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標題: | GCM1與人類絨毛膜性腺激素在相互作用下調控胎盤細胞之融合 Reciprocal regulation between GCM1 and human chorionic gonadotropin controls placental cell fusion |
作者: | Mei-Leng Cheong 張美玲 |
指導教授: | 陳宏文 |
關鍵字: | GCM1,TFAP2C,人類絨毛膜性腺激素,胎盤細胞融合,滋養層細胞, cell-cell fusion,GCM1,hCG,placenta,TFAP2C,trophoblast, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 人類絨毛膜性腺激素(hCG)在懷孕之初,由胎盤滋養葉融合層細胞所分泌。hCG對胎盤發育時期所須的黃體素、血管生成、免疫系統調節及滋養葉細胞分化扮演重要角色。母體hCG不足跟流產、胎兒發育不良及子癲前症之發生有關。hCG包括alpha及beta單元,後者是胎盤獨有並俱有代表性。哺乳動物GCM1是胎盤獨有的轉錄因子,調控胎盤細胞融合、入侵及血管生成。剔除GCM1基因之小鼠在懷孕10天即因胎盤發育不全而死亡。曾有報告指出hCGb受TFAP2及Sp1等轉錄因子所調控,但對GCM1而言是否可調控hCGb未曾被報導。
在本實驗我們透過ChIP-chip發現hCGb是GCM1的目標基因。TFAP2C、GCM1及hCGb三者在胎盤滋養葉融合層細胞有共同表現。hCGb也隨著GCM1的表現高低而同步增減。相反地,我們也證實hCG可以透過LH/CG接受器及cAMP訊息傳遞調控GCM1的轉錄、轉繹後修飾(磷酸化及乙醯化)、生物活性及其目標基因表現,包括syncytin-1及hCGb。證實GCM1對hCGb的自我調控是非常重要的。剔除LH/CG接受器或hCGb基因也會降低GCM1之表現。我們利用多種方法驗證並找出GCM1在hCGb promoter上的直接結合位置。雖然這位置與TFAP2C的結合位置相同,不過我們發現TFAP2C對hCGb的影響須經由GCM1作用。另外,LH/CG接受器-cAMP-GCM1-hCGb的訊息傳遞受到PKA抑制劑H89的阻撓,這表示cAMP-PKA為GCM1-hCGb互相調節迴路的主要路徑。最後,我們發現hCG促進BeWo細胞的融合也必須依賴GCM1才能達成。本實驗發現在hCGb調節及胎盤細胞分化上的一項新機制。 Human chorionic gonadotropin (hCG) is a hormone secreted mainly by syncytiotrophoblasts governing many crucial functions of placental development, such as progesterone production from corpus lutein of ovaries in the first trimester of pregnancy, angiogenesis of uterine vasculature, immune modulation and adaptation, and cytotrophoblast differentiation. hCG contains two components, alpha and beta subunits, and the latter is placenta-specific. A low maternal serum level of hCG in early pregnancy could indicate miscarriage, fetal growth retardation, and subsequent pre-eclampsia. Maternal serum hCG levels increase dramatically from the implantation of the embryo, and reach a peak at approximately 10 weeks of gestation. Mechanisms contributing to this phenomenon are not yet known. Mammalian glial cells missing 1 (GCM1) is a placenta-specific transcription factor involved in placental cell invasion, cell-cell fusion, and angiogenesis. A knockout of mouse GCM1 induces malformation of placenta and early embryonic lethality approximately 10 dpc. It is reported that hCGb is regulated by two transcriptional factors, Sp1 and TFAP2. Whether GCM1 regulates hCGb remains elusive. In this study, we have identified that hCGb is among the GCM1 target genes through ChIP-chip analysis. We further discovered that TFAP2C, GCM1, and hCGb are co-localized at the syncytiotrophoblasts through immunohistochemical study of the placental tissue. The protein expression and secretion levels of hCGb are up- and down-regulated by GCM1 overexpression and knockdown in choriocarcinoma cell lines, respectively. hCG treatment reciprocally enhances GCM1 transcription, post-translational modification (Serine269/275 phosphorylation and acetylation), and contributes to GCM1 stability through the LH/CG receptor (LH/CGR), cAMP signaling pathway and CBP transactivation, and further enhances the target genes of GCM1, including syncytin-1 and hCGb. These data indicate that GCM1 plays an important and positive role in hCGb production and auto-regulation; either knockdown of LH/CGR or hCGb suppress GCM1 and its target gene expressions. We have identified the precise binding region of GCM1 on the hCGb promoter using a luciferase reporter assay, ChIP, and electrophoretic mobility shift assay, which was previously found to be the same binding region of TFAP2, suggesting that GCM1 and TFAP2C simultaneously regulate hCGb. Indeed, the regulation of hCGb by TFAP2C is through GCM1. The LH/CGR-cAMP-GCM1-hCGb pathway is inhibited by the PKA inhibitor, H89, indicating that the cAMP-PKA pathway is the dominant pathway in the GCM1-hCGb positive loop. Finally, by use of a functional assay, the fusion of BeWo cells was enhanced by hCG treatment, which is GCM1-dependent. Our study reveals a novel mechanism for regulating hCGb and placental cell differentiation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51357 |
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顯示於系所單位: | 生化科學研究所 |
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