hCGβ受到轉錄因子GCM1及AP-2c的調控機制

Abstract

胎盤是主要提供母體與胎兒之間進行物質交換的場所，在胎盤樹狀絨毛的部分，絨毛膜內層的細胞性滋養層細胞 (cytotrophoblast, CTB) 可以經由細胞融合 (cell fusion) 分化成多細胞核的融合滋養層細胞 (syncytiotrophoblast, STB)，且融合滋養層細胞會製造與分泌生長因子以及荷爾蒙，調控母體與胎兒之間的物質交換。從過去實驗已知，胎盤專一性表現的轉錄因子glial cells missing 1 (GCM1)，可以藉由活化syncytin-1、syncytin-2來促進胎盤細胞融合。根據實驗室所做的染色質免疫沉澱結合晶片分析技術 (chromatin immunoprecipitation-on-chip (ChIP-chip) analysis) 找到GCM1轉錄因子相關的下游標的基因，發現GCM1轉錄因子會與hCGβ基因前方的啟動子結合。我們想了解GCM1轉錄因子調控hCGβ的機制，另外，GCM1與hCGβ皆會受到cAMP/PKA訊號傳遞的調控，是否會透過cAMP路徑活化GCM1進而去影響hCGβ的表現。而過去文獻指出轉錄因子activating protein 2 (AP-2) 家族會調控hCGβ的表現，在hCGβ啟動子上含有兩個AP-2的結合位置在-311到-275與-250到-200的區域，可以增加hCGβ的基因表現，本篇針對GCM1與AP-2轉錄因子之間的調控進行討論，幫助我們了解hCGβ受到GCM1與AP-2轉錄因子的調控機制。 首先，在BeWo建立穩定表現GCM1 shRNA的細胞株，由Q-PCR與西方墨點法的實驗結果發現GCM1會直接影響到由forskolin所刺激hCGβ基因的轉錄與蛋白質的表現。利用不含有內生性AP-2的人類肝癌細胞HepG2，進行冷光報導基因活性檢測實驗，發現在forskolin刺激下當GCM1與CBP coactivator同時存在時，GCM1可以增加hCGβ的啟動子活性。在hCGβ啟動子-1674~+103的區域中包含四個可能的GCM1結合區域 (GCM1-binding site, GBS)，為了確定四個GCM1結合區域是否會與GCM1有交互作用，將結合區域個別進行突變，發現GBS2為最重要的GCM1調控hCGβ啟動子活性的區域。進一步利用電泳遷移確定GCM1與AP-2c會直接結合在GBS2的位置上，另外，在BeWo建立穩定表現AP-2c shRNA的細胞株，結果發現GCM1與hCGβ的蛋白質表現明顯下降，且證明AP-2c會去調控GCM1的啟動子活性，由以上的結果知道在胎盤細胞中hCGβ的轉錄機制受到GCM1與AP-2c的調控。

Human cytotrophoblasts in the chorionic villi may undergo cell-cell fusion to form syncytiotrophoblasts to mediate gas and nutrient transport between mother and fetus. The placental transcription factor glial cells missing 1 (GCM1) has been shown to promote placental cell-cell fusion through transcriptional activation of membrane fusogenic proteins, syncytin-1 and -2. We recently performed chromatin immunoprecipitation-on-chip (ChIP-chip) analysis and identified the gene for human chorionic gonadotropin β (hCGβ) as a GCM1 target gene. A major function of hCGβ is to maintain the production of progesterone hormone and other growth factors in the corpus luteum until the 7th week of gestation. The activating protein 2 (AP-2) family has been shown to play an important role in hCGβ expression. The mechanism of regulation of hCGβ gene expression by GCM1and AP-2 is not clear. In this study, we first demonstrated that the hCGβ mRNA and protein levels are significantly decreased in the GCM1-konckdown BeWo cells by Q-PCR and Western blotting. Luciferase reporter assays were performed to study the regulation of hCGβ reporter activity by GCM1 in HepG2 cells, which are human hepatoma-derived cells without endogenous AP-2 activity. Both GCM1 and CBP coactivator are required for the stimulation of hCGβ reporter activity by forskolin in HepG2 cells. The reporter construct harbors the hCGβ promoter region, nt -1674~+103 relative to the transcriptional start site, containing four potential GCM1-binding sites (GBSs). By site-directed mutagenesis, we identified GBS2 as a critical element for GCM1 and CBP to upregulate hCGβ promoter activity in response to forskolin. We further demonstrated that both GCM1 and AP-2c can bind GBS2 by EMSA. Interestingly, the GCM1 protein level was decreased by AP-2c knockdown in BeWo cells. Correspondingly, AP-2c upregulates GCM1 promoter activity in luciferase reporter assays. Our study reveals an underlying mechanism for regulation of hCGβ expression by GCM1 and AP-2c.