ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism
ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism
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DOI：
10.1093/molehr/gay034
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发表时间：
2018-10-01
影响因子：
4
通讯作者：
Beristain, A. G.
中科院分区：
文献类型：
--
作者：
Le, H. T.;Atif, J.;Beristain, A. G.
研究问题：解整合素和金属蛋白酶8（ADAM 8）是否控制人胎盘发育早期绒毛外滋养层细胞（EVT）的分化和迁移？简要答复：ADAM 8 mRNA优先定位于浸润性HLA-G阳性滋养细胞，与EVT表型的获得相关，并通过需要β 1 -整合素的机制促进滋养细胞迁移。妊娠早期胎盘的建立需要滋养层祖细胞分化为侵袭性EVT，其产生多种蛋白酶，这些蛋白酶促进基质重塑和细胞因子的活化。在控制细胞迁移中起重要作用的信号通路。研究设计、大小、持续时间：孕早期胎盘绒毛和蜕膜（6-12周妊娠）、原代滋养层细胞和滋养层细胞系（JEG 3、EMT、Bewo、HTR 8/SVNeo）用于检测ADAM 8的表达、定位和功能。所有实验在至少三个独立的场合进行（n = 3）。对象/材料，地点，方法：使用IRB批准的早期妊娠胎盘（n = 24）和蜕膜（n = 4）的胎盘绒毛和原代滋养层细胞，通过原位RNAScope杂交，流式细胞术，定量PCR和免疫印迹分析来检测ADAM 8的定位和表达。原代滋养层细胞分化成EVT样细胞通过铺在纤连蛋白上，并通过免疫荧光显微镜和角蛋白-7，波形蛋白，表皮生长因子受体（EGFR），HLA-G和ADAM 8的免疫印迹分析进行评估。利用siRNA指导的沉默和过表达策略，在原发性EVT和滋养层细胞系中检查了ADAM 8功能。滋养层细胞迁移使用Transwell小室进行评估，细胞-基质结合使用纤连蛋白粘附试验进行测试，并通过免疫荧光显微镜、免疫共沉淀实验和功能促进/抑制抗体确定ADAM 8-β 1-整联蛋白相互作用。在孕早期胎盘组织中，ADAM 8优先定位于HLA-G（+）滋养层细胞，位于锚定柱和蜕膜中。在原代滋养层细胞和滋养层细胞系中的功能实验表明，ADAM 8通过独立于内在蛋白酶活性的机制促进滋养层细胞迁移。我们发现，ADAM 8定位于核周和细胞膜肌动蛋白丰富的结构，在细胞-基质附着和促进滋养层结合到纤维连接蛋白基质。此外，ADAM 8增强β 1-整联蛋白的激活，并通过依赖于β 1-整联蛋白function.LIMITATIONS，理由回避：本研究的主要限制是使用体外实验中检查ADAM 8功能，以及实施永生化滋养层细胞系。胎盘和蜕膜组织切片中的ADAM 8的组织学定位仅限于mRNA水平分析。此外，患者的信息，相应的组织通过选择性终止是不可用的。更广泛的影响的发现：新的非蛋白水解促迁移作用的ADAM 8在控制滋养层细胞迁移揭示了这项研究揭示了深入了解的重要性，ADAM 8在EVT生物学和胎盘发育。
STUDY QUESTION: Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development?SUMMARY ANSWER: ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring beta 1 -integrin.WHAT IS KNOWN ALREADY: Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown.STUDY DESIGN, SIZE, DURATION: First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3).PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-beta 1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies.MAIN RESULTS AND THE ROLE OF CHANCE: Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G(+) trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates beta 1-integrin activation and promotes cell migration through a mechanism dependent on beta 1-integrin function.LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available.WIDER IMPLICATIONS OF THE FINDINGS: The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development.