Regulation of Cytokine-Mediated Lung Inflammation

细胞因子介导的肺部炎症的调节

基本信息

批准号：
8557921
负责人：
Stewart Levine
金额：
$ 15.19万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/8557921
关键词：
A kinase anchoring protein ADP-Ribosylation Factors Acute Lung Injury Affect Aminopeptidase Apoptosis Ascaridil Asthma Binding Binding Proteins Biological Models Blood Brefeldin A Calcium Caspase Characteristics Cleaved cell Co-Immunoprecipitations Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytokine Receptors Cytoplasmic Vesicles Double-Stranded RNA Endothelial Cells Epithelial Cells Extracellular Space GTP Binding Gel Gel Chromatography Generations Genes Goals Guanine Nucleotide Exchange Factors Guanosine Triphosphate High Density Lipoproteins Human Immune response Immunoelectron Microscopy Immunoglobulins Immunology Inflammation Inflammatory Integral Membrane Protein Lead Length Ligands Liquid substance Low-Density Lipoproteins Lung Lung Inflammation Lung diseases Mediating Mediator of activation protein Membrane Metalloproteases Movement Pathway interactions Physiological Plasma Pneumonia Poly I-C Process Protein Binding Proteins Pulmonary Emphysema Pulmonary Fibrosis RNA Binding RNA Interference RNA Splicing Reactive Oxygen Species Receptor Cell Receptor Signaling Regulation Role Sarcoidosis Serum Signal Pathway Signal Transduction TLR3 gene TLR4 gene TLR7 gene TNFRSF1A gene Tertiary Protein Structure Toll-like receptors Translations Tumor Necrosis Factor Receptor Tumor Necrosis Factor-alpha Vascular Endothelial Cell Vesicle Viral Virus Diseases Western Blotting X Chromosome Yeasts Zinc analog base cytokine density extracellular genetic regulatory protein human IL27RA protein human subject insight interleukin-1 receptor type II nanovesicle novel nucleobindin particle protein function receptor research study trafficking tumor necrosis factor receptor 1A yeast two hybrid system

项目摘要

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 HUVEC constitutively release TNFR1 to the extracellular compartment primarily as a full-length, 55-kDa protein (PNAS 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. 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. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from HUVEC. 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 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 (JBC 2007; 282: 9591 - 9599). II. BIG2 contains three A kinase-anchoring protein (AKAP) domains that may coordinate cAMP and ARF regulatory functions. We hypothesized that BIG2 might regulate the release of TNFR1 exosome-like vesicles via its AKAP, as well as its Sec7 domains. We showed that 8-Br-cAMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a PKA-dependent mechanism. RNA interference experiments showed that RIIb modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIb to BIG2 AKAP domains B and C. This showed that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIb to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide-exchange protein that activates class I ARFs and as an AKAP for RIIb that localizes PKA signaling within cellular TNFR1 trafficking pathways (JBC 2008; 283: 25364-71). III. Co-immunoprecipitation experiments identified RBMX (RNA-binding motif gene, X chromosome)as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains (BBRC 2008; 371: 505-9). IV. Since TNFR1 exosome-like nanovesicles are released by HUVEC, we hypothesized that they may circulate in human blood and modulate TNF-mediated inflammation. TNFR1 exosome-like vesicles 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 co-segregated with the HDL fraction of human plasma, which suggests that TNFR1 exosome-like vesicles are distinct from typical exosomes. This shows that human plasma contains 48-kDa TNFR1 exosome-like vesicles that fractionate with, but are distinct from, LDL particles, and display unique characteristics as compared to plasma- or endothelial cell-derived exosome-like vesicles (BBRC 2008 366: 579 - 584). V. The role of Toll-like receptor (TLR) signaling pathways that mediate TNFR1 release to the extracellular space was investigated. We found that poly (I:C), a viral double-stranded RNA (dsRNA) analog, selectively induces the cleavage and shedding of 34-kDa soluble TNFR1 ectodomains, but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles from human airway epithelial cells. The poly (I:C)-mediated increases in sTNFR1 shedding are mediated via TLR3, whereas ligands for other toll-like receptors, including TLR4, TLR7 and NOD2, do not. Furthermore, we show that poly (I:C)-induced sTNFR1 release is mediated via two TLR3-TRIF-RIP1-dependent pathways. One pathway involves the Duox2-mediated generation of reactive oxygen species (ROS), while the second pathway is via caspase-mediated activation of apoptosis. We conclude that viral dsRNA-induced shedding of 34-kDa sTNFR1 ectodomains from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated (J Immunol 2011; 186:1180-1188).

可溶性 TNFR1 最初被定性为一种蛋白水解裂解的受体胞外域，由受体脱落酶释放。该项目已经确定了几种用于产生可溶性细胞因子受体的新调节机制，这些机制不涉及受体胞外域的蛋白水解切割。首先，我们假设存在调节 TNFR1 释放到细胞外区室的调节蛋白。利用酵母-两种杂交方法，我们确定了 ARTS-1（TNF 受体脱落的氨基肽酶调节剂）是一种 II 型整合膜蛋白，可结合全长 55 kDa TNFR1 并促进人气道和血管内皮细胞释放 TNFR1。 HUVEC）（JCI 2002；110：515-526）。其次，我们表明 HUVEC 主要以全长 55 kDa 蛋白质的形式将 TNFR1 持续释放到细胞外区室 (PNAS 2004；101：1297-302)。这一发现导致发现全长 TNFR1 通过锌金属蛋白酶依赖性过程在外泌体样囊泡的膜内释放，该过程不涉及受体脱落酶活性。因此，TNFR1 外泌体样囊泡的释放代表了细胞因子受体从细胞中释放的一种新颖的替代机制，该机制不同于受体胞外域的蛋白水解切割或替代剪接翻译产物的产生（JImmunology 2004；173：5343） -8）。这些观察结果的生理相关性通过在人类受试者中血清和支气管肺泡内壁液中的 TNFR1 外泌体样囊泡的证实得到证实。第三，我们表明ARTS-1促进可溶性裂解形式的IL-6Ra（J Biol Chem 2003；278：28677-85）和IL-1RII（J Nutrition 2003；171：6814-9）的释放。因此，ARTS-1 调节三个不同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 样受体超家族 (IL-1RII)。我们还发现核结合蛋白 2 (NUCB2, NEFA) 是一种钙依赖性 ARTS-1 结合蛋白，与胞质内 TNFR1 囊泡相关，并且是外泌体样囊泡膜内 TNFR1 组成型释放所必需的，以及IL-1b 介导的 TNFR1 诱导型蛋白水解裂解（JBC 2006； 281：6860-6873）。因此，NUCB2和ARTS-1调节细胞因子受体脱落的两种锌金属蛋白酶依赖性机制，即包含全长TNFR1受体的外泌体样囊泡的不依赖于脱落酶的组成型释放，以及受体胞外域的脱落酶依赖性的诱导性蛋白水解裂解。该项目已经确定了有关 TNFR1 释放到细胞外空间的一些新见解： I. TNFR1 释放途径的调节似乎涉及细胞质 TNFR1 囊泡的运输。囊泡运输由 ADP-核糖基化因子 (ARF) 控制，该因子在 GTP 结合状态下活跃，在与 GDP 结合时不活跃。鸟嘌呤核苷酸交换因子可催化 GTP 取代 GDP，从而增强 ARF 的激活。我们研究了布雷菲德菌素 A (BFA) 抑制的鸟嘌呤核苷酸交换蛋白 BIG1 和/或 BIG2 是否是 HUVEC 释放 TNFR1 所必需的。 RNA干扰（RNAi）显示BIG2而非BIG1调节TNFR1外泌体样囊泡的释放，而IL-1b诱导的TNFR1胞外域蛋白水解裂解不需要BIG2和BIG1。 BIG2 与 TNFR1 共定位于细胞质囊泡中，并且 BIG2 和 TNFR1 之间的关联被 BFA 破坏。与 BIG2 对 I 类 ARF 的优先激活一致，ARF1 和 ARF3 以非冗余和附加的方式参与 TNFR1 外泌体样囊泡的细胞外释放。因此，我们发现 BIG2 和 TNFR1 之间的关联通过 ARF1 和 ARF3 依赖性机制选择性调节 TNFR1 外泌体样囊泡的细胞外释放，但不影响 TNFR1 胞外域的诱导性蛋白水解切割 (JBC 2007; 282: 9591 - 9599）。二. BIG2 包含三个 A 激酶锚定蛋白 (AKAP) 结构域，可以协调 cAMP 和 ARF 调节功能。我们假设 BIG2 可能通过其 AKAP 及其 Sec7 结构域调节 TNFR1 外泌体样囊泡的释放。我们发现 8-Br-cAMP 通过 PKA 依赖性机制诱导外泌体样囊泡内全长 55-kDa TNFR1 的释放。 RNA 干扰实验表明，RIIb 调节 TNFR1 外泌体样囊泡的组成型释放和 cAMP 诱导的释放。与其 AKAP 功能一致，BIG2 是 cAMP 诱导的 PKA 依赖性 TNFR1 外泌体样囊泡释放所必需的，其机制涉及 RIIb 与 BIG2 AKAP 结构域 B 和 C 的结合。这表明组成型和 cAMP- TNFR1 外泌体样囊泡的诱导释放是通过 PKA 依赖性途径发生的，该途径受 RIIb 与 BIG2 锚定的调节因此，BIG2 通过两种不同的机制调节 TNFR1 外泌体样囊泡的释放，作为激活 I 类 ARF 的鸟嘌呤核苷酸交换蛋白，以及作为 RIIb 的 AKAP，将 PKA 信号定位在细胞 TNFR1 运输途径中。 JBC 2008；283：25364-71）。三．免疫共沉淀实验确定 RBMX（RNA 结合基序基因，X 染色体）是一种 ARTS-1 相关蛋白，可调节 TNFR1 外泌体样囊泡的组成型释放和 TNFR1 胞外域的诱导性蛋白水解切割 (BBRC 2008; 371: 505-9）。四．由于 TNFR1 外泌体样纳米囊泡是由 HUVEC 释放的，因此我们假设它们可能在人类血液中循环并调节 TNF 介导的炎症。通过免疫电子显微镜在人血清中证实了 TNFR1 外泌体样囊泡。人血浆蛋白质印迹显示 48 kDa TNFR1，这与膜相关受体一致。凝胶排阻色谱显示，人血浆中的 48 kDa TNFR1 不会与可溶性蛋白质分离，而是根据大小与 LDL 颗粒共分离。人血浆中的 48 kDa TNFR1 通过峰值密度与 LDL 颗粒独立分离，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与人血浆的 HDL 部分共分离，这表明 TNFR1 外泌体样囊泡与典型的外泌体不同。这表明人血浆中含有 48-kDa TNFR1 外泌体样囊泡，它们与 LDL 颗粒分离但又不同，并且与血浆或内皮细胞来源的外泌体样囊泡相比显示出独特的特征 (BBRC 2008 366: 579 - 584）。 V. 研究了介导 TNFR1 释放到细胞外空间的 Toll 样受体 (TLR) 信号通路的作用。我们发现，病毒双链 RNA (dsRNA) 类似物 Poly (I:C) 选择性诱导 34-kDa 可溶性 TNFR1 胞外域的裂解和脱落，但不会增强全长 55-kDa TNFR1 的释放。来自人气道上皮细胞的外泌体样囊泡。 Poly (I:C) 介导的 sTNFR1 脱落增加是通过 TLR3 介导的，而其他 Toll 样受体的配体（包括 TLR4、TLR7 和 NOD2）则不然。此外，我们发现聚 (I:C) 诱导的 sTNFR1 释放是通过两条 TLR3-TRIF-RIP1 依赖性途径介导的。一种途径涉及 Duox2 介导的活性氧 (ROS) 生成，而第二种途径是通过 caspase 介导的细胞凋亡激活。我们得出的结论是，病毒 dsRNA 诱导的人支气管上皮细胞 34-kDa sTNFR1 胞外域的脱落代表了一种调节对病毒感染的先天免疫反应的新机制 (JImmunol 2011; 186:1180-1188)。