轉錄因子GCM1及MTF1調控PGF基因機制之探討

PGF (Placental growth factor)為血管內皮生長因子家族其下的一員。在正常懷孕的期間PGF會高度表現在胎盤裡。過去的研究顯示PGF在血管新生(vasculogenesis)與血管的衍生(angiogenesis)上，藉由旁分泌作用在內皮細胞扮演著關鍵的角色。此外，PGF也在早期胚胎發育表現並控制早期胚胎發育。胎盤專一表現的轉錄因子GCM1 (Glial cells missing 1)對於胎盤細胞的分化過程很重要。最近，我們利用ChIP-chip (chromatin immunoprecipitation-on-chip)的實驗確認PGF為GCM1的目標基因。雖然在過去文獻中已知在胎盤細胞裡GCM1在轉錄上會調控PGF的表現，但PGF如何被調控的機制尚不清楚。在其他的研究顯示， MTF1 (metal-responsive transcription factor 1)參與在BeWo細胞(由滋養層絨毛膜所衍生的細胞株)裡，誘導PGF的表現。MTF1為反應於各種環境因子的轉錄因子，包括金屬負載、缺氧以及氧化壓力。因此，在我們的研究中也探討MTF1是否涉及PGF表在胎盤細胞裡。由我們實驗結果表明，PGF mRNA和蛋白表現在GCM1剔除以及缺氧處理下的BeWo細胞中都很顯著地減少；相反地，於JAR細胞過度表現GCM1時，其結果有增多的現象。此外，我們利用冷光報導基因試驗在HEK 293T (人類胚胎腎細胞株)細胞進行實驗，發現GCM1會促進位在-1000到-345以及+5到+100區域的PGF啟動子活性。進一步地藉由點突變的方式，我們進一步發現了對於GCM1上調PGF啟動子活性的GCM1結合位點。另一方面，在MTF1剔除和缺氧條件下的細胞，PGF mRNA表現有稍有下降。但是，在我們的結果裡，我們卻無法觀察到MTF1調節PGF啟動子活性。綜合以上的實驗結果，於本篇論文中得到以下推論。在胎盤細胞裡，可能主要由GCM1轉錄因子結合到PGF基因啟動子5端非編碼區內的位置後，去調控開啟PGF基因；而在非胎盤細胞裡，可能才是藉由MTF1轉錄因子去啟動PGF基因。

關鍵字

PGF ； GCM1 ； MTF1 ；胎盤；滋養層細胞

並列摘要

PGF is a member of VEGF family. PGF is highly expressed in placenta during normal pregnancy. Previous study showed that it plays a pivotal role for vasculogenesis and angiogenesis in endothelial cells via paracrine action. In addition, PGF is also expressed during early embryonic development and control early embryogenesis. The placenta-specific transcription factor GCM1 is critical for placental cell differentiation. We have recently identified PGF as a GCM1 target gene by ChIP-chip analysis. Although GCM1 has been showed to regulate PGF expression transcriptionally, the mechanism of PGF expression is still not clear. Other study demonstrate that MTF1 takes part in the induction of PGF expression in Bewo cell, the trophoblast-derived choriocarcinoma cell line. MTF1 is a transcription factor that responds to a variety of stresses including metal load, hypoxia and oxidative stress. Here, we also investigated whether MTF1 is involved in PGF expression. We demonstrated that the PGF mRNA and protein levels are significantly decreased in GCM1-knockdown and hypoxia Bewo cells. Conversely, the results were increased in GCM1-overexpressed JAR cells. Furthermore, GCM1 stimulated the PGF promoter activity by luciferase reporter construct harboring the PGF promoter region from -1000 to -345 and +5 to +100 relative to the transcriptional start site. By site-directed mutagenesis, we further identified a GCM1-binding site that is essential for GCM1 to upregulate PGF promoter activity. On the other hand, the PGF mRNA level was slightly decreased on MTF1 -knockdown and hypoxia condition. But we failed to observe that MTF1 regulate PGF promoter activity. Based on the above results, we get the following inference in this paper. In placental cells, it may mainly bind to PGF promoter 5’ UTR region and turn on PGF gene by GCM1 transcription factor. In the non-placental cells, MTF1 may transcriptionally regulate PGF gene.

並列關鍵字

PGF ； GCM1 ； MTF1 ； placenta ； trophoblast cell

參考文獻

1. Gude, N., et al., Growth and function of the normal human placenta. Thromb Res, 2004.114(5-6): p. 397-407.

2. Cross, J. C., Placental function in development and disease. Reprod Fertil Dev,2006.18(1-2): p. 71-6.

5.Hosoya, T., et al., glial cells missing: a binary switch between neuronal and glial determination in Drosophila. Cell, 1995. 82(6): p. 1025-36.

6.Jones, B.W., et al., glial cells missing a genetic switch that controls glial versus

7.Wegner, M., and Riethmacher, D., Chronicles of a switch hunt: gcm genes in development. Trends Genet, 2001. 17(5): p. 286-90.

國際替代計量

轉錄因子GCM1及MTF1調控PGF基因機制之探討

全文下載

主題瀏覽