Choi Jayoung, Dong Linda, Ahn Janice, Dao Diem, Hammerschmidt Matthias, Chen Jau-Nian
Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
Dev Biol. 2007 Apr 15;304(2):735-44. doi: 10.1016/j.ydbio.2007.01.023. Epub 2007 Jan 20.
Flk1 is the major receptor for VEGF on endothelial cells. During embryogenesis, flk1 is required for both vasculogenesis and angiogenesis and abnormally elevated flk1 expression is often associated with pathological conditions in adults. While the biological function of flk1 has been studied extensively, very little is known about how the flk1 gene is regulated at the transcriptional level. Our transgenic study led to the identification of a flk1 endothelial enhancer positioned approximately 5 kb upstream of the flk1 translation initiation site. Binding sites for FoxH1, scl, ets and gata factors are found in the zebrafish flk1 endothelial enhancer, as well as in upstream sequences of mouse flk1 and human kdr genes, suggesting that the regulatory machinery for flk1/kdr is conserved from fish to mammals. The roles of scl, ets and gata factors in hemangioblasts have been well defined, but the significance of FoxH1 in vessel formation has not been explored previously. Here we show that FoxH1 binds to the flk1 endothelial enhancer in vitro and functions as a repressor for flk1 transcription in cultured cells. Consistent with these findings, the expression level of flk1 is elevated in embryos lacking both maternal and zygotic FoxH1. We further show that overexpression of FoxH1 has a negative effect on vascular formation that can be counteracted by the down-regulation of smad2 activity in zebrafish embryos. Taken together, our data provide the first evidence that flk1 is a direct target of FoxH1 and that FoxH1 is involved in vessel formation in zebrafish.
Flk1是血管内皮生长因子（VEGF）在内皮细胞上的主要受体。在胚胎发育过程中，Flk1对于血管发生和血管生成都是必需的，而Flk1表达异常升高通常与成人的病理状况相关。虽然对Flk1的生物学功能已进行了广泛研究，但对于Flk1基因在转录水平上是如何被调控的却知之甚少。我们的转基因研究导致鉴定出一个位于Flk1翻译起始位点上游约5 kb处的Flk1内皮增强子。在斑马鱼Flk1内皮增强子以及小鼠Flk1和人类Kdr基因的上游序列中发现了FoxH1、scl、ets和gata因子的结合位点，这表明从鱼类到哺乳动物，Flk1/Kdr的调控机制是保守的。scl、ets和gata因子在成血管细胞中的作用已得到明确界定，但FoxH1在血管形成中的意义此前尚未被探索。在此我们表明，FoxH1在体外与Flk1内皮增强子结合，并在培养细胞中作为Flk1转录的抑制因子发挥作用。与这些发现一致，在缺乏母源和合子型FoxH1的胚胎中，Flk1的表达水平升高。我们进一步表明，FoxH1的过表达对血管形成有负面影响，而这种影响可通过下调斑马鱼胚胎中Smad2的活性来抵消。综上所述，我们的数据首次证明Flk1是FoxH1的直接靶点，且FoxH1参与斑马鱼的血管形成。
