An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding*

Xinle Cui, Farshid N. Rouhani {ddagger}, Feras Hawari {ddagger} and Stewart J. Levine §

From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590

Received for publication, January 15, 2003 , and in revised form, May 10, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6R{alpha}), we hypothesized that ARTS-1 might also regulate shedding of IL-6R{alpha}, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6R{alpha}, a size consistent with soluble IL-6R{alpha} generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6R{alpha} shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R{alpha} shedding, and decreased membrane-associated IL-6R{alpha} in cell lines overexpressing ARTS-1. The absence of basal IL-6R{alpha} shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6R{alpha} shedding. Furthermore, the mechanism of constitutive IL-6R{alpha} 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{alpha}) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6R{alpha} and TNFR1 shedding.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although interleukin-6 (IL-6)1 was originally identified as a B cell differentiation factor, it is now recognized to function as a pleiotropic cytokine capable of modulating a variety of immune and inflammatory responses (e.g. the hepatic acute phase response, T cell activation, bone metabolism, and hematopoiesis) (14). IL-6 production occurs in response to infection, trauma, and immunological challenge and may contribute to the pathogenesis of a number of diseases (e.g. autoimmune disorders, multiple myeloma, osteoporosis, Crohn's disease, rheumatoid arthritis, and congestive heart disease) (2, 3, 5). In addition to its pro-inflammatory effects, IL-6 has anti-inflammatory functions, such as the suppression of endotoxin-mediated neutrophil recruitment, induction of interleukin-1 receptor antagonist expression, and stimulation of shedding of the 55-kDa, type I tumor necrosis factor receptor (TNFR1, CD120a) as a soluble TNF-binding protein (68).

The IL-6 receptor complex comprises two distinct membrane-bound glycoproteins, the 80-kDa IL-6 {alpha}-receptor subunit (IL-6R{alpha}, CD 126) and the 130-kDa signal transducing subunit (gp130) (9). Following binding of IL-6 to IL-6R{alpha}, two IL-6/IL-6R{alpha} molecules form a complex with the homodimeric gp130, which mediates signaling via the activation of Janus kinases (JAK1, JAK2, and TYK2) that are constitutively associated with the gp130 intracytoplasmic tail (1012). Subsequent phosphorylation of members of the signal transducer and activator of transcription (STAT) family, such as STAT1 and STAT3, as well as the mitogen-activated protein kinase (MAPK) pathway, mediate activation of IL-6-responsive genes (1316).

Generation of soluble IL-6 receptors (sIL-6R{alpha}) represents an important mechanism by which IL-6 signaling can be amplified. Soluble IL-6 receptors bind IL-6 with an affinity similar to that of the membrane IL-6 receptor, thereby prolonging the half-life of IL-6 (17). Furthermore, binding of the sIL-6R{alpha}·IL-6 complex to membrane-bound gp130 confers IL-6 signaling capability to cells that do not express IL-6R{alpha}. Because of the ubiquitous expression of gp130, trans-signaling via the generation of sIL-6R{alpha}·IL-6 complexes can extend the repertoire of IL-6-responsive cell types (9, 18). For example, trans-signaling via sIL-6R{alpha}·IL-6 complexes has been identified as an important regulator of CXC and CC chemokine expression that contributes to the termination of neutrophil recruitment and the concurrent influx of mononuclear cells during acute inflammation secondary to bacterial infection (7). It is important to note, however, that the trans-signaling function of the sIL-6R{alpha}·IL-6 complex can be abrogated by the soluble form of gp130 (sgp130), which competes with membrane gp130 for sIL-6R·IL-6 complex binding (5, 19). Further, sIL-6R{alpha} can potentiate the antagonistic potential of sgp130. Therefore, soluble IL-6 receptors have the potential to serve either as IL-6 signaling agonists or, alternatively, as IL-6 signaling antagonists.

Soluble IL-6 receptors can be generated by two distinct pathways: proteolytic cleavage that sheds the membrane-bound IL-6R{alpha} ectodomain from the cell surface or differential mRNA splicing that results in the generation of an IL-6R{alpha} that lacks the transmembrane domain (2024). Proteolytic cleavage of IL-6R{alpha}, which occurs between Gln-357 and Asp-358, is strongly promoted by phorbol ester (22, 25). Experiments utilizing hydroxamic acid-based metalloprotease inhibitors have suggested that TACE (TNF-{alpha}-converting enzyme or ADAM 17), a member of the metalloprotease-disintegrin (ADAM) family of zinc metalloproteases, possesses IL-6R{alpha} sheddase activity (20, 2628). Further evidence supporting TACE-mediated IL-6R{alpha} shedding is the strong reduction in phorbol ester-induced IL-6R{alpha} shedding in TACE-deficient murine fibroblasts that can be rescued by reconstitution of TACE expression (25). However, the existence of additional IL-6R{alpha} sheddases has been suggested by a basal hydroxamate-sensitive IL-6R{alpha} shedding from TACE-deficient cells, as well as by IL-6R{alpha} shedding that is resistant to the hydroxamic acid-based metalloprotease inhibitor, TAPI (9, 14, 25).

The aminopeptidase regulator of TNFR1 shedding (ARTS-1) has recently been identified as a type II integral membrane protein that binds to the TNFR1 extracellular domain and promotes TNFR1 shedding (29). Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and IL-6R{alpha}, we hypothesized that ARTS-1 might also regulate IL-6R{alpha} shedding. By utilizing an arts-1 knock-out cell line (arts-1(/)), we demonstrate that ARTS-1 is required for constitutive IL-6R{alpha} shedding. Transfection of arts-1(/) cell lines with plasmids containing full-length ARTS-1 restored IL-6R{alpha} shedding, whereas transfection with ARTS-1 catalytic site mutants did not. These data indicate that the mechanism of constitutive IL-6R{alpha} shedding requires ARTS-1 catalytic activity. Furthermore, ARTS-1 directly binds to a 55-kDa IL-6R{alpha}, a size consistent with soluble IL-6R{alpha} generated by ectodomain cleavage of the membrane-bound receptor. Thus, ARTS-1 may modulate inflammatory events by promoting the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R{alpha}) and the TNF receptor superfamily (TNFR1).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ARTS-1/IL-6R{alpha} Co-immunoprecipitation and Immunoblotting—The NCI-H292 human pulmonary mucoepidermoid carcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% fetal bovine serum under 5% CO2 at 37 °C. Immunoprecipitation experiments were performed as described previously (29). Briefly, cells were harvested for membrane isolation by scraping and were disrupted by sonicating twice (for 10 s each) in lysis buffer (50 mM Tris-HCl, pH 7.2, 120 mM NaCl, 0.1% Triton X-100, and CompleteTM protease inhibitor (Roche Applied Science)), followed by centrifugation at 1,000 x g for 5 min to remove nuclei and cellular debris. Post-nuclear supernatants were centrifuged at 100,000 x g for1hto recover membrane pellets that were suspended by sonicating three times (for 2 s each) in lysis buffer. Protein concentrations were determined utilizing the BCA protein determination kit (Pierce). For immunoprecipitation, samples of membrane proteins (200 µg) were incubated overnight, 4 °C, with 20 µg of murine monoclonal anti-human IL-6R{alpha} antibody against the IL-6R{alpha} extracellular domain (R & D Systems) or 1 µl of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 µl of immobilized protein A/G beads (Pierce) for 2 h at room temperature. The ARTS-1 antibody recognizes a 17-amino acid epitope (RGRNVHMKQE-HYMKGSD) located in exon 11 (29). After the beads were washed 8 times, bound proteins were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated overnight (4 °C) with ARTS-1 immune or pre-immune serum diluted 1:20,000 or the murine anti-IL-6R{alpha} monoclonal antibody, 2 µg/ml. Detection was by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies.

For immunoblotting, samples of membrane proteins (20–40 µg) were separated via SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated overnight (4 °C) with either ARTS-1 immune serum, diluted 1:20,000, rabbit polyclonal anti-human IL-6R{alpha} (Santa Cruz Biotechnology), 1 µg/ml, or goat anti-MUC1 polyclonal antibody (Santa Cruz Biotechnology), 1 µg/ml. Detection was by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies.

ARTS-1 Cell Lines—Stably transfected NCI-H292 cell lines that expressed either full-length human ARTS-1 or antisense ARTS-1 (bases 61–213) were utilized, as described previously (29). To quantify membrane-associated IL-6R{alpha}, membrane fractions were prepared, as described above, and assays were performed on 300 µg of membrane proteins utilizing a sandwich ELISA with a sensitivity of 7.8 pg/ml (R & D Systems). sIL-6R{alpha} in cell culture supernatants was assayed by ELISA. Immunoblotting was performed on culture supernatants that were concentrated 30-fold utilizing a Centriprep filter with a 10-kDa exclusion (Amicon). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. TAPI-0, TAPI-1, and TAPI-2 were purchased from Peptides International. Statistical analysis was performed using a Student's t test with a Bonferroni correction for multiple comparisons. A p value of less than 0.05 was considered significant.

Construction of an arts-1 Knock-out NCI-H292 Cell Line—The arts-1 targeting vector was generated by assembling sequences flanking arts-1 exons 5 and 6 as the upstream and downstream arms of the PKO scrambler NTKV 1902 vector (Stratagene) (30). The following PCR primer pairs, which span exons 3 and 4, were utilized to generate the up-stream arm: primer A (5'-CCC-AAG-CTT-GGG-TTC-TCC-CTC-TGT-TAG-TCG-C-3') and primer B (5'-CCA-TCG-ATG-GTG-AAA-TGA-CAG-TTA-GAC-CCT-C-3'). Primer A contains a HindIII restriction site, whereas primer B contains a ClaI restriction site. The downstream arm, which spans exons 7 and 8, was generated utilizing the following primers: primer C (5'-CGG-GAT-CCC-GAT-TGT-TTC-TCC-AAA-GCA-TTC-GT-3'), which contains a BamHI restriction site, and primer D (5'-TCC-CCC-GGG-GGA-CAT-CAT-CTG-CCA-ACT-CCC-TTT-G-3'), which contains a SmaI restriction site.

Genomic DNA isolated from NCI-H292 cells was utilized as a template for PCR amplification of the 7468-bp upstream segments and the 2783-bp downstream segments utilizing Pfu turbo DNA polymerase (Stratagene). The PCR products were digested with the appropriate endonucleases to generate the 3841-bp upstream arm, based upon the presence of an internal HindIII restriction site, and the 2783 downstream arm. The cDNA segments were gel-purified and ligated into the polylinker region of the PKO scrambler NTKV 1902 vector, which contains both positive (neomycin phosphotransferase) and negative (thymidine kinase) selection markers. The neomycin phosphotransferase gene, in the antisense orientation, is driven by a phosphoglycerate kinase promoter, whereas the thymidine kinase gene, in the sense orientation, is driven by a polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. Sequences of the upstream and downstream arms were confirmed by DNA sequencing.

NCI-H292 cell lines were transfected with the arts-1 targeting vector using FuGENE 6 (Roche Applied Science) and maintained under selective pressure by addition of 20 mg/ml geneticin (Invitrogen) and 400 µM ganciclovir (Sigma) to medium followed by pH adjustment with NaHCO3. Transfected cells were cloned by limiting dilution, and clones containing homozygous deletions generated by homologous recombination were characterized by PCR and RT-PCR analysis of genomic DNA and mRNA utilizing the following primer pairs: Neo 1 sense, 5'-TTC-CTT-CCC-TGG-CAT-CTA-CCT-C-3', and arts-1 intron 8/9 antisense, 5'-TTC-CCG-CTT-CAG-TGA-CAA-CG-3'; Neo 2 sense, 5'-TCG-CCT-TCT-ATC-GCC-TTC-TTG-3', and arts-1 intron 10/11 antisense, 5'-AAA-AGA-ATG-TGC-TTG-GGG-GAA-C-3'; arts-1 exon 5/6 sense, 5'-CAG-TCA-TTG-TGA-TGC-CAA-G-3', and arts-1 exon 5/6 antisense, 5'-GCT-GTG-CCA-GAC-AAG-ATA-AAT-C-3'; arts-1 exon 15/17 sense, 5'-CTA-CTG-GGT-TCC-TGC-CAA-TGA-G-3', and arts-1 exon 15/17 antisense, 5'-TCA-ACT-ACT-ACT-CCT-CGC-CTG-TGT-G-3'; arts-1 exon 2/19 sense, 5'-CAT-GGT-GTC-AGA-GCA-CT-3', and arts-1 exon 2/19 antisense, 5'-CAT-ACG-TTC-AAG-CTT-TTC-3'; G3PDH sense, 5'-TGA-AGG-TCG-GAG-TCA-ACG-GAT-TTG-GT-3', and G3PDH antisense, 5'-CAT-GTG-GGC-CAT-GAG-GTC-CAC-CAC-3'.

RT-PCR of IL-6 mRNA was performed utilizing previously described primer pairs that span the IL-6R{alpha} transmembrane domain to generate a 398-bp product (24). The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers were purchased from Clontech. arts-1(/) cell lines were reconstituted by transient transfection with plasmids encoding either full-length ARTS-1 or ARTS-1 catalytic site mutants (H353P, H357V, and H353P/E354V), utilizing Gene Porter II and Booster (Gene Therapy Systems), as described previously (29).

IL-6R{alpha} Ectodomain Cleavage Assay—A model system was utilized to assess whether ARTS-1 catalyzes the proteolytic cleavage of the IL-6R{alpha} ectodomain. A recombinant glutathione S-transferase-ARTS-1 (GST-ARTS-1) fusion protein was synthesized in BL21 Escherichia coli transfected with a pGEX-6P-1 plasmid encoding the ARTS-1 extracellular domain and purified using glutathione-Sepharose 4B (Amersham Biosciences) (29). The GST-ARTS-1 was recovered from the insoluble fraction by denaturation with 6 M urea in phosphate-buffered saline and refolded by serial dialysis against phosphate-buffered saline containing decreasing urea concentrations. The GST-ARTS-1 fusion protein was demonstrated to be catalytically active and possess aminopeptidase activity against leucine, methionine, alanine, and phenylalanine p-nitroanilide model substrates. A 22-amino acid model peptide substrate (RDSANATSLPVQDSSSVPLPTF) containing the IL-6R{alpha} cleavage site was synthesized by Sigma-Genosys. Peptides corresponding to the 12-amino acid N-terminal cleavage product (RDSANATSLPVQ) and the 10-amino acid C-terminal cleavage product (DSSSVPLPTF) were also synthesized to serve as standards. The peptide substrate (2.5 µg/ml) was incubated with GST-ARTS-1 (200 ng/ml) for 2 h at 37 °C before transfer of 50 µl of reaction mixture to a Vydac (Hesperia, CA) C18 column (The Nest Group, Southborough, MA) equilibrated with solution A (0.1% trifluoroacetic acid, HPLC grade water). The mixture was then separated by gradient elution with solution B (0.1% trifluoroacetic acid, acetonitrile) at a flow rate of 0.8 ml/min: 100% solution A, 0–2 min, linear gradient 0–67.5% of solution B, 2–20 min. The absorbance of the eluate was recorded at 214 nm.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane-associated ARTS-1 Binds to IL-6R{alpha}Immunoprecipitation experiments were performed to assess whether an endogenous protein-protein interaction exists between ARTS-1 and IL-6R{alpha} in NCI-H292 cells. As shown in Fig. 1A, immunoprecipitation of membrane fractions with an anti-IL-6R{alpha} monoclonal antibody pulled down the 100-kDa ARTS-1 protein. In the reciprocal experiment (Fig. 1B), immunoprecipitation with ARTS-1 antiserum pulled down a 55-kDa IL-6R{alpha} species, which is consistent with the soluble form of IL-6R{alpha} generated by proteolytic cleavage of the IL-6R{alpha} extracellular domain. These experiments demonstrate that the 100-kDa membrane-associated ARTS-1 species binds the 55-kDa soluble form of IL-6R{alpha}.



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FIG. 1.
Binding of endogenous ARTS-1 to IL-6R{alpha} in NCI-H292 cells. A, co-immunoprecipitations (IP) were performed with either an anti-IL-6R{alpha} monoclonal antibody directed against the IL-6R{alpha} extracellular domain (+) or a murine IgG1 isotype control (IgG1) and immunoblotted (IB) with either anti-ARTS-1 preimmune (PI) or immune (+) serum. B, reciprocal co-immunoprecipitations were performed with either anti-ARTS-1 preimmune or immune serum and immunoblotted with either an anti-IL-6R{alpha} monoclonal antibody (+) or a murine IgG1 isotype control (IgG1).

 

Because both IL-6R{alpha} and TNFR1 can be co-immunoprecipitated with ARTS-1, we next assessed whether the binding of IL-6R{alpha} and TNFR1 to ARTS-1 is mutually exclusive. When immunoblots demonstrating that an anti-IL-6R{alpha} antibody coimmunoprecipitates ARTS-1 were stripped and re-probed with an anti-TNFR1 antibody, no TNFR1 was detected. Similarly, immunoblots demonstrating that an anti-TNFR1 antibody coimmunoprecipitates ARTS-1 were stripped and re-probed with an anti-IL-6R{alpha} antibody, no IL-6R{alpha} was detected (data not shown). When immunoblots demonstrating that an anti-ARTS-1 antibody co-immunoprecipitates IL-6R{alpha} were stripped and reprobed with an anti-TNFR1 antibody, TNFR1 was detected. These experiments are consistent with the conclusion that the bindings of IL-6R{alpha} and TNFR1 to ARTS-1 are mutually exclusive.

ARTS-1 Promotes IL-6R{alpha} Shedding—To determine whether ARTS-1 increases IL-6R{alpha} shedding, experiments were performed utilizing cell lines stably transfected with ARTS-1 cDNA in either the sense or antisense orientation (29). The ARTS-1 cell lines express full-length ARTS-1 coding sequence, whereas the antisense cell lines express ARTS-1 bases 61–213, which includes the putative translation start site and the intracellular and transmembrane domains. The effect of ARTS-1 protein expression on IL-6R{alpha} ectodomain shedding into culture supernatants was assessed by ELISA. As shown in Fig. 2A, the amount of sIL-6R{alpha} present in culture supernatants from cell lines overexpressing ARTS-1 was significantly greater than that of mock-transfected cells, whereas supernatants from cell lines transfected with antisense ARTS-1 had significantly less sIL-6R{alpha} than did mock-transfected cells.



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FIG. 2.
ARTS-1 promotes IL-6R{alpha} shedding. A, concentrations of sIL-6R{alpha} in 24-h culture supernatants from two antisense (AS) and two sense (ARTS-1) cell lines were determined by ELISA (n = 5). WT denotes wild type NCI-H292 cells. B, supernatants from ARTS-1 cell lines were concentrated 30-fold, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-IL-6R{alpha} antibodies. C, effect of ARTS-1 on membrane-associated IL-6R{alpha} in ARTS-1 cell lines. The amount of IL-6R{alpha} present in 300 µg of membrane proteins from ARTS-1 cell lines was determined by ELISA (n = 5) and reported as pg of membrane-associated IL-6R{alpha} (mIL-6R{alpha}) per mg of membrane protein. * p < 0.05 versus mock-transfected cells. D, membrane fractions of ARTS-1 cell lines (40 µg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-IL-6R{alpha} antibodies.

 

The quantity of soluble 55-kDa IL-6R{alpha} in culture supernatants was also analyzed by immunoblotting. As shown in Fig. 2B, the quantity of 55-kDa IL-6R{alpha} in culture supernatants from ARTS-1 cell lines was significantly greater than that from wild type NCI-H292 cells or mock-transfected cells. Reciprocally, the quantity of IL-6R{alpha} protein in supernatants from ARTS-1 antisense cell lines was markedly less. These experiments demonstrate that changes in ARTS-1 protein levels correlated directly with changes in soluble IL-6R{alpha} protein.

Experiments were also performed to assess whether ARTS-1 protein expression correlated with levels of membrane-associated IL-6R{alpha}. As shown in Fig. 2C, the quantity of membrane-associated IL-6R{alpha}, as determined by ELISA, was significantly greater in the ARTS-1 antisense cell lines than in mock-transfected cell lines, whereas the quantity of membrane-associated IL-6R{alpha} in the ARTS-1 cell lines was below the limit of detection. Similarly, as shown in Fig. 2D, the quantity of full-length 80-kDa IL-6R{alpha}, as determined by immunoblotting, was significantly greater in the ARTS-1 antisense cell lines than in mock-transfected cell lines, whereas the quantity of membrane-associated full-length 80-kDa IL-6R{alpha} in the ARTS-1 cell lines was significantly reduced. These experiments demonstrate that ARTS-1 protein expression is inversely correlated with levels of membrane-associated IL-6R{alpha}, consistent with the ability of ARTS-1 to promote IL-6R{alpha} shedding. Further, in contrast to the ARTS-1 pull-down experiments, no 55-kDa, cleaved IL-6R{alpha} was identified in crude membrane fractions. This suggests that IL-6R{alpha} ectodomain cleavage and binding to ARTS-1 require co-localization within cellular membranes, as occurs during immunoprecipitation.

NCI-H292 Cells Selectively Express the Membrane-Bound IL-6 Receptor—RT-PCR experiments were performed to characterize the expression of IL-6R{alpha} isoforms by NCI-H292 cells. Primers were utilized that spanned the IL-6R{alpha} transmembrane domain and could amplify mRNAs for both the membrane-bound IL-6R{alpha} species, as demonstrated by a 398-bp PCR product, and the alternatively spliced, soluble IL-6R{alpha} species, as demonstrated by a 304-bp product (24). As shown in bottom right panel of Fig. 3B, RT-PCR amplification of NCI-H292 cell mRNA revealed a single 398-bp PCR product that is consistent with expression of the membrane-bound IL-6R{alpha}. Identity of the membrane-bound IL-6R{alpha} was confirmed by DNA sequencing. The alternatively spliced, soluble IL-6R{alpha} mRNA species was not detected. These experiments demonstrate that NCI-H292 cells preferentially express the membrane-bound IL-6 receptor. Therefore, the soluble IL-6R{alpha} recovered from NCI-H292 cell culture supernatants is generated exclusively by ectodomain shedding and will be referred to as sIL-6R{alpha}.



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FIG. 3.
Generation and characterization of arts-1(/) cell lines. A, schematic representation of part of the arts-1 locus (top), the targeting vector (middle), and the targeted null locus (bottom). ARTS-1 contains a consensus zinc metalloprotease catalytic domain, HELAH(Y)18E, which is characteristic of aminopeptidase family members. Exon 6 encodes two zinc-binding sites (His-353 and His-357), whereas the first glutamic acid residue (Glu-354) is the active catalyst. The third zinc-binding site is encoded by the second glutamic acid (Glu-376) located in exon 7. The targeted null locus contains a substitution of a neomycin phosphotransferase cassette (Neo), in the antisense orientation, for arts-1 exons 5 and 6. A HindIII site is located in the intron between exons 3 and 4. Arrows indicate the locations of PCR primers. The locations of the arts-1 exon 15/17 primers are not shown. B, PCR amplification of genomic DNA (left panels) and RT-PCR amplification of mRNA (right panels) from wild type (WT) and three arts-1(/) cell lines. The IL-6R{alpha}-TM primers amplify a 398-bp segment of IL-6R{alpha} mRNA containing the transmembrane domain that is present in the proteolytically cleaved sIL-6R{alpha} but is absent in differentially spliced sIL-6R{alpha}.

 

IL-6R{alpha} Shedding Is Absent in Arts-1 Knock-Out Cell Lines— Knock-out arts-1 NCI-H292 cell lines (arts-1(/)) were generated by homologous recombination, which resulted in the substitution of a neomycin phosphotransferase cassette, in the antisense orientation, for arts-1 exons 5 and 6. As shown in Fig. 3A, the targeting construct contained homologous upstream (3841 bp) and downstream (2783 bp) sequences that flanked arts-1 exons 5 and 6. ARTS-1 contains a consensus zinc metalloprotease catalytic domain, HEXXH(Y)18E, which is characteristic of aminopeptidase family members. Exon 6 encodes two zinc-binding sites (His-353 and His-357), whereas the glutamic acid residue (Glu-354) is the active catalyst (31, 32). The third zinc-binding site is a glutamic acid (Glu-376) encoded in exon 7. Because the neomycin phosphotransferase cassette transgene is in the antisense orientation, the homozygous arts-1(/) cell lines should not express ARTS-1 mRNA or protein.

Following transfection and homologous recombination, homozygous arts-1(/) cells were positively selected by resistance to high dose geneticin (20 mg/ml) and negatively selected for random integrations by resistance to high dose ganciclovir (400 µM) (33). Homozygous arts-1(/) cell lines, cloned by limiting dilution, were verified by PCR of genomic DNA. As shown in the top left panel of Fig. 3B, only DNA from arts-1(/) clones was amplified when utilizing primers located within the neomycin phosphotransferase cassette and arts-1 intron 8–9, which generated a 1764-bp product. Similar results were obtained utilizing primers located within the neomycin phosphotransferase cassette and arts-1 intron 10–11, which is downstream from the introduced targeting sequence, that generated a 3210-bp product (2nd left panel). This experiment demonstrates that the arts-1(/) clones did not arise by random integration but instead were generated as a consequence of homologous recombination.

As shown in Fig. 3B, only DNA from wild type NCI-H292 cells was amplified when using a primer pair that spanned a 1329-bp region within arts-1 exons 5 and 6 (3rd left panel). As shown in the bottom left panel of Fig. 3B, DNA from both wild type NCI-H292 cells and arts-1(/) cells was amplified when using primers located in arts-1 exons 15 and 17, which generated a 2035-bp product. This was expected because exons 15 and 17 are not included in the targeting construct. These experiments verify that arts-1(/) cells contain a targeted deletion of arts-1 exons 5 and 6.

The homozygous arts-1(/) cell lines were analyzed at the mRNA level by RT-PCR. As shown in Fig. 3B, only mRNA from the wild type NCI-H292 cells was amplified using the primers spanning arts-1 exons 5 and 6 (top right panel) or primers spanning exons 2–19 (2nd right panel), which encodes amino acids 31–941 of the ARTS-1 extracellular domain. G3PDH mRNA amplified simultaneously is shown in the 3rd right panel of Fig. 3B as a control for RNA loading. These experiments verify the targeted deletion of ARTS-1 at the mRNA level.

Immunoblotting was used for verification of the homozygous arts-1(/) cell lines at the protein level. Because the epitope recognized by the anti-ARTS-1 antibody is encoded by exon 11, it should recognize either full-length ARTS-1 or a truncated version that lacks exons 5 and 6. As shown in Fig. 4A, membrane-associated ARTS-1 protein was detected in wild type NCI-H292 cells but not in arts-1(/) cell lines. There was no difference in membrane-associated MUC1 protein, a transmembrane mucin glycoprotein, demonstrating equivalency of protein loading. These experiments demonstrate that the arts-1(/) cell lines do not express a truncated ARTS-1 lacking exons 5 and 6. Instead, these data are consistent with a targeted deletion of arts-1 at the protein level. Taken together, these data demonstrate the successful targeting of both arts-1 alleles in the homozygous arts-1(/) cell lines.



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FIG. 4.
Knock-out arts-1(/) cell lines do not shed IL-6R{alpha}. A, duplicate samples (20 µg) of membrane proteins from wild type (WT) NCI-H292 cells and three arts-1(/) clones were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with ARTS-1 or MUC1 antibodies. B, concentrations of sIL-6R{alpha} in culture supernatants from wild type NCI-H292 cells and arts-1(/) cell lines, incubated for 24 h with or without 0.1 µM PMA, were quantified by ELISA (n = 5). *, p < 0.05 arts-1(/) cell lines versus wild type NCI-H292 cells. **, p < 0.05 PMA-treated wild type NCI-H292 cells versus untreated wild type NCI-H292 cells. ***, p < 0.05 PMA-treated arts-1(/) cell lines versus PMA-treated wild type NCI-H292 cells.

 

The homozygous arts-1(/) cell lines were utilized to assess the effect of ARTS-1 on IL-6 receptor shedding. As shown in Fig. 4B, wild type NCI-H292 cells demonstrated constitutive IL-6R{alpha} shedding, whereas soluble IL-6R{alpha} was not detected in the culture supernatants from arts-1(/) cell lines. Stimulation with 0.1 µM PMA for 24 h significantly increased soluble IL-6R{alpha} in culture supernatants from wild type NCI-H292 cells. PMA also increased IL-6R{alpha} shedding from arts-1(/) cell lines, but the quantity of shed receptor was significantly less than that from wild type NCI-H292 cells. These experiments demonstrate that ARTS-1 is required for constitutive IL-6R{alpha} shedding and that ARTS-1 significantly enhances PMA-induced IL-6R{alpha} shedding.

Reconstitution of arts-1(/) Cell Lines—To demonstrate further that ARTS-1 promotes IL-6 receptor shedding, experiments were performed utilizing arts-1(/) cell lines that had been reconstituted by transient transfection with plasmids encoding either the full-length ARTS-1 or full-length ARTS-1 catalytic site mutants, as described previously (29). The ARTS-1 catalytic site mutants contained either single or double amino acid replacements of key residues in the consensus aminopeptidase zinc metalloprotease catalytic site HEXXH(Y)18E. Mutations of either of the two histidines, His-353 and His-357, abolished both catalytic activity and zinc binding, whereas mutation of the first glutamic acid, Glu-354, can abolish enzymatic activity (34, 35). As demonstrated in Fig. 5A, both the 100- and 68-kDa membrane-associated ARTS-1 species were present in the arts-1(/) cells following reconstitution by transient transfection. As shown in Fig. 5B, reconstitution of arts-1(/) cell lines with full-length ARTS-1 cDNA restored IL-6R{alpha} shedding, whereas reconstitution with ARTS-1 catalytic site mutants did not. These experiments demonstrate that an intact ARTS-1 zinc metalloprotease catalytic site is required for ARTS-1-mediated IL-6R{alpha} shedding.



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FIG. 5.
IL-6R{alpha} shedding is reconstituted by ARTS-1 expression in arts-1(/) cell lines. A, duplicate samples (20 µg) of membrane proteins from wild type NCI-H292 cells (WT) and arts-1(/) cell lines that were transiently transfected with plasmids encoding either full-length ARTS-1 or ARTS-1 catalytic site mutants (H353P, H357V, H353P, and E354V) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with ARTS-1 antibodies. No ARTS-1 protein was detected in cells transfected with empty pTarget mammalian expression vector (Mock). B, concentrations of sIL-6R{alpha} in 24-h culture supernatants from wild type NCI-H292 cells and transiently transfected arts-1(/) cell lines were quantified by ELISA (n = 5). *, p < 0.05 arts-1(/) cell lines transfected with full-length ARTS-1 versus mock-transfected cells.

 

Additional experiments were performed to assess whether the TAPI family of hydroxamic acid-based zinc metalloprotease inhibitors could attenuate ARTS-1-mediated IL-6 receptor shedding. TAPI-0, TAPI-1, TAPI-2, are structural analogues that share similar affinities for zinc metalloproteases. As shown in Fig. 6, treatment with 25 µM TAPI-0, TAPI-1, or TAPI-2 significantly decreased IL-6R{alpha} shedding from wild type, mock-transfected, and ARTS-1 cell lines. Thus, a hydroxamic acid-sensitive zinc metalloprotease activity is required for IL-6 receptor shedding.



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FIG. 6.
TAPI inhibits ARTS-1-mediated increases in IL-6R{alpha} shedding. ARTS-1 cell lines were incubated for 24 h with or without TAPI-0, TAPI-1, or TAPI-2 (25 µM). Amounts of sIL-6R{alpha} present in cell culture supernatants were quantified by ELISA (n = 5). *, p < 0.05 versus no TAPI.

 

Recombinant ARTS-1 Does Not Cleave an IL-6R{alpha} Model Peptide Substrate—Ectodomain cleavage of IL-6R{alpha} occurs between Gln-357 and Asp-358 in the stalk region, located between the transmembrane and ligand-binding domains (22, 25). Because ARTS-1 zinc metalloprotease catalytic activity is required for IL-6R{alpha} shedding, additional experiments were performed to assess whether ARTS-1 directly catalyzes the proteolytic cleavage of the IL-6R{alpha} ectodomain. A catalytically active GST-ARTS-1 fusion protein that possesses aminopeptidase activity against leucine, methionine, alanine, and phenylalanine p-nitroanilide model substrates was synthesized from BL21 E. coli and purified via binding to a glutathione affinity column. A 22-amino acid peptide model substrate containing the IL-6R{alpha} cleavage site with the following sequence, RDSANATSLPVQDSSSVPLPTF, was synthesized to serve as a substrate for ARTS-1. We hypothesized that if ARTS-1 directly catalyzes IL-6R{alpha} ectodomain cleavage, it should cleave the IL-6R{alpha} model peptide substrate into peptides of 10 and 12 amino acids. As shown in Fig. 7, incubation of the model peptide substrate with recombinant ARTS-1 did not result in the cleavage of the IL-6R{alpha} model substrate. Further, the IL-6R{alpha} model peptide did not serve as a substrate for ARTS-1, which is consistent with our prior demonstration (29) that GST-ARTS-1 does not possess aminopeptidase activity toward arginine residues. Taken together, these experiments demonstrate that although ARTS-1 catalytic activity is required for ectodomain shedding, ARTS-1 does not directly catalyze IL-6R{alpha} ectodomain cleavage.



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FIG. 7.
GST-ARTS-1 does not cleave an IL-6R{alpha} peptide substrate. A model IL-6R{alpha} peptide substrate and purified GST-ARTS-1 were subjected to reverse phase HPLC utilizing a C18 column. Absorbance at 214 nm is shown as a function of elution time. A 22-amino acid model IL-6R{alpha} peptide substrate (RDSANATSLPVQDSSSVPLPTF) that contains the IL-6R{alpha} cleavage site (A) was synthesized to serve as a model substrate for GST-ARTS-1 (D). The GST-ARTS-1 fusion protein was catalytically active and had aminopeptidase activity against leucine, methionine, alanine, and phenylalanine p-nitroanilide model substrates. Both the expected N-terminal 12-amino acid (RDSANATSLPVQ) cleavage product (B) and the C-terminal 10-amino acid (DSSSVPLPTF) cleavage product (C) were synthesized to serve as standards. The IL-6R{alpha} model peptide substrate was incubated with catalytically active GST-ARTS-1 fusion protein for 2 h at 37 °C. As shown in E, the catalytically active GST-ARTS-1 did not cleave the IL-6R{alpha} model peptide substrate.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our laboratory had initially identified ARTS-1 based upon its ability to bind to the extracellular domain of the type I, 55-kDa TNF receptor and thereby promote its shedding as a soluble TNF receptor (29). ARTS-1 was characterized as a type II integral membrane protein that is present at the apical plasma membrane of polarized ciliated bronchial epithelial cells where it co-localizes with TNFR1. In addition, recombinant human ARTS-1 possesses aminopeptidase activity that is selective for non-polar N-terminal residues. These data are consistent with the classification of ARTS-1 as a member of the MA clan of the M1 family of zinc metalloproteases that contain a highly conserved catalytic motif (HEXXH(Y)18E) and utilize water, bound by a single zinc ion, as a nucleophile to mediate catalysis (36, 37).

Human and murine homologues of ARTS-1 have been independently identified (29, 30, 3640). For example, ARTS-1 has been identified as a soluble aminopeptidase (adipocyte-derived leucine aminopeptidase and puromycin-insensitive leucyl-specific aminopeptidase) that has substrate specificity for leucine and methionine, as well as for peptide hormones, such as angiotensin II and kallidin (30, 39, 40). Furthermore, an association between an adipocyte-derived leucine aminopeptidase K528R mutation and essential hypertension has been reported in a Japanese cohort (41). A murine homologue of ARTS-1 has also been identified (endoplasmic reticulum aminopeptidase associated with antigen processing, ERAAP) that processes antigens in the endoplasmic reticulum by trimming N-terminal lysine, leucine, tyrosine, and asparagine residues that are not followed by a proline (37). This identifies ERAAP as the aminopeptidase responsible for processing peptides to optimal lengths prior to their presentation in the context of major histocompatibility complex class I. Thus, ARTS-1 is a multifunctional protein that may play an important role in regulating innate and adaptive immune responses, as well as vasomotor tone.

Similar to TNFR1, the membrane-bound IL-6 receptor can be cleaved and released as a soluble protein. This is an important regulatory mechanism because trans-signaling via soluble IL-6 receptors can bestow IL-6 signaling capabilities upon IL-6R{alpha}-deficient cells that express gp130 (9, 18). Hydroxamic acid-based zinc metalloprotease inhibitors, such as TAPI, can attenuate both TNFR1 and IL-6R{alpha} shedding, consistent with ectodomain cleavage by hydroxamic acid-sensitive zinc metalloproteases. TAPI also inhibits ARTS-1 aminopeptidase activity and the ability of ARTS-1 to promote TNFR1 shedding (29). Therefore, we hypothesized that ARTS-1 might regulate IL-6 receptor shedding. Here we report that ARTS-1 binds to and promotes IL-6 receptor shedding. This is supported by the following: (i) an in vivo protein-protein interaction between endogenous membrane-associated ARTS-1 and the cleaved 55-kDa soluble IL-6 receptor; (ii) a direct correlation among increased membrane-associated ARTS-1 protein, increased IL-6 receptor shedding, and decreased membrane-associated IL-6R{alpha} levels; (iii) the absence of constitutive IL-6R{alpha} shedding from arts-1(/) cell lines; (iv) the attenuation of PMA-induced IL-6R{alpha} shedding from arts-1(/) cell lines; and (v) the reconstitution of IL-6R{alpha} shedding by arts-1(/) cell lines following transfection with an expression plasmid encoding full-length arts-1. Taken together, these data identify that ARTS-1 is required for constitutive IL-6 receptor shedding. Although the majority of PMA-induced IL-6R{alpha} shedding is dependent upon ARTS-1, an ARTS-1-independent pathway also exists as demonstrated by a low level of IL-6R{alpha} shedding from arts-1(/) cell lines following PMA stimulation. Therefore, PMA-induced IL-6R{alpha} shedding may be mediated by multiple mechanisms. Finally, ARTS-1 binds to the cleaved IL-6R{alpha} ectodomain, which is in contrast to TNFR1 where ARTS-1 binds the full-length receptor (29). This suggests that shedding of IL-6R{alpha} and TNFR1 may be regulated by different mechanisms.

The failure of ARTS-1 catalytic site mutants to reconstitute IL-6R{alpha} shedding from arts-1(/) cell lines demonstrates that ARTS-1 catalytic activity is required for IL-6R{alpha} shedding. Because the ARTS-1 antibody co-immunoprecipitates the 55-kDa cleaved IL-6R{alpha} ectodomain, we investigated whether ARTS-1 directly catalyzes IL-6R{alpha} ectodomain shedding. In a model assay system, a catalytically active GST-ARTS-1 did not exhibit endopeptidase activity toward an IL-6R{alpha} peptide substrate, which suggests that ARTS-1 does not function as a direct IL-6R{alpha} sheddase. This result is consistent with our previous report (29) that although ARTS-1 promotes TNFR1 shedding, it does not directly cleave the TNFR1 ectodomain. Similarly, we reported previously (29) that GST-ARTS-1 does not possess nonspecific endopeptidase activity toward human albumin, bovine serum albumin, rabbit myosin heavy chain, or human transferrin. In addition, ARTS-1 is a member of the MA clan of the M1 family of zinc metalloproteases that typically possess aminopeptidase but not endopeptidase activity. Therefore, we propose that ARTS-1 promotes IL-6R{alpha} ectodomain shedding via an indirect mechanism, whereby ARTS-1 enzymatic activity is required for the activation of other proteases that catalyze the cleavage of the IL-6R{alpha} ectodomain.

Experiments utilizing TACE-deficient fibroblasts have demonstrated that PMA-induced IL-6R{alpha} shedding is mediated by TACE (ADAM 17), a member of the metalloprotease-disintegrin (ADAM) family of zinc metalloproteases (25, 4245). Although constitutive IL-6R{alpha} shedding is reduced in TACE-deficient fibroblasts, a basal level IL-6R{alpha} shedding persists, consistent with the involvement of IL-6 receptor sheddases other than TACE or the existence of alternative mechanisms of receptor shedding (9, 14, 25). Because NCI-H292 cells express TACE, one possible mechanism by which ARTS-1 promotes IL-6R{alpha} receptor shedding is by modulating either the expression or maturation of TACE. In prior experiments, however, ARTS-1 expression did not increase TACE protein levels or enhance the processing of TACE to a mature, catalytically active form (29). Therefore, although it is possible that ARTS-1 can increase TACE activity via other mechanisms, we have not identified a role for ARTS-1 in TACE-mediated receptor shedding.

In summary, we have identified ARTS-1 as a key component of the IL-6 receptor shedding mechanism. Thus, ARTS-1 binds to and 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 multifunctional aminopeptidase that may modulate inflammatory events by promoting cytokine receptor shedding.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Pulmonary-Critical Care Medicine Branch, Bldg. 10, Rm. 6D03, MSC 1590, NHLBI, National Institutes of Health, Bethesda, MD 20892-1590. E-mail: levines{at}nhlbi.nih.gov.

1 The abbreviations used are: IL-6, interleukin-6; IL-6R{alpha}, interleukin-6 {alpha}-receptor; ARTS-1, aminopeptidase regulator of TNFR1 shedding; TNFR1, type I tumor necrosis factor receptor; sIL-6R{alpha}, soluble IL-6 receptors; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; STAT, signal transducer and activator of transcription; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high pressure liquid chromatography; RT, reverse transcriptase; gp, glycoprotein; sgp130, soluble form of gp130; TACE, TNF-{alpha}-converting enzyme; PMA, phorbol 12-myristate 13-acetate. Back


    ACKNOWLEDGMENTS
 
We thank Drs. Martha Vaughan, Joel Moss, and Vincent Manganiello for their helpful advice and critical review of the manuscript. We also thank Linda Stevens for expert advice and technical assistance regarding the HPLC experiments.



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