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• W2070157035 abstract "The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both 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 human umbilical vein endothelial cells. 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-1β-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 nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism. The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both 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 human umbilical vein endothelial cells. 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-1β-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 nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism. Tumor necrosis factor (TNF) 2The abbreviations used are: TNF, tumor necrosis factor; AP-1, adaptor protein 1; ARF, ADP-ribosylation factor; ARF-GEP, ADP-ribosylation factor guanine nucleotide-exchange protein; ARTS-1, aminopeptidase regulator of TNFR1 shedding; BFA, brefeldin A; BIG2, BFA-inhibited guanine nucleotide-exchange factor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEP, guanine nucleotide-exchange protein; GGA, Golgi-associated, γ-adaptin ear-containing, ARF-binding protein; HUVEC, human umbilical vein endothelial cells; IL-1β, interleukin-1β, NUCB2, nucleobindin 2 (NEFA); siRNA, small interfering RNA duplexes; TGN, trans-Golgi network; TNFR1, Type I 55-kDa TNF receptor (TNFRSF1A); PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; WT, wild type. is an important regulator of inflammation, apoptosis, and innate immune responses. TNF signals through the type I 55-kDa (TNFR1, TNFRSF1A, CD120a) and type II 75-kDa (TNFR2, TNFRSF1B, CD120b) TNF receptors (1Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Crossref PubMed Scopus (1505) Google Scholar, 2Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3038) Google Scholar, 3Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1910) Google Scholar). TNFR1, which contains death domains in its intracytoplasmic tail, is considered the major receptor for TNF signaling (3Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1910) Google Scholar, 4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1131) Google Scholar). TNFR1 is also released from cells to the extracellular space and thereby modulates TNF bioactivity. Release of TNFR1 to the extracellular space is mediated by two distinct mechanisms. The first involves the proteolytic cleavage of TNFR1 ectodomains by a receptor sheddase that results in the shedding of soluble TNFR1 ectodomains (5Engelmann H. Aderka D. Rubinstein M. Rotman D. Wallach D. J. Biol. Chem. 1989; 264: 11974-11980Abstract Full Text PDF PubMed Google Scholar, 6Nophar Y. Kemper O. Brakebusch C. Engelmann H. Zwang R. Aderka D. Holtmann H. Wallach D. EMBO J. 1990; 9: 3269-3278Crossref PubMed Scopus (288) Google Scholar, 7Olsson I. Lantz M. Nilsson E. Peetre C. Thysell H. Grubb A. Adolf G. Eur. J. Haematol. 1989; 42: 270-275Crossref PubMed Scopus (232) Google Scholar, 8Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Granger G.A. Lentz R. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (849) Google Scholar, 9Seckinger P. Isaaz S. Dayer J.M. J. Biol. Chem. 1989; 264: 11966-11973Abstract Full Text PDF PubMed Google Scholar). The major site of TNFR1 cleavage is in the spacer region adjacent to the transmembrane domain between Asn-172 and Val-173, and a minor site is between Lys-174 and Gly-175 (8Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Granger G.A. Lentz R. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (849) Google Scholar, 10Brakebusch C. Varfolomeev E.E. Batkin M. Wallach D. J. Biol. Chem. 1994; 269: 32488-32496Abstract Full Text PDF PubMed Google Scholar, 11Wallach D. Aderka D. Engelmann H. Nophar Y. Kemper O. Holtmann H. Brakebusch C. Villa S. Gondi F.G. Bucciarelli U. Brakebusch C. Osawa T. Bonavida B. Tumor Necrosis Factor: Structure-Function Relationship and Clinical Application. Karger, Basel1991: 47-57Google Scholar). TACE was identified as a TNFR1 sheddase because TACE-deficient cells had lower ratios of shed to cell surface TNFR1 than did TACE-reconstituted cells (12Reddy P. Slack J.L. Davis R. Cerretti D.P. Kozlosky C.J. Blanton R.A. Shows D. Peschon J.J. Black R.A. J. Biol. Chem. 2000; 275: 14608-14614Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Proteolytic cleavage and shedding of soluble TNFR1 can be induced by diverse stimuli, such as phorbol ester, interleukin-1β, and proteasome inhibitors (13Lantz M. Gullberg U. Nilsson E. Olsson I. J. Clin. Investig. 1990; 86: 1396-1402Crossref PubMed Scopus (229) Google Scholar, 14Levine S.J. Adamik B. Hawari F.I. Islam A. Yu Z.X. Liao D.W. Zhang J. Cui X. Rouhani F.N. Am. J. Physiol. Lung Cell Mol. Physiol. 2005; 289: L233-L243Crossref PubMed Scopus (21) Google Scholar, 15Levine S.J. Logun C. Chopra D.P. Rhim J.S. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 254-261Crossref PubMed Scopus (42) Google Scholar). The second mechanism is the constitutive release of full-length TNFR1 from cells within membranes of exosome-like vesicles (16Hawari F.I. Rouhani F.N. Cui X. Yu Z.X. Buckley C. Kaler M. Levine S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1297-1302Crossref PubMed Scopus (178) Google Scholar). Exosomes are small membrane-enclosed vesicles, 30–200 nm in diameter, that correspond to the internal vesicles of endolysosome-related multivesicular bodies and are released via exocytic fusion with the plasma membrane (17Denzer K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar, 18Gould S.J. Booth A.M. Hildreth J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10592-10597Crossref PubMed Scopus (470) Google Scholar, 19Raposo G. Nijman H.W. Stoorvogel W. Leijendekker R. Harding C.V. Melief C.J.M. Geuze H.J. J. Exp. Med. 1998; 183: 1161-1172Crossref Scopus (2517) Google Scholar, 20Théry C. Zitvogel L. Amigorena S. Nat. Rev. Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3835) Google Scholar). TNFR1 exosome-like vesicles were initially identified in conditioned medium from human umbilical vein endothelial cells (HUVEC), which contained 20–50-nm exosome-like vesicles that were pelleted by high speed centrifugation, sedimented to a density of 1.1 g/ml, and were capable of binding TNF (16Hawari F.I. Rouhani F.N. Cui X. Yu Z.X. Buckley C. Kaler M. Levine S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1297-1302Crossref PubMed Scopus (178) Google Scholar). Recent investigations have identified previously unrecognized pathways for regulation of TNFR1 release from cells to the extracellular space. For example, histamine has been reported to induce redistribution of TNFR1 to the cell surface from an intracellular storage pool, such as the Golgi system, which served as a source of proteolytically cleaved receptors (21Wang J. Al-Lamki R.S. Zhang H. Kirkiles-Smith N. Gaeta M.L. Thiru S. Pober J.S. Bradley J.R. J. Biol. Chem. 2003; 278: 21751-21760Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We showed that calcium-dependent formation of a complex comprising ARTS-1 (aminopeptidase regulator of TNF receptor shedding), a type II integral membrane aminopeptidase, and NUCB2 (nucleobindin 2), a putative DNA- and calcium-binding protein, associates with cytoplasmic TNFR1 prior to the commitment of TNFR1 to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains (22Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F.N. Miskinis D. Levine S.J. J. Clin. Investig. 2002; 110: 515-526Crossref PubMed Scopus (193) Google Scholar, 23Islam A. Adamik B. Hawari F.I. Ma G. Rouhani F.N. Zhang J. Levine S.J. J. Biol. Chem. 2006; 281: 6860-6873Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Taken together, these findings suggested the probable involvement of intracytoplasmic vesicular trafficking between ERGolgi and cell surface plasma membranes in these processes. Initiation of vesicle formation from a donor membrane (e.g. Golgi) requires activation of a 20-kDa ADP-ribosylation factor (ARF) by interaction with a guanine-nucleotide exchange protein (GEP) that accelerates release of GDP and thereby GTP binding (24Moss J. Vaughan M. J. Biol. Chem. 1998; 273: 21431-21434Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Accumulation of ARF-GTP and coatomer (plus other proteins) leads to membrane deformation or budding. A vesicle, released by sealing off the bud at its base, is translocated to and fuses with a target membrane. Vesicular transport from the Golgi or trans-Golgi network to the plasma membrane in mammalian cells is known to involve the class I ARFs 1 and 3, which are activated by the brefeldin A-inhibited ARF-GEPs, BIG1 and BIG2 (25Togawa A. Morinaga N. Ogasawara M. Moss J. Vaughan M. J. Biol. Chem. 1999; 274: 12308-12315Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 26Yamaji R. Adamik R. Takeda K. Togawa A. Pacheco-Rodriguez G. Ferrans V.J. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2567-2572Crossref PubMed Scopus (99) Google Scholar). The experiments reported here demonstrate that BIG2, but not BIG1, was required for the constitutive release of full-length TNFR1 in exosome-like vesicles from human vascular endothelial cells. Cells and Reagents—HUVEC (passages 3 and 8) and EGM-2 medium were purchased from Cambrex BioScience (Walkersville, MD). Recombinant human IL-1β wasfromR&D Systems (Minneapolis, MN) and brefeldin A (BFA) was from MP Biomedicals (Aurora, OH). Antibodies—Chicken polyclonal anti-NUCB2 antibodies were generated against a glutathione S-transferase fusion protein with sequence corresponding to amino acids 326–420 of the NUCB2 C-terminal leucine zipper domain (Sigma Genosys) (23Islam A. Adamik B. Hawari F.I. Ma G. Rouhani F.N. Zhang J. Levine S.J. J. Biol. Chem. 2006; 281: 6860-6873Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Murine IgG2b monoclonal (H5) and goat polyclonal (C20) antibodies that reacted with TNFR1 were from Santa Cruz Biotechnology (Santa Cruz, CA), as were antibodies against β-tubulin (D10). Rabbit polyclonal antibodies against ARTS-1, BIG1, and BIG2 were used as previously described (27Shen X. Xu K.F. Fan Q. Pacheco-Rodriguez G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2635-2640Crossref PubMed Scopus (49) Google Scholar). Specific murine monoclonal antibodies reactive with human ARF1, ARF3, ARF5, and ARF6, respectively, were from Stressgen Bioreagents (Victoria, Canada), BD Biosciences (Palo Alto, CA), Abnova Corp. (Taipei, Taiwan), and Chemicon International (Temecula, CA). RNA Interference and Quantitative Real Time RT-PCR—Individual siGENOME RNA duplexes for BIG1 and BIG2, siGENOME SMARTpool RNA duplexes for ARF1, ARF3, ARF5, and ARF6, as well as siCONTROL nontargeting siRNA 1 were purchased from Dharmacon (Lafayette, CO). HUVEC were transfected with siRNA (50–100 nm) using DharmaFECT 1 transfection reagent (Dharmacon) for 3 days. RNA was isolated with an RNeasy Mini kit (Qiagen, Valencia, CA), and cDNA templates were prepared with a ProSTAR Ultra-HF RT-PCR system (Stratagene, Cedar Creek, TX). Sequences of RT-PCR primers are listed in supplemental Table S1. Quantitative real time RT-PCR was performed using the iCycler iQ thermocycler, iQ SYBR green PCR supermix, and iCycler iQ data analysis software (Bio-Rad). BIG2, ARF1, and ARF3 Expression Plasmids—Mammalian expression plasmids encoding full-length human BIG2, ARF1, or ARF3 were purchased from Origene Technologies (Rockville, MD). The BIG2 cDNA clone contained 7 nucleotide substitutions that differed from the ARFGEF2 reference sequence (NCBI accession NM_006420). Among these putative polymorphisms, two represented silent mutations (C3663T, C4131T), while 5 represented nonsynonymous mutations (A619G, G620A, A2884G, A3145G, and A5287G) that resulted in 4 amino acid substitutions. The 5 nonsynonymous mutations were corrected by site-directed mutagenesis using the QuikChange Multi Site-directed Mutagenesis kit (Stratagene). All plasmids were sequence-verified. The T31N mutations were introduced using the Quik-Change Multi Site-directed Mutagenesis kit and the following primers; ARF1: 5′-CTGCAGGGAAGAACACGATCCTCTACAAG-3′ and ARF3: 5′-CGCAGGAACGAACACCATCCTATACAAG-3′. RNAi-resistant BIG2, ARF1, and ARF3 mutant plasmids, which contained synonymous mutations, were generated by site-directed mutagenesis using the following primers; BIG2: 5′-ATTTATGTCAATTATGATTGCGATTTAAATGCTGC-3′, ARF1: 5′-CAGCAATGACAGGGAACGAGTAAACGAGGCCCG-3′, and ARF3: 5′-CAGGAAAGACGACGATACTGTACAAGCTGAAACTGGGGG-3′. Mutations were confirmed by sequencing. HUVEC, grown in 6-well plates, were transfected with plasmids using FuGENE 6, according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN). For co-transfection, individual siGENOME siRNA duplexes (Dharmacon) targeting BIG2 (5′-CAACUACGACUGUGAUUUA-3′) ARF1 (5′-ACAGAGAGCGUGUGAACGA-3′), or ARF3 (5′-GGAAAGACCACCAUCCUAU-3′) were transfected at a concentration of 100 nm using the DharmaFECT 1 transfection reagent. Immunoblotting—HUVEC were lysed in buffer containing 1% Triton X-100, 1% n-octyl β-d -glucopyranoside, 50 mm Tris, pH 7.5, and 120 mm NaCl (Sigma), supplemented with Complete™ protease inhibitor (Roche Applied Science, Indianapolis, IN). For immunoblots of HUVEC-conditioned medium, cells were grown in medium that contained fetal bovine serum that had been depleted of exosomes by centrifugation at 175,000 × g at 4 °C for 16 h. Prior to immunoblotting, conditioned medium was cleared of cells and debris by sequential centrifugation at 200 × g for 10 min, 500 × g for 10 min, 1,200 × g for 20 min, and 10,000 × g for 30 min. Immunoblots of cellular proteins (50 μg per lane) or medium (26 μl per lane) were performed as previously described, using NIH Image Software (version 1.63) for densitometry (23Islam A. Adamik B. Hawari F.I. Ma G. Rouhani F.N. Zhang J. Levine S.J. J. Biol. Chem. 2006; 281: 6860-6873Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Immunoprecipitation—HUVEC were lysed in buffer containing 0.1% Triton X-100, 50 mm Tris, pH 7.5, and 120 mm NaCl (Sigma) supplemented with Complete™ protease inhibitor. Samples (200 μg) of HUVEC lysates were incubated for 2 h with 8 μg of rabbit anti-BIG2 antibodies immobilized on 100 μl of protein A/G beads (Pierce) that had been blocked with 1% ovalbumin in PBS. Controls were protein A/G beads incubated with preimmune serum or without bound antibodies. Beads were washed six times with cold lysis buffer, and immunoblots were performed as previously described (23Islam A. Adamik B. Hawari F.I. Ma G. Rouhani F.N. Zhang J. Levine S.J. J. Biol. Chem. 2006; 281: 6860-6873Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Proteins from HUVEC supernatants after immunoprecipitation were precipitated with 10% trichloroacetic acid for immunoblotting. Immunofluorescence Confocal Laser Scanning Microscopy—HUVEC grown on collagen I-coated slides (BD Biosciences) were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS, permeabilized with 0.1% saponin in PBS (5 min), washed three times with PBS, and blocked with PBS containing 10% donkey and 10% goat serum for 1 h. Cells were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 1% donkey and 1% goat serum, as follows: goat anti-TNFR1 (C20) polyclonal antibodies (2 μg/ml), rabbit polyclonal anti-BIG2 antibodies (diluted 1:500), and mouse monoclonal anti-p230 trans-Golgi antibody (1:200). After washing three times in PBS containing 0.1% bovine serum albumin and incubation with species-specific secondary antibodies conjugated to Alexa Fluor® 488 or Alexa Fluor® 568, (Molecular Probes, Eugene, OR, 1:200 dilution), cells were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and visualized using a Leica SP Laser Scanning Confocal Microscope (Leica, Heidelberg, Germany). Quantification of Extracellular TNFR1 by ELISA—HUVEC were transfected with siRNA for 3 days, and then fresh, exosome-depleted medium was added for 24 h. Medium cleared of cells and debris as described for immunoblotting was analyzed for TNFR1 using a Quantikine sandwich ELISA kit with a sensitivity of 7.8 pg/ml (R & D Systems). Statistical Analyses—Data were analyzed by a paired Student's t test with a Bonferoni correction for multiple comparisons or analysis of variance. A p value ≤0.05 was considered significant. BIG2 Regulates the Constitutive Release of TNFR1 Exosomelike Vesicles—The role of BIG1 and BIG2 in the constitutive release of TNFR1 exosome-like vesicles from HUVEC was assessed using RNA interference. Small interfering RNA duplexes (siRNA) specifically decreased levels of BIG1 and BIG2 mRNA and protein (Fig. 1, A and B), but did not alter the quantities of mRNA (data not shown) or protein (Fig. 1B) encoding NUCB2 or β-tubulin. As quantified by ELISA, TNFR1 constitutively released into the medium from cells transfected with siRNA targeting BIG2 was 63% less than that from cells transfected with control non-targeting siRNA (Fig. 1C). Similarly, Western blots showed that medium from cells transfected with siRNA targeting BIG2 contained 69% less 55-kDa TNFR1 than did medium from cells transfected with non-targeting siRNA (p = 0.017, n = 4) (Fig. 1D). A ∼40-kDa TNFR1 band was also seen, as has been previously described, which was similarly decreased (23Islam A. Adamik B. Hawari F.I. Ma G. Rouhani F.N. Zhang J. Levine S.J. J. Biol. Chem. 2006; 281: 6860-6873Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In contrast, no effects of siRNA targeting BIG1 were found. Neither BIG1 nor BIG2 Affects the IL-1β-induced Proteolytic Cleavage of TNFR1 Ectodomains—In experiments performed to assess the role of BIG1 or BIG2 in the inducible proteolytic cleavage of TNFR1 ectodomains, transfection of HUVEC with siRNA targeting BIG1 or BIG2 did not reduce the quantity of TNFR1 released into medium following 2-h stimulation with IL-1β, as measured by ELISA (Fig. 2A) or Western blotting (Fig. 2B). The quantity of full-length 55-kDa TNFR1 released in exosome-like vesicles into medium in 2 h was below the limit of detection by Western blotting. Endogenous BIG2 Associated with TNFR1 in HUVEC—To determine whether the action of BIG2 in the constitutive release of TNFR1 exosome-like vesicles involved an association between the endogenous proteins, BIG2 was immunoprecipitated from HUVEC homogenates, and the full-length, 55-kDa TNFR1 was detected by immunoblotting, but neither ARTS-1 nor NUCB2 were present (Fig. 3A). The BIG2 antibodies immunoprecipitated 48% of BIG2, but only 16% of the TNFR1 was pulled-down (Fig. 3B), consistent with the association of a subpopulation of endogenous TNFR1 with BIG2. The association between BIG2 and TNFR1 in HUVEC was also characterized by confocal microscopy. As shown in Fig. 3C, TNFR1 and BIG2 were co-localized in diffusely distributed cytoplasmic vesicles. Although BIG2 had been described in the trans-Golgi network, neither BIG2 nor TNFR1 co-localized with the trans-Golgi marker, p230 (data not shown). Because BIG2 is a BFA-inhibited activator of ARF, we looked for a BFA effect on the association between endogenous BIG2 and TNFR1. As shown in Fig. 4, no full-length, 55-kDa TNFR1 was found among proteins immunoprecipitated by BIG2 antibodies from cells incubated for 15 min with BFA. This showed that the association between endogenous BIG2 and TNFR1 was disrupted by BFA. BIG2-dependent Release of TNFR1 Exosome-like Vesicles Involves ARF1 and ARF3 in a Nonredundant and Additive Fashion—BIG2 activates both ARF1 and ARF3 (25Togawa A. Morinaga N. Ogasawara M. Moss J. Vaughan M. J. Biol. Chem. 1999; 274: 12308-12315Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 28Shin H.W. Morinaga N. Noda M. Nakayama K. Mol. Biol. Cell. 2004; 15: 5283-5294Crossref PubMed Scopus (107) Google Scholar). To assess the roles of individual ARFs in the constitutive release of TNFR1 exosome-like vesicles, ARF1, ARF3, ARF5, or ARF6 mRNA and protein were selectively depleted using RNA interference (Fig. 5, A and C), while neither TNFR1 nor β-tubulin mRNA (data not shown) and protein (Fig. 5C) were decreased. Treatment of HUVEC with siRNA targeting ARF1 or ARF3 decreased by 59 and 60%, respectively, the amount (quantified by ELISA) of TNFR1 released relative to that from cells treated with control, non-targeting siRNA (Fig. 5B). In contrast, siRNA-mediated depletion of ARF5 or ARF6 had no effect. Quantification of Western blots showed that the siRNA-mediated depletion of either ARF1 or ARF3 reduced the amount of constitutively released 55-kDa TNFR1 exosome-like vesicles into the culture medium by 67% (p = 0.008, n = 3) and 55% (p = 0.016, n = 3) respectively, from that released by cells transfected with non-targeting siRNA (Fig. 5C). We next assessed whether ARF1 and ARF3 function in an additive fashion to mediate the extracellular release of TNFR1 exosome-like vesicles. HUVEC transfected with siRNAs targeting both ARF1 and ARF3 showed specific depletion of both mRNAs (data not shown) and proteins (Fig. 5E), while β-tubulin mRNA (data not shown) and protein (Fig. 5E) were not decreased. As shown in Fig. 5D, the amount of TNFR1 constitutively released from cells transfected with siRNAs targeting both ARF1 and ARF3 was 82% less than that from cells transfected with control, non-targeting siRNA. The quantity of TNFR1 constitutively released into culture medium was also significantly less from cells transfected with siRNAs targeting both ARF1 and ARF3 than from cells treated with siRNAs individually targeting ARF1 or ARF3. Western blots (Fig. 5E) confirmed that the siRNA-mediated depletion of both ARF1 and ARF3 reduced the constitutive release of the 55-kDa TNFR1 exosome-like vesicles by 90% (p = 9.04 × 10–6, n = 3), from the level of cells treated with control, non-targeting siRNA. Similarly, Western blots (Fig. 5E) showed that the siRNA-mediated depletion of both ARF1 and ARF3 significantly reduced the constitutive release of the 55-kDa TNFR1 exosome-like vesicles as compared with cells treated with siRNAs individually targeting ARF1 or ARF3 (p = 0.00013, n = 3). To confirm that BIG2 activation of ARF1 and ARF3 mediates the release of TNFR1 exosome-like vesicles from HUVEC, cells were transfected with plasmids expressing either wild-type or guanine nucleotide exchange-defective (T31N) ARF1 or ARF3 mutants. The T31N mutants exhibit minimal GTP binding and are preferentially constrained to the GDP-bound inactive form (29Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar, 30Furman C. Short S.M. Subramanian R.R. Zetter B.R. Roberts T.M. J. Biol. Chem. 2002; 277: 7962-7969Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Quantification of Western blots of cell lysates (Fig. 6A) showed levels of immunoreactive ARF1 and ARF3, respectively, 205% (p = 0.015, n = 3) and 174% (p = 0.043, n = 3) those of cells transfected with the empty (control) plasmid. These were associated with increases of 106% (p = 0.0009, n = 3) and 116% (p = 0.0008, n = 3), respectively, in the amounts of 55-kDa TNFR1 exosome-like vesicles that were constitutively released into the culture medium (Fig. 6, A and B). Similarly, quantification by ELISA (Fig. 6C) showed that overexpression of ARF1 or ARF3 was associated with respective increases of 86% (p < 10–9, n = 6) and 89% (p < 10–12, n = 6) in the amount of TNFR1 released into the culture medium. In contrast, overexpression of the ARF1(T31N) or ARF3(T31N) plasmids did not alter TNFR1 exosome-like vesicle release. Immunoprecipitations were next performed to assess the effect of the ARF1(T31N) and ARF3(T31N) mutants on the association between BIG2 and TNFR1. Consistent with the lack of a dominant-negative effect on the release of TNFR1 exosome-like vesicles, the mutants did not disrupt the association between BIG2 and TNFR1 (Fig. 6D). Although ARF1(T31N) and ARF3(T31N) mutants have previously been demonstrated to function as dominant-negative constructs, which occurs via sequestering of relevant ARF-GEPs (29Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar), this was not the case in our system where their behavior was consistent with a GDP-bound inactive form. Our findings are similar to a prior report investigating secretory vesicle formation in pituitary cells, which also found that ARF1(T31N) functioned as an inactive ARF, rather than as a dominant-negative mutant (31Chen Y.G. Shields D. J. Biol. Chem. 1996; 271: 5297-5300Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). This study hypothesized that the T31N mutant could not undergo a GTP-induced conformational change, which resulted in impaired membrane localization, so that the mutant was inaccessible to compete with proteins binding to the ARF effector domain (31Chen Y.G. Shields D. J. Biol. Chem. 1996; 271: 5297-5300Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The reason why overexpression of ARF1(T31N) and ARF3(T31N) mutants did not produce a dominant-negative effect in our system is unclear. Our data suggest that a sufficient quantity of BIG2 was not sequestered by the ARF (T31N) mutants and therefore remained available to mediate the association between BIG2 and TNFR1, as well as the release of TNFR1 exosome-like vesicles. This interpretation is consistent with our finding that only a subset of BIG2 associates with TNFR1 in HUVEC. RNAi-resistant BIG2, ARF1, and ARF3 expression plasmids with 4 silent single base pair mutations in the region targeted by siRNA were used to confirm the specificity of the observed siRNA effects. BIG2, ARF1, and ARF3 proteins were detected in lysates from HUVEC co-transfected with siRNA and RNAi-resistant expression plasmids, whereas BIG2, ARF1, and ARF3 protein levels were markedly diminished in cells co-transfected with siRNA and plasmids expressing wild-type BIG2, ARF1, or ARF3 (Fig. 7, A–C). We next assessed if the effect of BIG2, ARF1, and ARF3 siRNAs on TNFR1 exosome-like vesicle release were specific and not caused by off-target effects. Medium from HUVEC co-transfected with the empty (control) plasmid and siRNA targeting BIG2, ARF1, or ARF3 contained significantly less TNFR1, quantified by ELISA, than those transfected with the empty (control) plasmid in the absence of siRNA (Fig. 7, D–F). Reductions in TNFR1 release by siRNAs targeting BIG2, ARF1, or ARF3 were rescued by co-transfection with RNAi-resistant, but not the wild-type, BIG2, ARF1, or ARF3 plasmids. Western blots confir" @default.
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• W2070157035 date "2007-03-01" @default.
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• W2070157035 title "The Brefeldin A-inhibited Guanine Nucleotide-exchange Protein, BIG2, Regulates the Constitutive Release of TNFR1 Exosome-like Vesicles" @default.
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• W2070157035 doi "https://doi.org/10.1074/jbc.m607122200" @default.
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