Tumor necrosis factor (TNF) is a multifunctional cytokine that may play an important role in the pathogenesis of airway inflammation in asthma. TNF bioactivity is regulated by soluble TNF receptors that serve as TNF binding proteins.
The specific aim of this project is to identify new mechanisms by which the release of soluble TNFR is regulated. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Utilizing a yeast two-hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNFR1 Shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by co-immunoprecipitation experiments utilizing human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells over-expressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing anti-sense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a GST-ARTS-1 fusion protein demonstrated selective aminopeptidase activity towards non-polar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These findings indicate that ARTS-1 is a multi-functional ectoprotein capable of binding to and promoting TNFR1 shedding. Therefore, we have proposed that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated. Since both release of both soluble TNFR1 and IL-6 receptor-alpha (IL-6R) can be inhibited by hydroxamic acid based zinc metalloprotease inhibitors, we hypothesized that ARTS-1 might also regulate IL-6R shedding. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6R, a size consistent with soluble IL-6R generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6R shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R shedding, and decreased membrane-associated IL-6R in cell lines overexpressing ARTS-1. The absence of basal IL-6R shedding from ARTS-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6R shedding. Furthermore, the mechanism of constitutive IL-6R shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multi-functional aminopeptidase that may modulate inflammatory events by promoting the release of soluble receptors from at least two cytokine receptor superfamilies. Therefore, ARTS-1 may play an important role in regulating inflammatory events in the airway. Ongoing investigations will be aimed at identifying the mechanism by which ARTS-1 promotes cytokine receptor shedding.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL002544-05
Application #
6809765
Study Section
(PCCM)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Adamik, Barbara; Islam, Aminul; Rouhani, Farshid N et al. (2008) An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release. Biochem Biophys Res Commun 371:505-9
Islam, Aminul; Shen, Xiaoyan; Hiroi, Toyoko et al. (2007) The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles. J Biol Chem 282:9591-9
Islam, Aminul; Adamik, Barbara; Hawari, Feras I et al. (2006) Extracellular TNFR1 release requires the calcium-dependent formation of a nucleobindin 2-ARTS-1 complex. J Biol Chem 281:6860-73
Buckley, Caitriona A; Rouhani, Farshid N; Kaler, Maryann et al. (2005) Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch. Am J Physiol Lung Cell Mol Physiol 288:L1132-8
Rouhani, Farshid N; Meitin, Catherine A; Kaler, Maryann et al. (2005) Effect of tumor necrosis factor antagonism on allergen-mediated asthmatic airway inflammation. Respir Med 99:1175-82
Levine, Stewart J; Adamik, Barbara; Hawari, Feras I et al. (2005) Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 289:L233-43
Levine, Stewart J (2004) Mechanisms of soluble cytokine receptor generation. J Immunol 173:5343-8
Hawari, Feras I; Rouhani, Farshid N; Cui, Xinle et al. (2004) Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors. Proc Natl Acad Sci U S A 101:1297-302
Cui, Xinle; Rouhani, Farshid N; Hawari, Feras et al. (2003) An aminopeptidase, ARTS-1, is required for interleukin-6 receptor shedding. J Biol Chem 278:28677-85
Cui, Xinle; Rouhani, Farshid N; Hawari, Feras et al. (2003) Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding. J Immunol 171:6814-9

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