From the a Tri-Institutional (Cornell/Rockefeller University/Memorial Sloan-Kettering Cancer Center) M.D./Ph.D. Training Program, New York, New York 10021, the b Cellular Biochemistry and Biophysics Program and i Molecular Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, the h Department of Molecular Biochemistry, Glaxo Wellcome Research and Development Inc., Research Triangle Park, North Carolina 27709, and the e Laboratory of Immunology, g Laboratory of Cellular Physiology and Immunology, and l Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021
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ABSTRACT |
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Tumor necrosis factor (TNF)-related
activation-induced cytokine (TRANCE), a member of the TNF family, is a
dendritic cell survival factor and is essential for osteoclastogenesis
and osteoclast activation. In this report we demonstrate (i) that
TRANCE, like TNF- Tumor necrosis factor
(TNF)1-related
activation-induced cytokine (TRANCE), a recently identified member of
the TNF family, is a dendritic cell survival factor that also has a
role in bone homeostasis (1-4). Like TNF- Similar to TNF- cDNA Constructs and Reagents--
A FLAG-tagged full-length
murine TRANCE expression vector (pFLAG-TRANCE) has been described (5).
hCD8-TRANCE was expressed in baculovirus and purified on an
Cell Cultures--
The T cell hybridoma KMLS-8.3.5.1 (5) was
grown in S-minimal essential medium supplemented with 10% fetal bovine
serum. COS-7 and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
Transient Transfections--
COS-7 cells were transiently
transfected with pcDNA3 vector or pFLAG-TRANCE vector with
LipofectAMINE (Life Technologies, Inc.) following the manufacturer's
suggestions. Human 293T cells were transiently transfected with similar
constructs using a standard calcium phosphate method.
Pulse-Chase Analysis--
COS-7 cells transiently transfected
with either pcDNA3 vector or pFLAG-TRANCE were subjected to
pulse-chase with 200 µCi of 35S-labeled methionine and
cysteine (EXPRESS, NEN Life Sciences) as described previously (16).
After chasing in Opti-MEM (Life Technologies, Inc.) for different
amounts of time, supernatant and lysate samples were spun at 13,000 rpm
in a Sorvall tabletop centrifuge for 15 min. Where indicated, the
hydroxamate-based metalloprotease-inhibitor batimastat (BB-94) (17),
the serine protease inhibitors leupeptin (Sigma),
N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK, Sigma), soybean trypsin inhibitor (STI, Sigma),
4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (Pefabloc SC, Roche Molecular Biochemicals), the cysteine protease inhibitor
L-trans-epoxysuccinyl-leucylamide-(4-guanidino)butane (E-64, Sigma), or the aspartate protease inhibitor pepstatin (Sigma) were included in the chase medium in the presence or absence of TPA (50 ng/ml). KMLS-8.3.5.1 cells were labeled overnight with 50 µCi of
EXPRESS label and then stimulated for 3 h in the presence of
ionomycin (500 ng/ml) and TPA (50 ng/ml). The cleared cell lysates were
immunoprecipitated with the M2 anti-Flag mAb or TR-Fc (as indicated),
and protein G beads, and the cleared supernatants were
immunoprecipitated with the TR-Fc and protein A beads. The immunoprecipitated material was recovered by boiling in sample loading
buffer, and separated by SDS-PAGE. Gels were fixed in 50% methanol,
10% acetic acid and incubated in 1 M salicylic acid for 15 min prior to drying and exposure to autoradiography film (Kodak XAR).
Western Blot Analysis--
Approximately 500 µg of cleared
lysates from COS-7, 293T, and THP-1 cells were incubated with
concanavalin A-Sepharose (Amersham Pharmacia Biotech). Bound
glycoproteins were eluted with sample loading buffer and boiling at
95 °C for 5 min. Western analysis was performed following SDS-PAGE
as described previously (18).
In Vitro Cleavage of Full-length TRANCE, hCD8-TRANCE, and
N-terminal Sequence Analysis--
To generate full-length TRANCE,
COS-7 cells transiently transfected with FLAG-TRANCE were pulse-labeled
and then chased for 3 h as described above. FLAG-TRANCE was
immunoprecipitated and washed three times in lysis buffer without
protease inhibitor, followed by one wash in PBS. After incubation
overnight at 37 °C with recombinant TACE (10) (2.5 µg/ml) or MMP-1
(19) (0.1 µg/ml), the samples were eluted from the beads by boiling
in sample loading buffer and subjected to SDS-PAGE. Proteolysis of
hCD8-TRANCE was accomplished by incubating 3 µg of purified
hCD8-TRANCE with addition of 1 µg of recombinant TACE in 30 µl of
PBS for 5 h at 37 °C. The TRANCE ectodomain, which is shed into
the culture supernatant of 293T cells transfected with FLAG-TRANCE, was
precipitated with TR-Fc and protein A. For N-terminal sequence
analysis, the samples were transferred to polyvinylidene difluoride
membranes (ProBlott, Applied Biosystems) after electrophoresis, stained
with Coomassie Blue R-250, destained in 50% methanol, 10% acetic
acid, and rinsed with double-distilled H2O. The bands of
interest were excised, and the N-terminal amino acid residues were
analyzed by automated Edman degradation, using an Applied Biosystems
477A sequenator, with instrument and procedure optimized for femtomole
level analysis as described (20).
Incubation of Substrate Peptides with TACE and MMP-1 and
Determination of Cleavage Sites and Kinetics--
Synpep (Dublin, CA)
synthesized peptides corresponding to the 12 amino acid residues
surrounding the reported cleavage sites for TNF- Dendritic Cell Survival and Osteoclastogenesis Assay--
Mature
murine bone-marrow derived dendritic cells (DCs) were isolated as
described previously (22) and incubated with either medium alone,
hCD8-TRANCE (1 µg/ml), or supernatants (1:50 dilution) from 293T
cells transfected with pFLAG-2 vector (Kodak) or pFLAG-TRANCE in
96-well plates. TR-Fc (10 µg/ml) was simultaneously added to certain
samples as indicated. 293T cell supernatants were centrifuged (100,000 × g, 1 h) and filtered through a
0.2-µm cutoff filter membrane to remove cell debris and membranes.
Amounts of released TRANCE into culture supernatants were estimated to
be approximately 3 µg/ml. This was determined by complete depletion
of ecto-TRANCE with TR-Fc, and subsequently comparing amounts of
precipitated ecto-TRANCE with bovine serum albumin standards on
SDS-PAGE after Coomassie staining (data not shown). Cell viability was
assessed by trypan blue staining 48-72 h after treatment as described
(1, 5). Osteoclastogenesis assays were performed as described (7).
To determine if TRANCE, like TNF-, is made as a membrane-anchored precursor, which
is released from the plasma membrane by a metalloprotease; (ii) that
soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-
convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a
fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8
fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed
from cells into the supernatant. We propose that the TRANCE ectodomain
is released from cells by TACE or a related
metalloprotease-disintegrin, and that this release is an important
component of the function of TRANCE in bone and immune homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, TRANCE is a type II
integral membrane glycoprotein of ~45 kDa with a long extracellular
stalk region followed by a receptor-binding core domain (5). TRANCE
expression in osteoblasts and stromal cells can be induced with vitamin
D3, prostaglandin E2, interleukin-1, or glucocorticoids
(4). In turn, TRANCE is known to induce differentiation and activation of osteoclasts. This suggests that TRANCE provides an important link
between the action of hormones and physiological cytokines and bone
resorption. TRANCE is also expressed on activated T cells (5), where it
induces dendritic cell survival, thereby enhancing T cell priming (1,
2). Therefore TRANCE may also regulate antigen presentation during an
immune response. TRANCE mediates its effects through the
membrane-anchored TRANCE receptor (TRANCE-R, also referred to as RANK
(receptor activator of nuclear factor-
B)), which results in
activation of c-Jun N-terminal kinase and nuclear factor-
B (2, 5).
Finally TRANCE is known to bind to a soluble receptor, termed
osteoprotegerin or osteoclast inhibitory factor (OPG/OCIF), which is a
member of the TNF-
receptor family (6). OPG/OCIF presumably
functions as a decoy receptor, since systemic overexpression or
injection of OPG/OCIF causes osteopetrosis in mice (7), whereas
OPG/OCIF deficiency results in osteoporosis (8).
, which is thought to be released from the plasma
membrane by the metalloprotease-disintegrin TNF-
convertase (TACE)
(9-11), TRANCE may also be shed by TACE or a related metalloprotease. Metalloproteases have been implicated in the shedding or release of
several different cell surface proteins from the plasma membrane. These
proteins include various cytokines, cytokine receptors, adhesion
proteins, and other proteins such as the
-amyloid precursor protein
(12). Shed membrane proteins may have very different properties
compared with the membrane-anchored forms. Solubilized Fas-ligand
(FasL), for example, has been shown to be a much weaker inducer of
apoptosis than its transmembrane form, suggesting that shedding
down-regulates the activity of FasL (13). In contrast, soluble TNF-
is a potent pro-inflammatory cytokine, and release of TNF-
into the
circulatory system contributes to its systemic effects and to
pathologic conditions such as septic shock (14). In this study, we
provide evidence that TACE or a related metalloprotease-disintegrin is
a likely candidate for the proteolytic release of the TRANCE ectodomain. Furthermore, we show that soluble TRANCE promotes dendritic
cell survival and osteoclast differentiation in tissue culture.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-hCD8-Sepharose column as described (1). Mouse TRANCE-R fused to
human IgG1 (TR-Fc) was cloned into the vector PVL1392 and expressed in
baculovirus. Purification was performed by binding to a protein
A-Sepharose column, eluting with glycine (0.2 M, pH 2.9),
and dialyzing against PBS. M2 anti-FLAG mAb was purchased from Sigma. A
cDNA fragment encoding for the human TACE cytotail (corresponding
to amino acids 695-824) was cloned in frame to the coding region of
GST in the pGEX-4T-1 vector (Amersham Pharmacia Biotech). The GST-TACE
cytotail fusion protein was expressed and purified from BL21 bacteria
and used as an immunogen to raise rabbit polyclonal antisera as
described previously (15).
and TRANCE. The
peptide sequences and the reported cleavage sites are presented in
Table I. TACE (600 nM) or the catalytic domain of MMP-1 (30 nM) was incubated with 50, 25, or 12.5 µM TRANCE peptide substrate in 10 mM HEPES, pH 7.2 containing
0.05% bovine serum albumin (Sigma). For the TRANCE and TNF-
peptide substrates, reactions were timed to allow approximately 5-20% turnover of the substrate. Reactions were quenched using 1%
heptafluorobutyric acid, and products were separated by high
performance liquid chromatography reverse phase chromatography (C18
column, Vydac, Hesperia, CA) with absorbance monitored at 350 nm.
Turnover was quantitated by integrating peak areas of the substrate and
product. Liquid chromatography-mass spectroscopy was used to determine
the masses of the product and therefore the cleavage site recognized by
either TACE or MMP-1. Briefly, digestion mixtures were passed over a Hypersil C18 column and, after UV detection at 350 nm, the sample was
routed into the ion spray source of a Sciex API-III triple quadrupole
mass spectrometer. Specificity constants were calculated from initial
velocities using the equation:
kcat/Km = (% turnover/100)/([E] × time). Conditions of
kcat/Km were verified by
running the reactions at more than one substrate concentration. Enzyme
concentrations were determined utilizing a potent hydroxamate-type
inhibitor of TACE by the methods described in Ref. 21.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, is shed from the plasma
membrane by a protease, pulse-chase analysis was performed in COS-7
cells transiently expressing full-length TRANCE with a cytoplasmic FLAG
epitope tag (FLAG-TRANCE). Immediately after the pulse labeling, two
closely co-migrating bands of 46 and 48 kDa could be immunoprecipitated from the extracts with an anti-FLAG mAb (Fig.
1, lane 2). After deglycosylation of an identical sample with
peptide:N-glycosidase F, only a 35-kDa band was detected,
corresponding to the predicted molecular weight of membrane-anchored
TRANCE (data not shown). As the duration of the chases increased from 3 to 12 h (Fig. 1A, lanes 3-5),
the relative amount of 46- and 48-kDa proteins immunoprecipitated with
anti-FLAG mAb decreased. Simultaneously, a 26-kDa band, which could be
precipitated from the culture supernatant using a soluble TRANCE-receptor-Fc fusion protein (TR-Fc), increased in intensity (Fig.
1A, lanes 7-9). N-terminal sequence
analysis (see below) confirmed that the 26-kDa protein is a soluble
form of TRANCE (referred to as ecto-TRANCE hereafter). After a 12-h
chase period, an additional band of 24 kDa, which most likely
represents a minor shed TRANCE product, could also be seen in the
supernatant (Fig. 1A, lane 9). These
observations indicate that TRANCE, like TNF-
, can be released from
the cell surface.
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Fig. 1.
Release of a soluble TRANCE ectodomain from
transfected COS-7 cells and from a T cell hybridoma. A,
pulse-chase analysis of COS-7 cells transiently transfected with
pcDNA3 (lanes 1 and 6) or
FLAG-TRANCE (lanes 2-5 and 7-9). At
the indicated time points, full-length TRANCE was immunoprecipitated
from cell lysates with the anti-FLAG mAb (lanes
1-5), and soluble TRANCE was precipitated from supernatants
with TR-Fc (lanes 6-9). B, T cell
hybridoma cells (KMLS-8.3.5.1) were stimulated for 3 h with 500 ng/ml ionomycin and 50 ng/ml phorbol 12-myristate 13-acetate following
labeling overnight with [35S]methionine/cysteine.
Supernatant (lanes 3 and 4) and lysate
samples (lanes 1 and 2) were collected
and incubated with TR-Fc followed by protein A (lanes
2 and 4), or protein A beads alone
(lanes 1 and 3). All samples were
reduced prior to electrophoresis.
To confirm that TRANCE shedding also occurs in non-transfected cells, a similar experiment was performed using the T cell hybridoma KMLS-8.3.5.1 (5) from which TRANCE was originally cloned. Hybridoma cells were labeled overnight with [35S]methionine/cysteine and then stimulated for 3 h with ionomycin and phorbol 12-myristate 13-acetate to induce shedding. Immunoprecipitation with TR-Fc revealed TRANCE proteins in the media (24 and 26 kDa; Fig. 1B, lane 4) and lysate (24, 26, 46, and 48 kDa; Fig. 1B, lane 2). The observed membrane-anchored and soluble forms of TRANCE were thus similar in molecular weight to those seen in COS-7 cells expressing TRANCE. The 24- and 26-kDa proteins in the cell lysate may represent cleaved ectodomains that reside in the intracellular secretory pathway, or that are still bound to uncleaved TRANCE molecules on the cell surface.
To address whether ecto-TRANCE is indeed functional, its activity was
tested in osteoclast differentiation and in DC survival assays. Bone
marrow precursors cultured in the presence of macrophage colony-stimulating factor or mature bone-marrow derived DCs were incubated with 293T supernatants containing ecto-TRANCE (see Fig. 3C) at an estimated final concentration of 60 ng/ml (see
"Materials and Methods"). Fig.
2A demonstrates that bone
marrow progenitors incubated with supernatants containing ecto-TRANCE,
but not supernatants from cells transfected with vector alone, induced
high levels of tartrate-resistant acid phosphatase activity, indicating
that ecto-TRANCE mediates the differentiation of osteoclasts from
precursors. This activity was dependent on a TRANCE/TRANCE-R
interactions because its effect could be blocked with saturating doses
of a soluble receptor, TR-Fc. Fig. 2B shows that
ecto-TRANCE-containing supernatants enhanced dendritic cell survival
and this effect could also be inhibited by the addition of TR-Fc,
indicating that the shed form of TRANCE is functionally active as a
survival factor for mature DCs.
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Since shedding of many cell surface proteins can be stimulated with
phorbol esters and inhibited with metalloprotease inhibitors (12), we
tested how these factors affect TRANCE shedding in transiently
transfected COS-7 cells. In a pulse-chase experiment, a 15-min (data
not shown) or 6-h treatment with the phorbol ester TPA increased the
amount of ecto-TRANCE released into the supernatant (Fig.
3A, lower
panel, lane 1) compared with constitutive
shedding for 15 min (data not shown) or 6 h (Fig. 3A,
upper panel, lane 1).
Treatment of TPA stimulated cells with BB-94 (17), a hydroxamic acid-based metalloprotease inhibitor, strongly decreased TPA-stimulated shedding of TRANCE (Fig. 3A, lower
panel, lane 2), but did not detectably affect
constitutive shedding (Fig. 3A, upper
panel, lane 2). Serine protease
inhibitors (leupeptin, STI, Pefabloc SC, and TPCK; Fig. 3A,
upper and lower panels,
lanes 3-6), a cysteine protease inhibitor (E-64,
10 µM; data not shown), and an aspartate protease
inhibitor (pepstatin, 10 µM; data not shown) had no
apparent effect on the TPA-dependent or constitutive
shedding of TRANCE from COS-7 cells.
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Titrating the amount of BB-94 in the cell-based assay revealed
significant inhibition of TRANCE shedding at 10 nM BB-94,
and maximal inhibition at 100 nM (Fig. 3B). This
is consistent with the Ki of 11 nM that
has been determined for the inhibition of TACE by BB-94 in
vitro (21). We note that a residual component TRANCE shedding is
not inhibited even by high doses of BB-94 in the TPA-treated sample.
Since BB-94 also does not inhibit TRANCE shedding in unstimulated
cells, other proteases besides metalloproteases may play a role in the
constitutive release of TRANCE into the supernatant. A similar
observation has been reported for the -amyloid precursor protein
(
APP) (23). In fibroblasts lacking TACE, the phorbol 12-myristate
13-acetate-dependent shedding of
APP is abolished, while
a low level of constitutive shedding of
APP, which is not inhibited
by the hydroxamate-based metalloprotease inhibitor TAPI-2, is still
present. Only a small percentage of total TRANCE was released, as
levels of TRANCE in the cell lysate did not decrease with TPA
stimulation compared with untreated cells (Fig. 3A,
lane 1) or increase in BB-94-treated cells. Taken together, these results provide the first evidence that TRANCE, like
TNF-
,
APP, and other shed proteins can be released in response to
phorbol esters (12, 24, 25), and that this release can be inhibited by
a hydroxamate-based metalloprotease inhibitor.
To test for a potential role of TACE in the shedding of TRANCE, metabolically labeled full-length TRANCE was immunoprecipitated and incubated in vitro with recombinant TACE. This treatment yielded polypeptides of 23 and 26 kDa (Fig. 3C, lane 3) that were not visible in the untreated sample (Fig. 3C, lane 2). A likely explanation for the relatively inefficient processing of immunoprecipitated TRANCE in Nonidet P-40 by soluble TACE is that TACE and its substrate may both need to be membrane-anchored for optimal cleavage to occur. The 26-kDa band generated in vitro by TACE co-migrated with the ecto-TRANCE isolated from the supernatant of transfected COS-7 cells (Fig. 3C, lane 1), whereas the 23-kDa product did not. As a control for specificity, immunoprecipitated full-length TRANCE was also incubated with MMP-1, a member of the matrix metalloprotease family (26). MMP-1 produced two polypeptides of 25.5 and 24.5 kDa (Fig. 2B, lane 6). The 25.5-kDa band generated with MMP-1 did not co-migrate with ecto-TRANCE in the supernatant of TRANCE-expressing COS-7 cells (data not shown), or with TRANCE polypeptides resulting from TACE cleavage (Fig. 2B, lane 5). These in vitro cleavage results are consistent with the idea that TACE, or a protease with a similar substrate specificity, may be involved in cleaving TRANCE. Western blot analysis confirmed the presence of TACE in COS-7 cells (Fig. 3D, lane 2) and 293T cells (Fig. 3D, lane 6).
To compare the in vitro cleavage site for TACE in TRANCE to
the N terminus of ecto-TRANCE in the supernatant of 293T-transfected cells, a soluble fusion protein of the TRANCE ectodomain with human CD8
(hCD8-TRANCE, see Fig. 4C and
"Materials and Methods") was incubated in vitro with
TACE. This treatment resulted in at least three visible cleavage
products (Fig. 4A, lane 2) that were not present in the TACE sample (Fig. 4A, lane
3) or in the hCD8-TRANCE sample (Fig. 4A,
lane 1) incubated separately. N-terminal sequence analysis of the 26-kDa band, which co-migrated with ecto-TRANCE, revealed that it contained two polypeptide species. One of the two
sequences corresponded to the N terminus of ecto-TRANCE (see below and
Ref. 3), whereas the other corresponded to the N terminus of human CD8
after removal of its signal sequence. N-terminal sequence analysis of
the 24-kDa band (Fig. 4A, lane 2)
revealed only the hCD8 N terminus (data not shown). The higher
molecular weight bands seen in Fig. 4A (lane
2) presumably represent intermediate in vitro
cleavage products. N-terminal sequencing of ecto-TRANCE released into
the supernatant of transfected 293T cells (Fig. 4A,
lane 5) confirmed the previously reported
cleavage site of TRANCE, and was identical to the cleavage site
generated by TACE (Fig. 4B and Ref. 3). These results
demonstrate that a major in vitro cleavage site for TACE in
the ectodomain of TRANCE is identical to the cleavage site of the
protease that releases ecto-TRANCE from 293T cells (see diagram in Fig.
4C).
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To further evaluate the cleavage of TRANCE, we determined the cleavage
site and kinetics for processing of TRANCE and TNF- peptides by
TACE. The TRANCE peptide was cleaved in the correct position by TACE,
but the specificity constant was 1000-fold lower than for the TNF-
peptide (Table I). MMP-1 did not cleave
the TRANCE peptide (data not shown). This is consistent with the
observation that TACE cleavage of immunoprecipitated pro-TRANCE
produced a protein that co-migrated with TRANCE shed from cells,
whereas cleavage with MMP-1 did not (see Fig. 2C). With
respect to the peptide cleavage specificity of
metalloprotease-disintegrins, we note that both ADAM10 (KUZ/MADM) and
TACE cleave a TNF-
peptide at the physiological position (9, 10,
27), while MDC9 has a clearly distinct specificity compared with TACE
(21). The correct cleavage of the TRANCE peptide by TACE therefore
suggests that TACE, or a metalloprotease with a similar substrate
specificity, cleaves TRANCE in cells. The difference in specificity
constants of TACE for the TRANCE and TNF-
peptides (1000-fold) is
similar to the reported difference in peptide cleavage efficiency of
TACE for TNF-
and the putative TACE substrate L-selectin (2250-fold) (11). A general question raised by these observations is whether TACE
substrate recognition in cells involves additional targeting events
between the protease and the substrate (11, 21, 28, 29), or
alternatively if TRANCE is actually cleaved by another related
metalloprotease with a higher specificity constant for the TRANCE
cleavage site.
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The results presented here demonstrate that a phorbol ester-inducible
metalloprotease can release ecto-TRANCE from the plasma membrane. The
finding that incubation of the hCD8-TRANCE ectodomain or of a TRANCE
peptide with recombinant TACE generates a fragment with the identical N
terminus as ecto-TRANCE suggests that TACE, or a related
metalloprotease, mediates TRANCE shedding. Soluble TRANCE has potent
functional activity in promoting dendritic cell survival and osteoclast
differentiation. In analogy to the regulation of TNF- function by
shedding from the plasma membrane (9, 10), our results suggest that
TRANCE shedding may be an important aspect of the functional regulation
of this protein. We propose that released TRANCE may mediate signaling
as a soluble cytokine, and that the release of TRANCE may be an
important factor in the TRANCE/TRANCE-R/osteoprotegerin signaling axis.
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ACKNOWLEDGEMENTS |
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We thank Masha Vologodskaia and Angela Santana for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by a grant from Glaxo-Wellcome (to C. P. B.), by National Institutes of Health NIAID Grant AI44264 (to Y. C.), and by National Science Foundation Grant DBI-942013 (to P. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c Supported in part by National Institutes of Health MSTP Training Grant 5T32GM07739-17 and the Robert Wood Johnson Jr. Charitable Trust Endowment Fund,
d The first and second authors contributed equally to this report.
f Supported by National Institutes of Health Medical Scientist Training Program Training Grant 5T32GM07739-17.
j Supported in part by National Institutes of Health Medical Scientist Training Program Training Grant 5T32GM07739-17 and the Louis and Rachel Rudin Family Foundation.
k Supported by Memorial Sloan-Kettering Cancer Center Support Grant NCI-P30-CA-08748.
m An investigator of the Howard Hughes Medical Institute.
n To whom correspondence should be addressed: Cellular Biochemistry and Biophysics Program, Sloan-Kettering Inst., Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2915; Fax: 212-717-3047; E-mail: c-blobel{at}ski.mskcc.org.
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor necrosis
factor;
TRANCE, TNF-related activation-induced cytokine;
TRANCE-R, TRANCE receptor;
TACE, TNF- convertase;
OPG/OCIF, osteoprotegerin/osteoclast inhibitory factor;
APP,
-amyloid
precursor protein;
PBS, phosphate-buffered saline;
GST, glutathione
S-transferase;
mAb, monoclonal antibody;
DC, dendritic cell;
PAGE, polyacrylamide gel electrophoresis;
STI, soybean trypsin
inhibitor;
TPCK, N-tosyl-L-phenylalanine
chloromethyl ketone;
TPA, 12-O-tetradecanoylphorbol-13-acetate.
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