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.
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