Noy Peter J, Swain Rajeeb K, Khan Kabir, Lodhia Puja, Bicknell Roy
Angiogenesis Laboratory, Institutes for Cardiovascular Sciences and Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
Angiogenesis Laboratory, Institutes for Cardiovascular Sciences and Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
FASEB J. 2016 Jun;30(6):2311-23. doi: 10.1096/fj.201500122R. Epub 2016 Mar 3.
C-type lectin family 14, member A (CLEC14A), is a single-pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid-like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid-like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or -3. Site-directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2-fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3-fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.-Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).
C型凝集素家族14成员A（CLEC14A）是一种单次跨膜糖蛋白，在肿瘤内皮细胞中过表达，它通过与细胞外基质蛋白相互作用促进芽生血管生成并调节内皮功能。在此，我们表明CLEC14A被类菱形蛋白2（RHBDL2）切割，RHBDL2是3种具有催化活性的哺乳动物类菱形（RHBDL）蛋白酶之一，但它不被RHBDL1或-3切割。定点诱变确定了RHBDL2切割CLEC14A的精确位点，并且在人内皮细胞中靶向敲低内源性CLEC14A和RHBDL2的小干扰RNA验证了RHBDL2切割CLEC14A的特异性。内源性切割的CLEC14A缺失使内皮迁移增加2倍，而添加重组切割的CLEC14A在几种体外模型中抑制人和鼠内皮细胞的芽生3倍。我们使用啮齿动物皮下海绵植入模型评估了切割的CLEC14A在血管生成中的体内作用，我们发现CLEC14A蛋白使血管密度降低>50%。最后，我们表明切割的CLEC14A与芽生的内皮尖端细胞结合。我们的数据表明CLEC14A的胞外域调节芽生血管生成，并提示RHBDL2在内皮功能中的作用。-诺伊，P.J.，斯温，R.K.，汗，K.，洛迪亚，P.，比克内尔，R.芽生血管生成受类菱形蛋白2（RHBDL2）催化的C型凝集素家族14成员A（CLEC14A）胞外域脱落调节。
