探討GATA3參與在由GCM1所調控的滋養層細胞分化之機制

GATA3 participates in the GCM1-mediated placental trophoblast cell differentiation

邱越荷(Yueh-Ho Chiu)

指導教授 ： 陳宏文

國立臺灣大學/生命科學院/生化科學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10242

摘要

在人類胎盤滋養層細胞 (trophoblast cell) 的分化過程中，專ㄧ性表現在胎盤的GCM1 (glial cells missing 1) 轉錄因子會藉由啟動下游不同的目標基因來促使具有幹細胞特性的細胞性滋養層細胞 (cytotrophoblast, CTB) 分化成兩種不同的特化細胞： 融合滋養層細胞 （syncytiotrophoblast, STB) 及絨毛外滋養層細胞 (extravillous trophoblasts, EVT)。GCM1藉由活化syncytin-1的表現來促使細胞進行細胞融合形成多核的融合滋養層細胞 (STB)，這層多核的STB主要功能為控制胎兒與母體之間物質與氣體的交換；另一方面，若GCM1提高另一條路徑的目標基因HtrA4 (high-temperature requirement protein A4) 的表現量，細胞將會分化為具有入侵能力的絨毛外滋養層細胞 (EVT)，EVT入侵到母體子宮壁上蛻膜 (decidua) 的這個動作會使子宮內膜的螺旋動脈 (spiral arteries) 的血流量增加，提供足夠的血液幫助胎兒順利生長。GCM1在胎盤發育過程中扮演了舉足輕重的角色，由上述我們可以知道GCM1所調控的這兩條CTBs分化路徑對胎兒的生長來說是不可或缺的，他們提供了足夠的養分和血液使胎兒可以順利發育，但GCM1是如何決定活化下游哪一個目標基因其機制目前還不清楚。
GATA3 (GATA-binding protein 3 ) 轉錄因子已知會參與在許多器官與組織的發育之中，而胎盤也是其中之ㄧ，並且文獻指出GATA3會參與在胚胎發育以及滋養層幹細胞 (trophoblast stem cell) 的分化當中，因此，我們想知道GATA3會不會參與在由GCM1所調控的HtrA4表現路徑當中，更進一步，我們希望能藉由GATA3轉錄因子來告訴我們HtrA4大量表現在胎盤extravillous位置的原因。在我們的實驗結果中，藉由Luciferase reporter assay的結果我們發現無論是在HEK 293T細胞 (人類胚胎腎細胞株) 或是研究人類胎盤發育常用的人類絨毛膜癌細胞株BeWo以及JAR細胞中，GATA3的存在皆會降低HtrA4的表現量。利用共同免疫沉澱法我們發現GATA3和GCM1存在著交互作用的關係，並且GATA3會藉由結合在GCM1 transactivation domain (TAD) 來降低GCM1的轉錄活性。更進一步的，我們減緩BeWo及JAR細胞中GATA3的表現量，發現HtrA4的表現量及細胞入侵能力會隨之上升。綜合以上結果，我們發現GATA3轉錄因子會藉由透過和GCM1轉錄因子之間的交互作用來調控GCM1轉錄因子的轉錄活性，進而影響HtrA4蛋白的表現量，且證實胎盤細胞中GATA3表現量的下降會造成HtrA4表現量的上升，而這樣的結果會造成胎盤細胞侵襲能力的提高。

關鍵字

GCM1 ； HtrA4 ； GATA3 ； 胎盤 ； 滋養層細胞

並列摘要

The glial cells missing 1 (GCM1) transcription factor regulates two trophoblast differentiation pathways in placental development. Syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs) are two types of specialized trophoblasts developed from the stem cell-like cytotrophoblasts (CTBs) in the chorionic villi. GCM1 activates syncytin-1 expression to mediate cell-cell fusion for the formation of multinucleated STBs that mediate nutrient-gas exchange between mother and fetus. On the other hand, GCM1 up-regulates high-temperature requirement protein A4 (HtrA4) in the invasive EVTs to facilitate EVT invasion into decidua and adequate transformation of the spiral arteries. These events ensure sufficient blood flow to the placenta, which is essential for fetal growth. Given that GCM1 plays an important role in placental development, the mechanism of how GCM1 regulates HtrA4 expression in EVTs is not clear.
The transcription factor GATA-binding protein 3 (GATA3) is an essential regulator for several tissue developments including placenta. Previous studies have shown that GATA3 is able to induce trophoblast fate in embryonic stem cells and drive trophoblast differentiation in trophoblast stem cells. Here we investigated whether GATA3 is involved in HtrA4 expression. We showed that GATA3 down-regulates HtrA4 promoter activity in both choriocarcinoma cell lines, BeWo and JAR. Co-immunoprecipitation revealed that GATA3 interacts with GCM1 via its transactivation domain and therefore impairs GCM1 activity. Correspondingly, we observed that GATA3 knockdown in BeWo and JAR cells stimulates HtrA4 expression and therefore BeWo and JAR cell invasion. Taken together, our results indicated that GATA3 participates in HtrA4 expression and trophoblast invasion through interaction with GCM1.

並列關鍵字

GCM1 ； HtrA4 ； GATA3 ； placenta ； trophoblast cell

參考文獻

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