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.
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ABSTRACT |
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INTRODUCTION |
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The IL-6 receptor complex comprises two distinct membrane-bound
glycoproteins, the 80-kDa IL-6 -receptor subunit (IL-6R
, CD 126)
and the 130-kDa signal transducing subunit (gp130)
(9). Following binding of IL-6
to IL-6R
, two IL-6/IL-6R
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) 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
·IL-6 complex to membrane-bound gp130 confers IL-6
signaling capability to cells that do not express IL-6R
. Because of the
ubiquitous expression of gp130, trans-signaling via the generation of
sIL-6R
·IL-6 complexes can extend the repertoire of
IL-6-responsive cell types (9,
18). For example,
trans-signaling via sIL-6R
·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
·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
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 ectodomain
from the cell surface or differential mRNA splicing that results in the
generation of an IL-6R
that lacks the transmembrane domain
(2024).
Proteolytic cleavage of IL-6R
, 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-
-converting enzyme or ADAM 17), a member of the
metalloprotease-disintegrin (ADAM) family of zinc metalloproteases, possesses
IL-6R
sheddase activity
(20,
2628).
Further evidence supporting TACE-mediated IL-6R
shedding is the strong
reduction in phorbol ester-induced IL-6R
shedding in TACE-deficient
murine fibroblasts that can be rescued by reconstitution of TACE expression
(25). However, the existence
of additional IL-6R
sheddases has been suggested by a basal
hydroxamate-sensitive IL-6R
shedding from TACE-deficient cells, as well
as by IL-6R
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, we hypothesized that ARTS-1 might also regulate IL-6R
shedding. By utilizing an arts-1 knock-out cell line
(arts-1(/)),
we demonstrate that ARTS-1 is required for constitutive IL-6R
shedding.
Transfection of
arts-1(/)
cell lines with plasmids containing full-length ARTS-1 restored IL-6R
shedding, whereas transfection with ARTS-1 catalytic site mutants did not.
These data indicate that the mechanism of constitutive IL-6R
shedding
requires ARTS-1 catalytic activity. Furthermore, 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. Thus, ARTS-1 may modulate
inflammatory events by promoting the shedding of two cytokine receptor
superfamilies, the type I cytokine receptor superfamily (IL-6R
) and the
TNF receptor superfamily (TNFR1).
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MATERIALS AND METHODS |
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For immunoblotting, samples of membrane proteins (2040 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 (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 LinesStably transfected NCI-H292 cell lines
that expressed either full-length human ARTS-1 or antisense ARTS-1 (bases
61213) were utilized, as described previously
(29). To quantify
membrane-associated IL-6R, 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
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 LineThe 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 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 Ectodomain Cleavage AssayA model
system was utilized to assess whether ARTS-1 catalyzes the proteolytic
cleavage of the IL-6R
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
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, 02 min, linear gradient
067.5% of solution B, 220 min. The absorbance of the eluate was
recorded at 214 nm.
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RESULTS |
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Because both IL-6R and TNFR1 can be co-immunoprecipitated with
ARTS-1, we next assessed whether the binding of IL-6R
and TNFR1 to
ARTS-1 is mutually exclusive. When immunoblots demonstrating that an
anti-IL-6R
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
antibody, no
IL-6R
was detected (data not shown). When immunoblots demonstrating
that an anti-ARTS-1 antibody co-immunoprecipitates IL-6R
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
and TNFR1 to ARTS-1 are mutually exclusive.
ARTS-1 Promotes IL-6R SheddingTo determine
whether ARTS-1 increases IL-6R
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 61213, which includes the
putative translation start site and the intracellular and transmembrane
domains. The effect of ARTS-1 protein expression on IL-6R
ectodomain
shedding into culture supernatants was assessed by ELISA. As shown in
Fig. 2A, the amount of
sIL-6R
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
than did mock-transfected cells.
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The quantity of soluble 55-kDa IL-6R in culture supernatants was
also analyzed by immunoblotting. As shown in
Fig. 2B, the quantity
of 55-kDa IL-6R
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
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
protein.
Experiments were also performed to assess whether ARTS-1 protein expression
correlated with levels of membrane-associated IL-6R. As shown in
Fig. 2C, the quantity
of membrane-associated IL-6R
, 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
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
, 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
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
,
consistent with the ability of ARTS-1 to promote IL-6R
shedding.
Further, in contrast to the ARTS-1 pull-down experiments, no 55-kDa, cleaved
IL-6R
was identified in crude membrane fractions. This suggests that
IL-6R
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
ReceptorRT-PCR experiments were performed to characterize the
expression of IL-6R isoforms by NCI-H292 cells. Primers were utilized
that spanned the IL-6R
transmembrane domain and could amplify mRNAs for
both the membrane-bound IL-6R
species, as demonstrated by a 398-bp PCR
product, and the alternatively spliced, soluble IL-6R
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
. Identity of the membrane-bound IL-6R
was
confirmed by DNA sequencing. The alternatively spliced, soluble IL-6R
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
recovered from NCI-H292 cell culture supernatants is
generated exclusively by ectodomain shedding and will be referred to as
sIL-6R
.
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IL-6R 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 89, which generated a 1764-bp product. Similar results were obtained utilizing primers located within the neomycin phosphotransferase cassette and arts-1 intron 1011, 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 219 (2nd right panel), which encodes amino acids 31941 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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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 shedding, whereas soluble IL-6R
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
in culture supernatants from wild type NCI-H292
cells. PMA also increased IL-6R
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
shedding and that ARTS-1 significantly
enhances PMA-induced IL-6R
shedding.
Reconstitution of
arts-1(/)
Cell LinesTo 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 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
shedding.
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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 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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Recombinant ARTS-1 Does Not Cleave an IL-6R Model
Peptide SubstrateEctodomain cleavage of IL-6R
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
shedding,
additional experiments were performed to assess whether ARTS-1 directly
catalyzes the proteolytic cleavage of the IL-6R
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
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
ectodomain cleavage, it should cleave the IL-6R
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
model substrate. Further, the IL-6R
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
ectodomain cleavage.
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DISCUSSION |
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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-deficient cells that express gp130
(9,
18). Hydroxamic acid-based
zinc metalloprotease inhibitors, such as TAPI, can attenuate both TNFR1 and
IL-6R
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
levels; (iii) the absence of constitutive IL-6R
shedding
from
arts-1(/)
cell lines; (iv) the attenuation of PMA-induced IL-6R
shedding from
arts-1(/)
cell lines; and (v) the reconstitution of IL-6R
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
shedding is dependent upon ARTS-1, an
ARTS-1-independent pathway also exists as demonstrated by a low level of
IL-6R
shedding from
arts-1(/)
cell lines following PMA stimulation. Therefore, PMA-induced IL-6R
shedding may be mediated by multiple mechanisms. Finally, ARTS-1 binds to the
cleaved IL-6R
ectodomain, which is in contrast to TNFR1 where ARTS-1
binds the full-length receptor
(29). This suggests that
shedding of IL-6R
and TNFR1 may be regulated by different
mechanisms.
The failure of ARTS-1 catalytic site mutants to reconstitute IL-6R
shedding from
arts-1(/)
cell lines demonstrates that ARTS-1 catalytic activity is required for
IL-6R
shedding. Because the ARTS-1 antibody co-immunoprecipitates the
55-kDa cleaved IL-6R
ectodomain, we investigated whether ARTS-1
directly catalyzes IL-6R
ectodomain shedding. In a model assay system,
a catalytically active GST-ARTS-1 did not exhibit endopeptidase activity
toward an IL-6R
peptide substrate, which suggests that ARTS-1 does not
function as a direct IL-6R
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
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
ectodomain.
Experiments utilizing TACE-deficient fibroblasts have demonstrated that
PMA-induced IL-6R shedding is mediated by TACE (ADAM 17), a member of
the metalloprotease-disintegrin (ADAM) family of zinc metalloproteases
(25,
4245).
Although constitutive IL-6R
shedding is reduced in TACE-deficient
fibroblasts, a basal level IL-6R
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
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.
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FOOTNOTES |
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Both authors contributed equally to this work.
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,
interleukin-6
-receptor; ARTS-1, aminopeptidase regulator of TNFR1
shedding; TNFR1, type I tumor necrosis factor receptor; sIL-6R
, 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-
-converting enzyme; PMA,
phorbol 12-myristate 13-acetate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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