Department of Biochemistry, Kanazawa Medical University School of Medicine, 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan.
Biochem J. 2011 Jun 1;436(2):399-407. doi: 10.1042/BJ20101490.
sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O₂) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O₂ concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3'-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3'-end processing.
sFlt-1（可溶性 Flt-1）通过与 VEGF（血管内皮生长因子）的细胞外结合强烈抑制血管生成。在本研究中，我们报道低氧下调 HMVEC（人微血管内皮细胞）中 sFlt-1 的表达，HMVEC 是发生血管生成的微血管的组成部分。低氧（5-1% O₂）增加 HMVEC 中的 VEGF 表达。相比之下，随着 O₂浓度的降低，sFlt-1mRNA 和蛋白的水平显著下降，而 mFlt-1（膜结合 Flt-1）mRNA 和蛋白则保持不变。这表明低氧选择性调节 sFlt-1 前体 mRNA 的替代 3'-末端加工。我们还证明，慢病毒构建物感染的 HMVEC 中 sFlt-1 的过表达抵消了 VEGF 诱导的内皮细胞生长。接下来，我们使用人 Flt-1 小基因鉴定了 HMVEC 中参与 sFlt-1 mRNA 加工的顺式元件，并发现两个不连续的 AUUAAA 序列作为聚（A）信号。此外，我们鉴定了一个位于内含子 13 中的顺式元件，该元件调节 sFlt-1 mRNA 的加工。内含子 13 中富含 U 的区域发生突变会导致小基因转染的 HMVEC 中可溶性形式/膜形式 RNA 比值显著降低。这些结果表明，低氧引起的 sFlt-1 表达降低有助于低氧诱导的血管生成，并揭示了通过替代 mRNA 3'-末端加工调节血管生成的新机制。
