TRAF7 is an essential regulator of vascular integrity

Targeted deletion of TRAF7 revealed that it is a crucial part of shear stress-responsive MEKK3-MEK5-ERK5 signaling pathway induced in endothelial cells by blood flow. Similarly, to Mekk3-, Mek5- or Erk5-deficient mice, Traf7-deficient embryos died in utero around midgestation due to impaired endothelial cell integrity. They displayed significantly lower expression of transcription factor Klf2, an essential regulator of vascular hemodynamic forces downstream of the MEKK3-MEK-ERK5 signaling pathway. Deletion of Traf7 in endothelial cells of postnatal mice was also associated with severe cerebral hemorrhage. Here, we show that besides MEKK3 and MEK5, TRAF7 associates with a planar cell polarity protein SCRIB. SCRIB binds with an N-terminal region of TRAF7, while MEKK3 associates with the C-terminal WD40 domain. Downregulation of TRAF7 as well as SCRIB inhibited fluid shear stress-induced phosphorylation of ERK5 in cultured endothelial cells. These findings suggest that TRAF7 and SCRIB may comprise an upstream part of the MEKK3-MEK5-ERK5 signaling pathway. Objective: to present first in vivo experimental evidence of TRAF7 function by using global and endothelium-specific TRAF7 knockout mice and comparing transcriptomes of developing embryos. RNA-seq was performed to determine and compare transcriptome profiles in whole mouse embryos of different genotypes at E9.5. Genotypes include wild type, global, and endothelium-specific TRAF7 knockouts.针对TRAF7 (TNF receptor-associated factor 7) 的靶向缺失实验表明，其是血流在内皮细胞 (endothelial cells) 中诱导产生的、响应剪切应力的MEKK3-MEK5-ERK5信号通路的关键组成部分。与Mekk3缺陷、Mek5缺陷或Erk5缺陷的小鼠类似，Traf7基因敲除的胚胎会在妊娠中期于子宫内死亡，诱因是内皮细胞完整性受损。此类胚胎的转录因子Klf2 (Kruppel-like factor 2) 表达水平显著降低，而Klf2是MEKK3-MEK-ERK5信号通路下游调控血管血流动力学的关键调节因子。在出生后小鼠的内皮细胞中敲除Traf7，同样会引发严重的脑出血。本研究证实，除MEKK3和MEK5外，TRAF7还可与平面细胞极性蛋白SCRIB (Scribble) 结合：SCRIB通过TRAF7的N端区域与之结合，而MEKK3则结合于TRAF7的C端WD40结构域 (WD40 domain)。下调TRAF7或SCRIB的表达，均可抑制培养的内皮细胞中流体剪切应力 (fluid shear stress) 诱导的ERK5 (extracellular signal-regulated kinase 5) 磷酸化。上述研究结果提示，TRAF7与SCRIB可能构成MEKK3-MEK5-ERK5信号通路的上游组分。本研究的目标为：通过构建全身及内皮细胞特异性TRAF7敲除小鼠，首次提供TRAF7功能的体内实验证据，并对比分析发育中胚胎的转录组。实验采用RNA测序 (RNA-seq)，以测定并比较胚胎发育第9.5天 (E9.5)、不同基因型（野生型、全身TRAF7敲除型及内皮细胞特异性TRAF7敲除型）的完整小鼠胚胎的转录组谱。