Regulation of Cytokine-Mediated Lung Inflammation

细胞因子介导的肺部炎症的调节

• 批准号: 7321602
• 负责人: Stewart J Levine
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7321602
• 关键词: Regulation; Cytokine; Mediated; Lung; Inflammation

This project has the goal of identifying mechanisms that regulate the generation of soluble cytokine receptors and their role in inflammatory lung disorders. Since tumor necrosis factor (TNF) is an important regulator of inflammation, apoptosis, and innate immune responses, we have elected to use the type I, 55-kDa tumor necrosis factor receptor (TNFR1, TNFRSF1A) as a model system to study pathways that mediate the release of soluble cytokine receptors in the lung and vasculature. Soluble TNFR1 was originally characterized as a proteolytically cleaved receptor ectodomain that is released by a receptor sheddase. This project has identified several new regulatory mechanisms for generation of soluble cytokine receptors that do not involve the proteolytic cleavage of receptor ectodomains. First, we hypothesized the existence of regulatory proteins that modulate TNFR1 release to the extracellular compartment. Utilizing a yeast-two hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNF Receptor Shedding) as a type II integral membrane protein that binds the full-length 55-kDa TNFR1 and promotes TNFR1 release from human airway and vascular endothelial cells (HUVEC) (JCI 2002; 110: 515-526). Second, we showed that human vascular endothelial cells constitutively release TNFR1 to the extracellular compartment primarily as a full-length, 55-kDa protein (Proc Natl Acad Sci U S A. 2004; 101: 1297-302). This finding lead to the discovery that full-length TNFR1 is released within the membranes of exosome-like vesicles via a zinc metalloprotease-dependent process that does not involve receptor sheddase activity. Thus, the release of TNFR1 exosome-like vesicles represents a novel, alternative mechanism for the release of cytokine receptors from cells that is distinct from the proteolytic cleavage of receptor ectodomains or the generation of alternatively spliced translation products(J Immunology 2004; 173: 5343-8). The physiological relevance of these observations was confirmed by the demonstration in human subjects of TNFR1 exosome-like vesicles in serum and bronchoalveolar lining fluid. Third, we showed that ARTS-1 promotes the release of soluble, cleaved forms of IL-6Ra (J Biol Chem 2003; 278: 28677-85) and IL-1RII (J Immunology 2003; 171: 6814-9). Thus, ARTS-1 regulates the release of three distinct cytokine receptor superfamilies, the TNF receptor superfamily (TNFR1), the class I cytokine receptor superfamily (IL-6Ra), and the immunoglobulin/Toll-like receptor superfamily (IL-1RII). We have also identified nucleobindin 2 (NUCB2, NEFA) as a calcium-dependent, ARTS-1-binding protein that associates with intracytoplasmic TNFR1 vesicles and is required for the constitutive release of TNFR1 within the membranes of exosome-like vesicles, as well the IL-1b-mediated, inducible proteolytic cleavage of TNFR1 (JBC 2006; 281: 6860-6873). Therefore, NUCB2 and ARTS-1 regulate two zinc metalloprotease-dependent mechanisms of cytokine receptor shedding, the sheddase-independent, constitutive release of exosome-like vesicles containing full-length TNFR1 receptors and the sheddase-dependent, inducible proteolytic cleavage of receptor ectodomains. Since the last annual report, this project has identified several new insights regarding the release of TNFR1 to the extracellular space: I. The regulation of TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. Therefore, we investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from HUVEC. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1b-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a non-redundant and additive fashion. Thus, we identified that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism, but did not affect the inducible proteolytic cleavage of TNFR1 ectodomains. II. We investigated whether additional ARTS-1-associated proteins exist that regulate TNFR1 release. RBMX (RNA-binding motif gene, X chromosome; heterogeneous nuclear ribonucleoprotein G), a 43-kDa heterogeneous nuclear ribonucleoprotein (hnRNP) co-immunoprecipitated with ARTS-1 from NCI-H292 cells and was identified by MALDI-MS peptide mass mapping. The association between endogenous RBMX and ARTS-1 in HUVEC lysates was increased by RNase digestion, which suggests that the association with ARTS-1 involves a subset of RBMX that is not bound to RNA. Confocal microscopy co-localized RBMX and ARTS-1 to a population of diffusely distributed cytoplasmic vesicles. RNA interference was utilized to specifically knockdown RBMX expression and show that RBMX regulates both the constitutive release of exosome-like vesicles that contain a full-length 55-kDa TNFR1 and the inducible proteolytic cleavage of soluble TNFR1 ectodomains in response to IL-1b stimulation. Thus, RBMX participates in the regulation of both TNFR1 release pathways. These findings define a new and unexpected function for an hnRNP by identifying RBMX as a novel ARTS-1-interacting protein that regulates extracellular TNFR1 release. III. We assessed whether blood from healthy human volunteers contains TNFR1 exosome-like vesicles that function as TNF-binding structures. TNFR1 exosome-like vesicles, with a diameter of 27- to 36-nm, were demonstrated in human serum by immunoelectron microscopy. Western blots of human plasma showed a 48-kDa TNFR1, which is consistent with a membrane-associated receptor. Gel exclusion chromatography revealed that the 48-kDa TNFR1 in human plasma did not fractionate with soluble proteins, but instead co-segregated with LDL particles on the basis of size. The 48-kDa TNFR1 in human plasma segregated independently from LDL particles by peak density, which demonstrates that TNFR1 exosome-like vesicles are distinct from LDL particles. Known exosome-associated proteins, ICAM-1, LAMP-1, and LAMP-2, co-segregated with the HDL fraction of human plasma, which suggests that TNFR1 exosome-like vesicles are also distinct from typical exosomes. The reduced size of the 48-kDa exosome-associated TNFR1, as compared with the 55-kDa TNFR1 associated with human vascular endothelial cells, reflected a reduced content of N-linked carbohydrates. Furthermore, the 48-kDa TNFR1 exosome-like vesicles in human plasma bound TNF. Thus, we have demonstrated that plasma from healthy human volunteers contains 48-kDa TNFR1 exosome-like vesicles that fractionate with, but are distinct from, LDL particles and function as TNF-binding structures despite a reduced content of N-linked carbohydrates.

该项目的目的是确定调节可溶性细胞因子受体产生的机制及其在炎症性肺部疾病中的作用。 Since tumor necrosis factor (TNF) is an important regulator of inflammation, apoptosis, and innate immune responses, we have elected to use the type I, 55-kDa tumor necrosis factor receptor (TNFR1, TNFRSF1A) as a model system to study pathways that mediate the release of soluble cytokine receptors in the lung and vasculature. 可溶性TNFR1最初的特征是由受体SHEDDase释放的蛋白水解裂解受体外生域。该项目已经确定了几种新的调节机制，用于生成不涉及受体外生域蛋白水解裂解的可溶性细胞因子受体。首先，我们假设将TNFR1释放到细胞外室的调节蛋白的存在。利用酵母 - 两种混合方法，我们确定了ARTS-1（TNF受体脱落的氨基肽酶调节剂）为II型积分膜蛋白，该膜蛋白结合了全长55-KDA TNFR1，并促进了TNFR1的TNFR1，并促进了人类气道和血管内皮细胞和血管内皮细胞（HUBEC）（HUBEC）（jCI 2002）。其次，我们表明人血管内皮细胞组成型将TNFR1释放到细胞外室，主要是全长为55 kDa蛋白（Proc Natl Acad Sci u S A. 2004; 101：1297-302）。这一发现导致发现，发现全长TNFR1通过不涉及受体SHEDDase活性的锌金属蛋白酶依赖性过程在外泌体样囊泡的膜中释放。因此，TNFR1外生囊泡的释放代表了一种新型的，用于从细胞中释放细胞因子受体的替代机制，该机制与受体外生域的蛋白水解裂解不同或产生了剪接的翻译产物（J免疫学2004; 173：5343-8）。这些观察结果的生理相关性通过人类在血清和支气管肺泡内膜中的TNFR1外泌体样囊泡中的示范证实。第三，我们表明ARTS-1促进了IL-6RA的可溶性，分裂形式的释放（J Biol Chem 2003; 278：28677-85）和IL-1RII（J Immunology 2003; 171：6814-9）。因此，ARTS-1调节了三种不同的细胞因子受体超家族的释放，TNF受体超家族（TNFR1），I类细胞因子受体超家族（IL-6RA）和免疫球蛋白/Toll-toll-toll-like受体超级印记（IL-1RII）。我们还确定了核仁素2（nucb2，nefa）是一种依赖钙的ARTS-1结合蛋白，它与内胞浆内TNFR1囊泡相关，是在本质中释放TNFR1在膜中的膜中释放TNFR1所必需的。 2006; 281：6860-6873）。因此，NUCB2和ARTS-1调节了细胞因子受体脱落的两种锌金属蛋白酶依赖性机制，即与Sheddase独立的，含有全长TNFR1受体的外泌体样囊泡的组成型释放，具有全长TNFR1受体以及SHEDDase依赖性的，可诱导的，可诱导的蛋白质蛋白质裂解。 自上次年度报告以来，该项目已经确定了有关将TNFR1发布到细胞外空间的几个新见解： I. TNFR1释放途径的调节似乎涉及细胞质TNFR1囊泡的运输。囊泡运输受ADP-核糖基化因子（ARF）的控制，该因子在GTP结合状态下活跃，并且与GDP结合时不活跃。鸟嘌呤核苷酸交换因子增强了ARF激活，从而催化GTP替换GDP。因此，我们研究了BREFELDIN A（BFA）抑制的鸟嘌呤核苷酸 - 交换蛋白Big1和/或Big2是否需要从HUVEC释放TNFR1。特异性RNA干扰（RNAI）的影响表明，BIG2而不是BIG1调节TNFR1外泌体样囊泡的释放，而IL-1B诱导的TNFR1胞外域的蛋白水解裂解并不需要BIG2和BIG1。 BIG2与TNFR1在扩散分布的细胞质囊泡中共定位，BFA破坏了BIG2和TNFR1之间的关联。与BIG2，ARF1和ARF3对I类ARF的优先激活一致，以非冗余和添加的方式参与TNFR1外泌体样囊泡的细胞外释放。因此，我们确定了BIG2和TNFR1之间的关联选择性调节TNFR1外泌体样囊泡的细胞外释放通过ARF1-和ARF3依赖性机制，但不影响TNFR1 eCtodomain的诱导型蛋白水解裂解。 ii。我们研究了是否存在调节TNFR1释放的其他ARTS-1相关蛋白。 RBMX (RNA-binding motif gene, X chromosome; heterogeneous nuclear ribonucleoprotein G), a 43-kDa heterogeneous nuclear ribonucleoprotein (hnRNP) co-immunoprecipitated with ARTS-1 from NCI-H292 cells and was identified by MALDI-MS peptide mass mapping. RNase消化增加了HUVEC裂解物中内源性RBMX与ARTS-1之间的关联，这表明与ARTS-1的关联涉及与RNA不结合的RBMX子集。共聚焦显微镜共定位的RBMX和ARTS-1与分布的细胞质囊泡的群体。 RNA干扰用于特异性敲低的RBMX表达，并表明RBMX调节外泌体样囊泡的组成型释放，其中包含全长55 kDa TNFR1，以及对IL-1B刺激的响应响应IL-1B刺激的响应响应的溶解性TNFR1型TNFR1型TNFR1 tnfr1 cectodomain。因此，RBMX参与了两个TNFR1释放途径的调节。这些发现通过将RBMX鉴定为一种调节细胞外TNFR1释放的新型ARTS-1相互作用蛋白来定义HNRNP的新功能。 iii。我们评估了健康的人类志愿者的血液是否含有TNFR1外泌体样囊泡，这些囊泡起着TNF结合结构的作用。通过免疫电子显微镜在人血清中证明了TNFR1外泌体样囊泡，直径为27至36 nm。人血浆的蛋白质印迹显示出48 kDa TNFR1，与膜相关受体一致。凝胶排除色谱法表明，人血浆中的48 kDa TNFR1不是与可溶性蛋白分馏，而是根据大小与LDL颗粒共隔离。人等离子体中的48 kDa TNFR1通过峰密度独立于LDL颗粒隔离，这表明TNFR1外泌体样囊泡与LDL颗粒不同。已知的外泌体相关蛋白ICAM-1，LAMP-1和LAMP-2与人血浆的HDL分数共聚，这表明TNFR1外泌体样囊泡也与典型的外生体不同。与与人血管内皮细胞相关的55 kDa TNFR1相比，与48 kDa外泌体相关的TNFR1的尺寸减小，反映了N-链接的碳水化合物的含量降低。此外，人血浆结合的TNF中的48 kDa TNFR1外泌体样囊泡。因此，我们已经证明，健康人类志愿者的血浆中包含48 kDa TNFR1外泌体样囊泡，尽管N-连接碳水化合物的含量降低，但与LDL颗粒分馏但与LDL颗粒的分数相同，但与TNF结合结构的作用。