Regulation of endothelial cell activation by TGF-β family signaling

我们已经证明，Id1和Herp2分别促进和阻断内皮细胞的血管生成反应。在本研究中，我们试图通过酵母双杂交法分离调控Id1和Herp2功能的Id1和Herp2相互作用分子。在分离到的分子中，E2-2、Id2、CGI-128、FHL2和FLJ13861在哺乳动物细胞中都能与Id1或Herp2相互作用。其中，我们在进一步的实验中重点研究了E2-2，因为E2-2和Id1之间的异二聚体复合体的形成是最强烈的。事实上，Id1有效地抑制了E2-2诱导的荧光素酶活性。当E2-2在内皮细胞中过表达时，血清诱导的内皮细胞增殖和网络形成受到抑制，而Id1在内皮细胞中的表达则相反。为了阐明E2-2阻断内皮细胞血管生成反应的机制，我们检测了内皮细胞…中VEGFR2的表达，这种表达是在内皮激活过程中被诱导的E2-2在细胞内表达时，LLS较多。正如预期的那样，VEGFR2的表达被E2-2抑制，而E2-2介导的VEGFR2表达的降低可以通过Id1在细胞中得到改善。与VEGFR2基因表达下调一致，E2-2可阻断VEGFR2-荧光素酶活性。因此，Id1可能通过与E_2形成异二聚体，通过阻断血管内皮生长因子2的转录而显著抑制血管内皮细胞的活化，从而增强血管生成反应。Alk5基因敲除小鼠与Alk5基因敲除小鼠一样，在E10.5死亡。Alk5基因敲除小鼠卵黄囊内未见成熟血管形成。Alk5基因敲除小鼠卵黄囊的表型与Alk5基因敲除小鼠的卵黄囊表型非常相似。然而，与Alk5基因敲除小鼠相比，Alk5基因敲除小鼠的胎盘中可以检测到迷路的形成。因此，转化生长因子β/alk1信号通路可能改善了alk5基因敲除小鼠迷路形成的缺陷。较少

We have already shown that Id1 and Herp2 promote and block angiogenic responses in endothelial cells, respectively. In present study, we tried to isolate Id1-and Herp2-interacting molecules which modulate function of Id1 and Herp2 by yeast two hybrid method. Among molecules isolated, E2-2, Id2, CGI-128, FHL2 and FLJ13861 could interact with either Id1 or Herp2 in mammalian cells. Out of them, we focused on E2-2 in further experiments because the heterodimer complex formation between E2-2 and Id1 was the strongest. Indeed, Id1 efficiently inhibited E2-2-induced luciferase activity. When E2-2 was over-expressed in endothelial cells, serum-induced proliferation and network formation in endothelial cells was suppressed in contrast with expression of Id1 in endothelial cells. To elucidate the mechanism by which E2-2 blocks angiogenic responses in endothelial cells, we tested the expression of VEGFR2, of which expression is known to be induced during endothelial activation, in endothelial ce … More lls when E2-2 was expressed in cells. As expected, expression of VEGFR2 mRNA was inhibited by E2-2, whereas E2-2-mediated decrease of VEGFR2 expression was improved by introduction of Id1 in the cells. Consistent with reduction of VEGFR2 mRNA, VEGFR2-lucifease activity was blocked by E2-2. Thus, it is possible that Id1 potentiates angiogenic responses in endothelial cells due to making heterodimer with E2-2 which substantially suppresses endothelial cell activation by blocking of VEGFR2 transcript.We also made ALK5 knock-in mice which can not transduce TGF-β/ALK5 signaling, but still possess TGF-β/ALK1 signaling. ALK5 knock-in mice die at E10.5 like ALK5 knock-out mice. We could not observe any mature vessel formation in yolk sac in ALK5 knock-in mice. The phenotype of yolk sac from ALK5 knock-in mice was quite similar to that from ALK5 knock-out mice. However, labyrinth formation in placenta from ALK5 knock-in mice could be detected in contrast with ALK5 knock-out mice. Thus, TGF-β/ALK1 signaling might improve defect of labyrinth formation seen in ALK5 knock-out mice. Less