncytium results in a decrease, but not disappearance, of expression of caveolin-1 and a marked reduction of the caveolae.
小窝是在许多细胞类型中发现的质膜烧瓶状内陷，尤其是在内皮细胞中。一个主要的结构成分是膜蛋白小窝蛋白-1，它与许多信号分子相关联，包括内皮型一氧化氮合酶（eNOS）。小窝蛋白-1在内皮细胞制备物中与eNOS共免疫沉淀，调节eNOS活性，使其保持无活性状态。目前关于滋养层细胞中小窝和小窝蛋白-1的存在存在争议，因此进行了这项研究，以检查人合体滋养层细胞中高表达的eNOS是否与小窝蛋白-1相关，并查明绒毛细胞滋养层细胞和合体滋养层细胞中是否存在小窝。足月胎盘的免疫组织化学显示，尽管终末绒毛血管内皮细胞染色强烈，但合体滋养层细胞中小窝蛋白-1的标记仅为弱阳性。通过电子显微镜观察，在绒毛毛细血管内皮细胞中发现了大量小窝，但在合体滋养层细胞中极为罕见。小窝蛋白-1染色在纯化的、分离的足月绒毛细胞滋养层细胞中广泛存在，通过细胞涂片分析和流式细胞术证实了这些细胞角蛋白阳性细胞的纯度。在纯化的细胞滋养层细胞的超微结构切片中清楚地显示出小窝。研究了绒毛细胞滋养层细胞在培养中分化为合体滋养层细胞过程中小窝蛋白-1和eNOS表达的时间进程。蛋白质免疫印迹分析表明，在第1天全细胞裂解物中明显的小窝蛋白-1表达在第3天细胞融合时下降，并在第6天进一步下降，而肌动蛋白（对照）水平保持较高。同一样本中eNOS的表达遵循不同的模式，第1天细胞中的低水平在培养3天时大幅增加，在第6天再次下降。在第1天和第3天的滋养层细胞培养物中，eNOS与小窝蛋白-1的关联通过证明eNOS与小窝蛋白-1共免疫沉淀以及反之亦然得到证实。我们得出结论，人绒毛细胞滋养层细胞表达小窝蛋白-1，其组装成小窝。分化为合体滋养层细胞导致小窝蛋白-1表达减少，但并未消失，并且小窝显著减少。
