Qiao Huan, Liu Yan, Veach Ruth A, Wylezinski Lukasz, Hawiger Jacek
From the Departments of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine and.
Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
J Biol Chem. 2014 Aug 8;289(32):21973-83. doi: 10.1074/jbc.M114.588723. Epub 2014 Jun 23.
A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists.
炎症的一个标志是血管通透性增加，它由多种激动剂通过与CARMA3信号小体的刺激偶联组装在内皮细胞中诱导产生，CARMA3信号小体包含衔接蛋白BCL10。此前，我们报道免疫细胞中的BCL10被“死亡”衔接蛋白CRADD/RAIDD（CRADD）靶向，CRADD负向调节T细胞中核因子κB（NFκB）依赖性细胞因子和趋化因子的表达（Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493 - 2497）。这种新的抗炎CRADD - BCL10轴促使我们分析CRADD在非免疫细胞中的表达及其潜在的抗炎作用。我们将研究重点放在微血管内皮细胞上，因为它们在炎症中起关键作用。我们发现，CRADD缺陷的小鼠内皮细胞在响应脂多糖（LPS）和凝血酶时，表现出由BCL10介导的多效性促炎细胞因子白细胞介素 - 6（IL - 6）和趋化因子单核细胞趋化蛋白 - 1（MCP - 1/CCL2）表达增强。此外，与野生型（cradd(+/+)）原代小鼠内皮细胞相比，这些激动剂也会使cradd(-/-)细胞的通透性显著增加。CRADD缺陷细胞显示出更多的F - 肌动蛋白聚合，同时黏附连接被破坏。反过来，通过递送一种新型的重组细胞穿透性CRADD蛋白（CP - CRADD）来增加细胞内CRADD，可恢复内皮屏障功能，并抑制LPS和凝血酶诱导的IL - 6和MCP - 1的产生。同样，CP - CRADD增强了CRADD充足的内皮细胞的屏障功能。这些结果表明，内源性CRADD的缺失会损害内皮细胞对炎症信号的屏障功能。因此，我们定义CRADD在内皮细胞中的一种新功能为BCL10的诱导性抑制剂，BCL10是对促炎激动剂反应的关键介质。
