CLIC function in angiogenesis

描述（由申请人提供）：CLIC家族由六种与秀丽隐杆线虫Exc4相关的人类蛋白（1-6）组成，这对管腔的形成至关重要。我们研究了CLICs作为血管生成调节剂的作用，发现CLICs 1和CLICs 4在内皮细胞中表达，并且是培养内皮细胞增殖、迁移和管腔形成所必需的。我们培育了具有异常视网膜血管生成的Clic4基因敲除小鼠。Clic1/Clic4双突变胚胎在子宫内死亡，血管发育有缺陷，提示Clic1和Clic4在内皮细胞中功能冗余。我们假设小鼠Clic1和Clic4在发育性和病理性血管生成中起作用。为了验证这一假设，我们将在Clic1-/-基因型的背景下，使用内皮特异性驱动因子（Clic4ECKO）删除Clic4，并分析胚胎、出生后视网膜和缺氧驱动视网膜血管生成过程中的血管表型。虽然Clic基因编码氯离子通道，但哺乳动物Clic蛋白的分子作用尚不明确。我们最近发现鞘氨醇-1-磷酸（S1P）驱动的内皮细胞迁移、粘连结形成和肌动蛋白应力纤维形成需要CLIC1或CLIC4。通过在内皮细胞中选择性地敲低CLIC，我们记录了CLIC1和CLIC4的重叠和不同的内皮功能，突出了S1P受体对CLICs的特异性活性。因此，我们提供了第一个证据，证明CLIC蛋白作为已知血管生成途径S1P信号的一部分起作用，并且我们的功能研究将CLICs与G蛋白偶联受体信号联系起来。我们建议通过体外内皮研究来探索CLICs与S1P通路之间的分子相互作用。我们将确定内皮细胞是否需要CLIC1或CLIC4来介导s1p驱动的增殖、存活、迁移、屏障形成、胶原凝胶侵袭和管腔形成。我们假设CLIC1和CLIC4通过PI3K/RAC1和PI3K/Akt通路促进S1P1信号转导。我们假设CLIC1是S1P激活S1P2-RhoA/ROCK通路所必需的，而CLIC4是S1P激活S1P1- RAS/ERK通路所必需的。我们将评估CLICs与S1P受体或其下游细胞内信号成分的结合。S1P1在视网膜血管新生过程中起作用。内皮特异性S1P1缺失导致视网膜发芽增加，S1P1功能获得导致发芽减少。通过在小鼠中激活或灭活S1P1信号，使用药理学和遗传学方法，以及内皮细胞Clic4或Clic1-/-小鼠的损失，我们将解决S1P信号需要内皮细胞CLICs来调节血管新生发芽的假设。

DESCRIPTION (provided by applicant): The CLIC family consists of six human proteins (1-6) related to C. elegans Exc4, which is essential for lumen formation. We investigated the roles of CLICs as angiogenic regulators and found that CLIC1 and CLIC4 are expressed in endothelial cells and are required for proliferation, migration and lumen formation of cultured endothelial cells. We developed Clic4 knockout mice that had aberrant retinal angiogenesis. Clic1/Clic4 double mutant embryos die in utero and have defects in vascular development, suggesting Clic1 and Clic4 are functionally redundant in the endothelium. We hypothesize that murine Clic1 and Clic4 function in developmental and pathological angiogenesis. To test this hypothesis, we will delete Clic4 using endothelial specific drivers (Clic4ECKO), in the background of the Clic1-/- genotype and analyze vascular phenotypes in the embryo, postnatal retina and during hypoxia-driven retinal angiogenesis. Although Clic genes encode putative chloride channels, the molecular action of mammalian CLIC proteins is poorly defined. We recently discovered that sphingosine-1-phosphate (S1P)-driven endothelial cell migration, adhesion junction formation, and actin stress fiber formation requires CLIC1 or CLIC4. By selective CLIC knockdowns in endothelial cells, we document both overlapping and distinct endothelial functions for CLIC1 and CLIC4, highlighting S1P receptor specific activities for CLICs. Thus, we provide the first evidence that CLIC proteins function as part of a known angiogenic pathway, S1P signaling, and our functional studies link CLICs to G protein coupled receptor signaling. We propose to use in vitro endothelial studies to explore the molecular interactions between CLICs and the S1P pathway. We will determine whether endothelial cells require CLIC1 or CLIC4 to mediate S1P-driven proliferation, survival, migration, barrier formation, collagen gel invasion, and lumen formation. We hypothesize that CLIC1 and CLIC4 promote S1P1 signaling via the PI3K/RAC1 and PI3K/Akt pathways. We hypothesize that CLIC1 is uniquely required for S1P activation of the S1P2-RhoA/ROCK pathway, while CLIC4 is required for S1P activation of the S1P1- RAS/ERK pathway. We will assess binding of CLICs to S1P receptors or their downstream intracellular signaling components. S1P1 functions during sprouting angiogenesis of the retina. Endothelial specific S1P1 loss caused increased retinal sprouting and S1P1 gain-of-function decreased sprouting. By activating or inactivating S1P1 signaling in mice, using both pharmacologic and genetic methods, along with endothelial loss of Clic4 or Clic1-/- mice, we will address the hypothesis that S1P signaling requires endothelial CLICs to regulate angiogenic sprouting.