From the Cellular Biochemistry and Biophysics
Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021 and § Tri-Institutional
(Cornell University/Rockefeller University/Sloan-Kettering Cancer
Center) MD-PhD Training Program, New York, New York 10021
Received for publication, November 28, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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A number of structurally and functionally diverse
membrane proteins are released from the plasma membrane in a process
termed protein ectodomain shedding. Ectodomain shedding may activate or
inactivate a substrate or change its properties, such as converting a
juxtacrine into a paracrine signaling molecule. Here we have characterized the activities involved in protein ectodomain shedding of
the tumor necrosis factor family member TRANCE/OPGL in different cell types. The criteria used to evaluate these activities include (a) cleavage site usage, (b) response to
activators and inhibitors of intracellular signaling pathways, and
(c) sensitivity to tissue inhibitors of metalloproteases
(TIMPs). At least two TRANCE shedding activities emerged, both of which
are distinct from the tumor necrosis factor A diverse set of transmembrane proteins is known to undergo
proteolysis in their juxtamembrane region leading to the release of
their extracellular domains into the surrounding milieu (reviewed in
Refs. 1-4). This process, which has been termed ectodomain shedding,
affects a wide variety of proteins, including growth factors and
cytokines, growth factor receptors, adhesion molecules, and
ectoenzymes. Ectodomain shedding has been shown to profoundly affect
the biological activity of its target proteins and plays a critical
role in the development and physiology of multicellular organisms.
Ectodomain shedding regulates signaling via epidermal growth
factor receptor ligands (5, 6), axonal guidance (7-9), and
Notch-mediated lateral inhibition (10-17). Discovering the molecular
identity of sheddases should lead to a better understanding of the
mechanisms that regulate their function and may provide targets for
therapeutic interventions in diseases that are caused by misregulated
ectodomain shedding.
Based on inhibitor studies, ectodomain shedding is predominantly
mediated by metalloproteases (3). Specifically, several members of the
ADAM (a disintegrin and
metalloprotease; also known as metalloprotease-disintegrins
and MDC proteins) family of metalloproteases have been implicated as
ectodomain sheddases (reviewed in Refs. 18-20).
TNF In this study, we used the TNF family member TRANCE
(TNF-related
activation-induced
cytokine; also known as osteoprotegrin ligand, OPGL, RANK ligand, RANKL, and osteoclast differentiation factor) as a model to ask whether one or more sheddases are
involved in its release from the membrane and to define the properties of this sheddase or these sheddases. TRANCE is required in osteoclast differentiation and activation, in T and B cell maturation and dendritic cell survival, and in mammary gland development during pregnancy (31-38). We have previously shown that TRANCE is
proteolytically released from the plasma membrane in a number of cell
lines and that the solubilized ligand is functional in in
vitro osteoclast differentiation and dendritic cell survival
assays (39). We have now further characterized the TRANCE sheddase
activities in CHO cells and embryonic fibroblasts using several
biochemical and pharmacological criteria. These criteria include
cleavage site selection, response to activators and inhibitors of
intracellular signaling pathways, and the inhibition by tissue
inhibitors of matrix metalloproteases (TIMPs). Based on these results
we have identified two distinct TRANCE sheddase activities that differ markedly from the TNF Materials--
Phorbol-12 myristate 13-acetate, hydrogen
peroxide, sodium vanadate, and anti-FLAG M2 monoclonal antibody were
purchased from Sigma. Pervanadate was generated immediately prior to
each experiment by mixing sodium vanadate and hydrogen peroxide to a
final concentration of 100 mM of each. Protein A- and
protein G-Sepharose were purchased from Amersham Pharmacia Biotech.
Restriction enzymes and Taq polymerase were purchased from
Roche Molecular Biochemicals. Pfu Turbo DNA polymerase was
obtained from Stratagene. The MEK 1 and 2 inhibitor U0126 and p38 MAP
kinase inhibitor SB202190 were purchased from CalBiochem.
Sulfo-NHS-biotin was purchased from Pierce. Anti-huTNF Constructs and Mutagenesis--
The TRANCE-FLAG cDNA (40)
was provided by B. Wong and Y.-W. Choi. The human TNF Cell Culture, Transfection, and Metabolic Label--
CHO cells
were grown in Ham's F-12 medium supplemented with 5% fetal calf serum
and 1% penicillin/streptomycin and glutamine. COS-7 cells were grown
in DME with identical supplements. CHO and COS-7 cells were transfected
in six-well tissue culture plates with LipofectAMINE (Life
Technologies, Inc.) following the manufacturer's recommendations.
Cells were allowed to recover for at least 4 h prior to metabolic
labeling. For metabolic labeling, cells were washed twice with PBS and
then incubated in DME lacking cysteine and methionine supplemented with
10% dialyzed calf serum and 200 µCi/ml of [35S]
Pro-mix (Amersham Pharmacia Biotech). Cells were labeled overnight for
10-14 h. After labeling, the cells were washed twice with PBS, pH 7.4, and chased for one hour in OptiMEM (Life Technologies, Inc.) with the
indicated additives. The conditioned medium was collected. The cells
were washed a further two times with PBS and lysed in lysis buffer
(TBS, pH 7.4, 1% (v/v) Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM
1,10-phenanthroline and protease inhibitors (2 µg/ml leupeptin, 0.4 mM benzamidine, 10 µg/ml soy-bean trypsin inhibitor, 0.5 mM iodoacetamide)). In experiments examining the MAP kinase
inhibitors U0126 and SB202190, the cells were preincubated in OptiMEM
with the indicated concentration of inhibitor for 2 h. The cells
were then washed twice in PBS and chased in OptiMEM with additives for
1 h. The medium and lysate were then processed as above. All
lysates and supernatants were spun on a tabletop centrifuge (Sorvall)
for 15 min at 13,000 rpm to remove nuclei and cellular debris.
For immunoprecipitations, the TRANCE receptor Fc fusion protein (TR-Fc)
or a rabbit TNF
TACE-deficient embryonic fibroblasts immortalized by infection with a
retrovirus encoding ras and myc (E3 clone) (5,
23) were kindly provided by P. Reddy, J. Peschon, and R. Black
(Immunex, Seattle, WA) and cultured in DME supplemented with 2% fetal
calf serum and 1% penicillin/streptomycin and glutamine. SV40
transformed embryonic fibroblasts from ADAM 10-deficient mice and their
wild type littermates were the generous gift of D. J. Pan (16).
These cells were maintained in DME supplemented with 10% fetal calf serum and 1% penicillin/streptomycin and glutamine. Primary
fibroblasts were isolated from day E13.5 murine embryos homozygous for
both ADAM 9 and ADAM 15 gene
disruptions2 or ADAM 9, ADAM
12, and ADAM 15 gene
disruptions,3 homozygous for
a gene trap insertion in the ADAM 19 gene,4 or having a wild type
background. Primary murine embryonic fibroblasts were cultured in DME
supplemented with 10% fetal calf serum, nonessential amino acids, and
1% penicillin/streptomycin and glutamine. Embryonic fibroblasts were
either electroporated using a Bio-Rad GenePulser II with a capacitance
extender and 0.4-cm cuvettes at 270 V, 960 microfarads, or transfected
using a standard calcium phosphate precipitation technique. Cells were
allowed to recover and metabolically labeled overnight in DME without
cysteine and methionine, 10% dialyzed fetal calf serum, and 100 µCi/ml of [35S] Pro-mix (Amersham Pharmacia Biotech).
After two washes in PBS, pH 7.4, cells were chased in OptiMEM with the
indicated additions. Conditioned medium was collected after 1 h,
and cell debris was removed by centrifugation. Cells were lysed in
lysis buffer and spun at 13,000 × g for 20 min
followed by a further 100,000 × g spin for 45 min.
TRANCE was immunoprecipitated from the medium and lysate as above. In
experiments utilizing the TACE-deficient fibroblasts, lysates were
split into two aliquots; one was used to isolate FLAG-tagged TRANCE,
and the other was used to immunoprecipitate TACE utilizing an anti-TACE
cytoplasmic domain polyclonal antiserum and protein A-Sepharose. In all
cases, immunoprecipitations were incubated at 4 °C for at least
6 h followed by washing as above. Samples were then analyzed by
SDS-PAGE and autoradiography as described above.
TRANCE Cleavage Site Determination--
15-cm-diameter plates of
CHO cells were transfected with LipofectAMINE and the TRANCE-FLAG
plasmid or control vector. COS-7 cells were electroporated with the
TRANCE-FLAG or control plasmid using an 0.4-cm cuvette in a Bio-Rad
GenePulser II with a capacitance extender and 0.4-cm cuvettes at 200 V,
500 microfarads, and plated onto 15-cm diameter plates. Both cell types
were cultured in OptiMEM for 2 days. The conditioned medium was
collected, supplemented with protease inhibitors, and spun at
13,000 × g for 20 min. The supernatant was incubated
with 1.5 µg of TR-Fc and protein A-Sepharose beads overnight at
4 °C. The beads were washed three times for 10 min in PBS, pH 7.4, 0.05% (v/v) Nonidet P-40. Bound protein was recovered by boiling the
beads in sample loading buffer with dithiothreitol for 5 min, loaded
onto a NuPAGE 10% Bis-Tris gel (Novex), and transferred onto
polyvinylidene difluoride membrane. The membrane was stained with
Coomassie Blue R-250, destained in 50% methanol, 10% acetic acid, and
rinsed in double-distilled water. The ~27-kDa bands representing
soluble TRANCE ectodomain 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 (43).
TNF In agreement with previously published observations (39, 44-47), both
TRANCE and TNF convertase. One of the
TRANCE sheddases is induced by the tyrosine phosphatase inhibitor
pervanadate but not by phorbol esters, whereas the other is refractory
to both of these stimuli. Furthermore, the pervanadate-regulated
sheddase activity is sensitive to TIMP-2 but not TIMP-1, which is
consistent with the properties of a membrane type matrix
metalloprotease. This study provides insights into the properties of
different activities involved in protein ectodomain shedding and has
implications for the functional regulation of TRANCE by potentially
more than one protease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 convertase (TACE,
ADAM 17) has a role in the shedding of TNF
(21, 22), TGF
,
L-selectin, both TNF receptors (5), interleukin-1 receptor II (23), HER4 (24), and Notch (17) and can act as a phorbol
ester-responsive APP
-secretase (25). Kuzbanian (KUZ, ADAM 10, MADM,
SUP-17) is required for Notch signaling (10-13), can cleave the Notch
ligand Delta (15), as well as ephrin A2 (9), and has been shown to have
APP
-secretase activity (26). ADAM 9 (MDC 9) has been implicated in
the phorbol ester-stimulated shedding of heparin binding
epidermal growth factor-like growth factor (27). Finally, ADAM 19 has
been linked to shedding of the epidermal growth factor receptor-ligand
neuregulin-
1 (28). In addition to the ADAM proteases, at least one
matrix metalloprotease, MMP-7 (matrilysin) has a functionally relevant
role in shedding TNF
(29) and FasL (30). For the majority of
proteins subjected to ectodomain shedding, the proteases responsible
have not been identified. Furthermore, in most cases it is not clear
whether one or several proteases can target the same protein or whether the same protease mediates shedding in response to distinct stimuli.
convertase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
polyclonal
antibody was obtained from Endogen. The recombinant TRANCE-receptor Fc
fusion protein, TR-Fc (39), was the generous gift of Y.-W. Choi, B. Wong, and J. Arron. The metalloprotease inhibitor BB-94 was kindly
provided by J. D. Becherer (Glaxo-Wellcome, Research Triangle
Park, North Carolina). Recombinant human TIMP-1 and -2 were the
kind gift of Gillian Murphy.
cDNA was
kindly provided by M. Milla. The coding region of TNF
was amplified
by polymerase chain reaction using primers containing NotI
and XbaI sites in the 5' and 3' primers, respectively. The
resulting fragment was subcloned in frame into the NotI and
XbaI sites of pFLAG CMV2 to allow for the expression of
N-terminally tagged TNF
. TRANCE-TNF chimeras were generated by
polymerase chain reaction using standard techniques (41). The sequence
of all polymerase chain reaction-generated constructs were confirmed by
DNA sequencing (The BioResource Center, Cornell University, Ithaca,
NY). A full-length murine TACE cDNA cloned into the eukaryotic
expression vector pEE12 (42) was the generous gift of G. Murphy. The
MT1-MMP cDNA was provided by M. Roderfeld and H. Tschesche.
antiserum, together with protein A-Sepharose, was
added to the medium. FLAG M2 monoclonal antibody and protein
G-Sepharose were used to immunoprecipitate full-length TRANCE and
TNF
from the lysates. The resulting suspensions were incubated at
4 °C for 2 h to overnight, and the beads were washed three
times with lysis buffer (for lysates) or PBS, pH 7.4, 0.05% (v/v)
Nonidet P-40 (for supernatants). 2× sample loading buffer supplemented
with 5 mM dithiothreitol was added, and the samples were
incubated at 95 °C for 5 min prior to SDS-PAGE analysis. Gels were
fixed in 50% (v/v) methanol, 10% (v/v) acetic acid for 15 min,
rehydrated in water for 15 min, dried, and exposed to Kodak BioMAX XR
film. Quantification was performed using a Fuji BAS2500 Bio Imaging analyzer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TRANCE both undergo metalloprotease-mediated ectodomain
shedding. To compare the properties of the TRANCE and TNF
shedding
activities, the release of soluble TRANCE and TNF
ectodomains was
examined in CHO cells. Cells were transiently transfected with
N-terminally FLAG-tagged TNF
or TRANCE and metabolically labeled.
The cells were chased for 1 h with or without the addition of
several potential modulators of shedding. Labeled TNF
and TRANCE in
the conditioned medium and lysate was then isolated by
immunoprecipitation and analyzed by SDS-PAGE.
ectodomains are released into the medium by a
mechanism sensitive to the hydroxamate-based metalloprotease inhibitor
BB-94 (Fig. 1, A and
B). The release of TRANCE is not further enhanced by
treating CHO cells with PMA, a phorbol ester, but is significantly
enhanced in the presence of pervanadate, a tyrosine phosphatase
inhibitor (Fig. 1A, top panel).
Pervanadate-stimulated shedding is inhibited by the presence of BB-94.
In contrast, TNF
ectodomain release is increased in CHO cells
treated with PMA, as previously described (Fig. 1B,
top panel). In addition, we found that pervanadate is also
effective at stimulating TNF
processing. In all cases, TNF
release is sensitive to inhibition by BB-94.
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Fig. 1.
Comparison of constitutive and PMA- and
pervanadate-stimulated shedding of TRANCE and TNF
in CHO and COS-7 cells. A, CHO (upper
panel) or COS-7 (lower panel) cells were transfected
with FLAG-TRANCE and metabolically labeled overnight. Cells were placed
in fresh medium with (lanes 2, 4, and
6) or without (lanes 1, 3, and
5) 2 µM BB-94 in addition to 25 ng/ml PMA
(lanes 3 and 4), 100 µM pervanadate
(lanes 5 and 6), or no further additives
(lanes 1 and 2), and chased for 1 h. Soluble
TRANCE was then immunoprecipitated from supernatants and analyzed by
SDS-PAGE autoradiography. B, TNF
shedding was assessed in
CHO (upper panel) and COS-7 (lower panel) cells.
Cells were transfected with pFLAG-TNF
and treated as in A
above. TNF
ectodomain was recovered from the medium and analyzed by
SDS-PAGE autoradiography. C, CHO (open bars) and
COS-7 (shaded bars) cells transiently overexpressing TRANCE
were metabolically labeled and then incubated in medium supplemented
with carrier (
), 25 ng/ml PMA, or 100 µM PV for 1 h. Soluble TRANCE ectodomain from the conditioned medium was
immunoprecipitated with TR-Fc and separated on SDS-PAGE. The amount of
TRANCE recovered was quantified using a Fuji Film BAS imager and is
shown as a percentage of TRANCE shed by the untreated cells. Results
represent the averages ± S.D. of three independent samples. In
both cell lines pervanadate treatment lead to increased shedding
compared with control cells. *, p < 0.025; **,
p < 0.0001 by two-sample Student t test.
D, TNF
shedding from CHO (open bars) and COS-7
(shaded bars) cells was assessed as for TRANCE in
C above. Results represent the averages ± S.D. of
three independent samples. *, p < 0.01; **,
p < 0.0001 by two-sample Student t test.
E, specificity of pervanadate stimulation. CHO cells
transfected with TRANCE (upper panel) or TNF
(lower
panel) were metabolically-labeled and then chased for 1 h
with fresh medium alone (lane 1) or medium containing 100 µM sodium vanadate (lane 2), 100 µM hydrogen peroxide (lane 3), or 100 µM pervanadate (lane 4). Soluble ectodomain
was then immunoprecipitated from the supernatant.
The inability of PMA to affect the rate of ectodomain shedding of
TRANCE is at odds with our previous findings in COS-7 cells (39). We
therefore examined the shedding of TNF and TRANCE in COS-7 cells
under conditions identical to those used above (Fig. 1, A
and B, lower panels). The release of TNF
in
COS-7 cells resembles that seen in CHO cells, with both PMA and
pervanadate increasing ectodomain shedding (Fig. 1B, compare
upper and lower panels). As in CHO cells,
constitutive and stimulated shedding is inhibited by BB-94. TRANCE
processing in COS-7 is also a BB-94-sensitive process but, in contrast
to our previous study, is minimally affected by PMA (Fig.
1A, lower panel). We cannot at present explain
this discrepancy, which may be due to previous experimental error or may reflect a difference in the COS-7 cells used in this study compared
with the previous one. Pervanadate treatment leads to only a marginal
increase in TRANCE ectodomain shedding in COS-7 cells, in sharp
contrast to its effect on CHO cells. The difference in
pervanadate-mediated TRANCE shedding in CHO and COS-7 cells was
confirmed by quantifying the TRANCE released from cells treated with
either PMA, pervanadate, or carrier as control (Fig. 1C). Although pervanadate-treated COS-7 cells release more TRANCE than control treated COS-7 cells (172% of control), it was considerably lower than the amount released by CHO cells in response to pervanadate (484% of control). In contrast, PMA and pervanadate induced a similar
increase in the release of TNF
in both COS-7 and CHO cells (Fig.
1D).
To control for the specificity of pervanadate stimulation, the effect
of equal concentrations of sodium vanadate, hydrogen peroxide, and
pervanadate on both TNF and TRANCE shedding was evaluated (Fig.
1E). Neither sodium vanadate nor hydrogen peroxide is able
to enhance shedding above that seen in unstimulated cells, whereas
pervanadate enhances the shedding of both proteins. Thus, although both
TNF
and TRANCE ectodomain shedding is sensitive to BB-94, TNF
processing is stimulated by both PMA and pervanadate, whereas TRANCE
shedding is only enhanced in the presence of pervanadate.
To further investigate the difference between TRANCE shedding in CHO
and COS-7 cells, we compared the soluble TRANCE ectodomain released
from these cells as well as the membrane stubs generated by shedding.
Interestingly, CHO cells produce at least two forms of TRANCE
ectodomain, whereas only the slower migrating form is seen in the
medium from COS-7 cells (Fig.
2A). Immunoprecipitation of
cell lysates with an anti-FLAG antibody recovers a small amount of both
cleaved ectodomain as well as membrane stub in addition to the
full-length TRANCE protein (Fig. 2B). Although COS-7 cells contain only one form of TRANCE ectodomain and a corresponding membrane
stub, in CHO cell lysates a slightly smaller form of ectodomain and a
correspondingly larger membrane stub is also detected. To confirm that
the differently migrating forms of TRANCE ectodomain are generated by
proteolysis at distinct sites, TRANCE was purified from the conditioned
medium of either CHO or COS-7 cells overexpressing FLAG-tagged TRANCE
and subjected to N-terminal sequencing. Only a single N terminus was
detected in TRANCE from COS-7 cells, which corresponded to cleavage at
the previously reported site between Arg138 and
Phe139 (33). In contrast, three forms of soluble TRANCE
differing at their N terminus were isolated from CHO conditioned
medium, corresponding to cleavage between Gln137 and
Arg138, Arg138 and Phe139, and
Met145 and Met146 (Fig. 2C).
Therefore, CHO cells shed TRANCE by cleaving the membrane bound protein
at three distinct sites, one of which corresponds to the previously
reported site used in 293 and COS-7 cells.
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Because CHO cells produce several distinct forms of TRANCE, we wished
to determine whether this is due to the activity of a single protease
or of different proteases. We therefore examined the ability of the
metalloprotease inhibitors TIMP-1 and TIMP-2 to inhibit the production
of the distinct forms of TRANCE (Fig. 3A). TIMP-2, but not TIMP-1,
was able to inhibit the production of the low molecular weight form of
TRANCE and the high molecular weight form but did not effect the
release of the intermediate form. In addition, pervanadate stimulation
appears to specifically enhance the production of the high and low
molecular weight forms of soluble TRANCE but not the intermediate form
(Fig. 1A, top panel). In the presence of TIMP-2,
constitutive and pervanadate-stimulated shedding are indistinguishable,
suggesting that pervanadate specifically enhances the activity of the
TIMP-2-sensitive protease.
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TIMP-2 was able to efficiently inhibit pervanadate-induced TRANCE shedding at concentrations in the low nanomolar range (Fig. 3B) with maximum inhibition seen by 18 nM TIMP-2. No further inhibition of the remaining constitutive sheddase activity was seen with concentrations of TIMP-2 up to 90 nM. TIMP-1 had no effect on either constitutive or stimulated shedding of TRANCE at any concentration tested, up to 75 nM (Fig. 3A and data not shown). In comparison, TRANCE shedding in COS-7 cells was not affected by either TIMP (Fig. 3A, lower panel). Taken together, these results indicate that two distinct proteases can shed TRANCE in CHO cells. One is a TIMP-2-sensitive protease that is stimulated by treatment with pervanadate. The second protease is not affected by either PMA or pervanadate treatment. Furthermore, it is resistant to TIMP-1 and TIMP-2 inhibition, although it is possible that the protease acts in a subcellular compartment that is inaccessible to the TIMPs. The TRANCE sheddase in COS-7 cells shares the characteristics of this second protease, because it is not stimulated by pervanadate and is not inhibited by either TIMP-1 or TIMP-2.
As a comparison with the shedding of TRANCE, the effect of TIMP-1 and
-2 on TNF shedding was assessed (Fig. 3C). Neither constitutive nor PMA or pervanadate-stimulated shedding of TNF
was
altered by TIMP-1 or -2 in CHO cells. These results are consistent with
the likely role of TACE, an ADAM metalloprotease, as a TNF
sheddase
(21, 22). TACE has previously been shown to be sensitive to TIMP-3, but
not to TIMP-1 or -2 (42). Furthermore, these results indicate that
MMP-7, implicated as a TNF
sheddase in macrophages (29), is likely
not involved in TNF
shedding in CHO cells.
The ectodomain shedding of several proteins has been shown to involve
the MEK/ERK and p38 MAP kinase pathways. In particular, constitutive
shedding of TGF, a TACE substrate, requires the p38 MAP kinase
pathway, whereas the MEK/ERK MAP kinase pathway mediates the increase
in TGF
shedding in response to phorbol ester and epidermal growth
factor and platelet-derived growth factor receptor activation (48). To
determine whether these signaling pathways are also required for TRANCE
shedding, the effect of MAP kinase pathway inhibitors on constitutive
and pervanadate-stimulated shedding was assessed in CHO cells (Fig.
4). Neither the MEK1/2 inhibitor U0126
nor the p38 MAP kinase inhibitor SB202190 affected the shedding of
TRANCE. In contrast, under similar conditions the metalloprotease
mediated shedding of interleukin-2 receptor
, and the low affinity
p75 NGFR was impaired by these inhibitors, confirming that the
inhibitors are active.5 These
results further emphasize that the shedding of TRANCE is distinct from
previously described shedding events.
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Through the use of cells generated from various "knock-out" mice,
including embryonic fibroblasts, it has been possible to examine the
importance of several proteases as potential sheddases (5, 16, 17, 21,
23-25, 29, 30). We therefore assessed whether murine embryonic
fibroblasts might offer a system for analyzing TRANCE shedding (Fig.
5). TRANCE shedding in fibroblasts from
wild type embryos closely resembles that seen in CHO cells. Shedding is
enhanced by pervanadate but not PMA (Fig. 5A). In addition,
TIMP-2, but not TIMP-1, inhibits the pervanadate-stimulated sheddase
(Fig. 5B). Thus, identical or very similar proteases are
likely active in TRANCE shedding in both CHO cells and embryonic fibroblasts.
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Previously, we demonstrated that TACE is capable of cleaving TRANCE
in vitro between amino acids Arg138 and
Phe139 (39), a site used in COS-7, 293 fibroblasts, and CHO
cells (Fig. 2C and Refs. 33 and 39). To determine whether
TACE is involved in TRANCE shedding in cells, FLAG-tagged TRANCE was
overexpressed in embryonic fibroblasts derived from TACE-deficient
mice, either alone or together with full-length murine TACE (Fig.
6A). TRANCE shedding by cells
lacking or expressing TACE is indistinguishable. Similar to COS-7
cells, the TACE-deficient fibroblasts show only a small increase in
TRANCE shedding in response to pervanadate stimulation; coexpression of
TACE does not alter this response. Thus, although TACE is capable of
cleaving TRANCE in vitro, it is not required for the
constitutive shedding of TRANCE in fibroblasts. The inability of TACE
overexpression to induce pervanadate-stimulated shedding does not in
itself rule out a role for TACE in this process. However, two aspects
of pervanadate-stimulated shedding of TRANCE argue against a role for
TACE. First, the pervanadate-responsive protease in CHO cells cleaves
TRANCE at a site distinct from that targeted by TACE in
vitro. Secondly, the pervanadate-stimulated sheddase is inhibited
by TIMP-2 (Fig. 3A), whereas TACE is not (42).
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Kuzbanian (KUZ, ADAM 10, MADM) has been reported as a candidate
sheddase for several of the same proteins as TACE, particularly TNF
and APP (26, 49, 50). The potential role of KUZ as a TRANCE sheddase
was assessed by comparing the release of TRANCE ectodomain from
fibroblasts derived from KUZ-deficient murine embryos and their wild
type littermates (Fig. 6B). No appreciable difference in
TRANCE shedding is observed in these two cell lines. Similar to results
using the TACE knock-out cells and COS-7 cells, TRANCE shedding was not
significantly enhanced in response to pervanadate in either cell type.
Furthermore, based on the different TIMP sensitivities (KUZ is
inhibited by TIMP-1 and -3 (51), and the pervanadate-stimulated TRANCE
sheddase is inhibited by TIMP-2 but not TIMP-1), it is unlikely that
KUZ is involved in pervanadate-stimulated TRANCE shedding. Thus,
neither TACE nor KUZ are required for the constitutive or
pervanadate-stimulated release of TRANCE in fibroblasts.
In addition to TACE and KUZ, several other ADAM metalloproteases with a catalytic site consensus sequence (ADAM 1, 8, 9, 12, 15, and 19) are currently known to be widely expressed. Because the TIMP inhibition profile of most of these potential TRANCE sheddases has not yet been established, we addressed the role of ADAM 9, 12, 15, and 19 in TRANCE shedding using primary embryonic fibroblasts lacking one or more of these ADAMs. Fig. 6C shows a representative experiment where TRANCE shedding is compared between fibroblasts from wild type embryos, embryos homozygous for a gene trap insertion in the ADAM 19 gene, and ADAM 9-, 12-, and 15-deficient animals. TRANCE shedding in the absence of stimuli and in response to pervanadate is very similar in all three cell populations. Similar observations were made using embryonic fibroblasts derived from ADAM 9/15-deficient mice (data not shown). In the case of ADAM 19, the gene trap gives rise to a truncated and thus most likely nonfunctional ADAM 19 protein lacking parts of the ectodomain and all of its cytoplasmic domain. These results argue against a role for ADAM 9, 12, 15, and 19 as constitutive or inducible TRANCE sheddases.
The TIMP inhibition profile of the pervanadate-stimulated TRANCE
sheddase is similar to that of several of the MT-MMPs. Furthermore, based on Northern blot analysis, CHO cells express MT1-MMP, whereas COS-7 cells express little or no MT1-MMP (data not shown). To assess
whether MT1-MMP is capable of acting as a TRANCE sheddase, MT1-MMP was
coexpressed with TRANCE in both COS-7 and CHO cells (Fig.
7). Overexpression of MT1-MMP led to a
large increase in the amount of TRANCE ectodomain released from both
CHO and COS-7 cells. At least some increase in TRANCE release was
observed when cells were treated with pervanadate. Furthermore, the
TRANCE ectodomain released from cells overexpressing MT1-MMP appears to
comigrate with the lower molecular weight forms of TRANCE released by
CHO cells.
|
To assess whether MT1-MMP targets the same peptide bonds as the pervanadate-induced sheddase in CHO cells, soluble TRANCE ectodomain was purified from COS-7 cells cotransfected with TRANCE and MT1-MMP and subjected to N-terminal sequencing as above. Although N-terminal sequencing of ecto-TRANCE released from control COS-7 cells confirmed the result shown in Fig. 2C, a second N-terminal sequence was seen in ecto-TRANCE released from COS-7 cells that had been cotransfected with MT1-MMP. This sequence (MEGSXLD) matches one of the sequences of ecto-TRANCE released from CHO cells exactly (Fig. 2C). The results from this analysis are consistent with a role of MT1-MMP or of another MT-MMP with a similar cleavage site specificity in PV-dependent TRANCE shedding in CHO cells.
The finding that two type II transmembrane proteins belonging to the
TNF protein family, TNF and TRANCE, are shed by different metalloproteases raises the question of which determinant in each substrate is necessary for recognition and cleavage by a specific protease. For several type I transmembrane proteins, including TGF
,
the juxtamembrane sequence surrounding the cleavage site has been shown
to be sufficient to target the protein for regulated shedding (24,
52-54). To assess whether this is also true for TRANCE and TNF
,
several chimeric proteins were generated. Specifically, the constructs
were designed to test the importance of the cytoplasmic domain and
transmembrane sequence, the juxtamembrane region, and the TNF domain.
The TNF-TRANCE chimeras (see schematic in Fig. 8A) were overexpressed and
metabolically labeled in CHO cells, and the release of ectodomain under
constitutive and PMA or pervanadate stimulation was examined (Fig.
8B). In all cases, the origin of the juxtamembrane region
determines the shedding profile of the chimeric protein. The shedding
of proteins containing the juxtamembrane sequence of TNF
(chimeras 1 and 4) is increased by both PMA and pervanadate treatment, whereas
chimeras containing the TRANCE juxtamembrane region (chimeras 2 and 3)
underwent enhanced shedding in the presence of pervanadate only.
Furthermore, for chimeras containing the TRANCE juxtamembrane region,
pervanadate specifically enhances the production of the high and low
molecular weight forms of the soluble ectodomain but not the
intermediate form (e.g. chimera 2), similar to its effect on
TRANCE (Figs. 1A and 3A). Therefore, the
juxtamembrane sequences of TNF
and TRANCE appear to specify their
distinct shedding profiles. Furthermore, the cytoplasmic domain of
TRANCE, which is tyrosine phosphorylated upon pervanadate stimulation
(data not shown), does not appear to be required for ectodomain
shedding of TRANCE.
|
In the case of several shed proteins, including TNF (55), it has
been proposed that the distance between the membrane and the
proteolytic site is a critical factor for shedding (53, 56, 57). TNF
and TRANCE differ significantly in the length of their juxtamembrane
regions and in the distance between their transmembrane domains and the
site of proteolysis. In addition, the longer juxtamembrane domain of
TRANCE contains two cysteine residues that presumably form a disulfide
bond. To address the importance of this region in TRANCE shedding, a
deletion mutant was constructed that lacks the first 44 amino acids of
the juxtamembrane domain, including both cysteine residues (TRANCE
JM), placing the cleavage sites at the same distance from the
membrane as for TNF
. This deletion mutant undergoes ectodomain
shedding, but pervanadate stimulation is not observed (Fig.
8B). Furthermore, only one form of TRANCE ectodomain is
visualized by SDS-PAGE. These data suggest that the
pervanadate-responsive sheddase requires the intact juxtamembrane
sequence to target TRANCE for proteolysis. However, whether the
protease relies on the overall length of the juxtamembrane region or
whether particular sequences deleted in TRANCE
JM recruit the
protease remains to be determined.
![]() |
DISCUSSION |
---|
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---|
Several members of the TNF family of cytokines undergo
metalloprotease-mediated ectodomain shedding, including TNF
(44-47), FasL (58, 59), and TRANCE (33, 39). We have previously shown
that TACE is capable of cleaving the osteoclast differentiation and
dendritic cell survival factor TRANCE/OPGL in vitro at the major cleavage site that is used in COS-7 and human 293 cells (33, 39).
Here we have extended our studies by examining TRANCE shedding in CHO
cells and in fibroblasts, both cell types that are commonly used for
studies of protein ectodomain shedding. We found that at least two
distinct metalloproteases are involved in shedding TRANCE in these
cells. These enzymes are distinct from TACE and differ from one another
in their responsiveness to known stimulators of ectodomain shedding,
their cleavage site selection, and their sensitivity to TIMPs.
At the outset of the present study, we tested the hypothesis that TACE
may have a role in shedding TRANCE by directly comparing several
properties of TRANCE and TNF sheddases. We found several lines of
evidence indicating that TACE is in fact not a TRANCE sheddase. In
primary embryonic fibroblasts and CHO cells, TRANCE shedding is
stimulated by pervanadate but not PMA. In contrast, in most cases where
TACE has been shown to play a role as a sheddase, it has been in the
context of phorbol ester-stimulated shedding (5, 23-25). In addition,
pervanadate stimulation of CHO cells appears to enhance a protease
activity that cleaves TRANCE at sites distinct from those targeted by
TACE in vitro. Finally, the pervanadate-stimulated sheddase
is inhibited by TIMP-2, but not TIMP-1, whereas TACE is sensitive to
TIMP-3, but not TIMP-1 or -2 (42). These results strongly argue against
a role for TACE in pervanadate-activated TRANCE shedding.
The matrix metalloprotease inhibitors TIMP-1 and -2 not only helped distinguish between TACE and TRANCE sheddases but also revealed the presence of a constitutive TRANCE sheddase that is distinct from the pervanadate-stimulated activity. (It should be noted that although both shedding activities are present in unstimulated CHO cells, we use the term constitutive TRANCE sheddase to refer to the protease(s) that does not respond to PMA or pervanadate stimulation.) The constitutive activity depends on a metalloprotease, as it is sensitive to BB-94 but, unlike the pervanadate-stimulated activity, is not inhibited by TIMP-1 or -2. Furthermore, the constitutive and pervanadate-stimulated sheddases cleave TRANCE at distinct sites within the juxtamembrane region, generating slightly different forms of soluble TRANCE. Only the constitutive TRANCE sheddase in COS-7 and CHO cells targets the same site that is cleaved by TACE in vitro. Yet TACE is not necessary for this activity either, because embryonic fibroblasts lacking TACE still shed TRANCE constitutively. These findings demonstrate the presence of two distinct shedding activities for TRANCE in the same cell type.
With respect to the existence of the two distinct TRANCE sheddases
observed here, it is interesting to point out that TNF can be
released by at least three different proteases in a functionally relevant manner, albeit in different cells and under different conditions. TACE is the major TNF
converting enzyme in T cells and
most likely also in several other cell types (21, 23). Yet matrilysin
(MMP-7) can also release TNF
from its membrane anchor, and this
activity has been shown to be physiologically relevant in a herniated
disc resorption model (29). Finally, the serine proteinase 3 can
produce functional soluble TNF
, at least in inflammatory foci (60).
In addition to these three proteases, ADAM 10 (49) and MT4-MMP (61) are
capable of acting as TNF
sheddases when overexpressed in cells.
For the majority of shed proteins, the protease or proteases responsible for their release are still unknown. To date, only five metalloproteases, TACE, KUZ, ADAM 9, ADAM 19, and MMP-7 have been implicated as sheddases. With respect to the activities that are involved in TRANCE shedding in CHO cells and primary fibroblasts, our results have excluded these five sheddases and have substantially narrowed down the remaining list of candidate metalloproteases.
All soluble matrix metalloproteases examined to date are sensitive to both TIMP-1 and TIMP-2, ruling out the currently known soluble MMPs, including MMP-7 (62, 63), as constitutive or stimulated TRANCE sheddases. Furthermore, the TIMP inhibition profile of the pervanadate-stimulated TRANCE sheddase not only rules out TACE (see above), but also rules out KUZ (ADAM 10), which is sensitive to TIMP-1 and -3 (51). Constitutive shedding of TRANCE is intact in fibroblasts lacking TACE and in cells lacking KUZ, indicating that these ADAMs are not required as constitutive TRANCE sheddases.
Besides TACE and KUZ, only five currently known ADAMs have a catalytic site consensus sequence and are expressed in a number of somatic tissues outside of the testis (ADAM 1, 9, 12, 15, 19). Of these, ADAMs 9, 12, and 15 can be excluded as TRANCE sheddases because the pervanadate-stimulated and constitutive shedding activities are still present in primary embryonic fibroblasts lacking these ADAMs. ADAM 19 can most likely also be ruled out because embryonic fibroblasts homozygous for a gene trap insertion in the ADAM 19 gene still have both TRANCE shedding activities. Thus, the majority of the currently known ADAMs predicted to be catalytically active and widely expressed have been excluded as candidate TRANCE sheddase.
Interestingly, the sensitivity of the pervanadate-stimulated TRANCE sheddase to TIMP-2 but not TIMP-1 matches the TIMP inhibitor profile of several MT-MMPs (63-66), which thus emerge as good candidate TRANCE sheddases in CHO cells and primary fibroblasts. In fact, overexpression of MT1-MMP in both CHO and COS-7 cells leads to a dramatic increase in TRANCE shedding, and this effect is further enhanced by pervanadate treatment. In addition, although CHO cells clearly express MT1-MMP, COS-7 cells express little if any detectable MT1-MMP mRNA (data not shown). Finally, at least one of the TRANCE cleavage sites that is used in CHO cells but not COS-7 cells is cleaved in COS-7 cells when MT1-MMP is coexpressed. These results are consistent with a role of MT1-MMP in pervanadate-stimulated TRANCE shedding. Nevertheless, further studies will be necessary to address whether or not other MT-MMPs may also meet the same set of criteria for candidate pervanadate-dependent TRANCE sheddases.
The ability to shed TRANCE in response to pervanadate stimulation
appears to be limited to certain cell types. Although CHO cells and
primary embryonic fibroblasts show a robust increase in TRANCE shedding
in response to pervanadate, COS-7 cells and three independently derived
immortalized embryonic fibroblast cell lines
(TACEZn, ADAM 10
/
, and ADAM 10 wt) do
not. It is likely that the difference between CHO and COS-7 cells is
due to a lack of a protease, such as an MT-MMP, or alternatively due to
a lack of a signaling pathway that is necessary to activate the
protease. Our results also do not rule out that the inability of COS-7
cells to process TRANCE at the two additional sites used in CHO cells
is due to a species specificity. The different TRANCE shedding seen in
the primary and immortalized embryonic fibroblasts is intriguing. The
primary fibroblast cultures likely represent a more diverse population of cell types compared with the immortalized cell lines. The expression of the pervanadate-stimulated sheddase may be limited to only some of
the cells in a preparation of embryonic fibroblasts. Alternatively, the
process of immortalization could interfere with the pathway responsible
for pervanadate-induced TRANCE shedding.
TRANCE is only one of a few proteins whose shedding is not enhanced by phorbol ester. For the majority of shed proteins, phorbol ester treatment induces a rapid increase in ectodomain processing. Indeed, it has been proposed that a general shedding mechanism exists that is responsible for the proteolysis of numerous structurally and functionally distinct proteins in response to PMA (67). Although pervanadate is also able to enhance the shedding of various proteins, in most of these cases PMA has a similar effect. Furthermore, in the case of erbB-4/HER4 shedding, a single protease, TACE, has been shown to be necessary for both phorbol ester and pervanadate-stimulated shedding (24). Thus, although PMA and pervanadate may initially activate distinct signaling pathways, they both can lead to the activation of a common sheddase(s). The finding that the ectodomain shedding of TRANCE (this study) and HER2/neu/erbB-2 (68) is enhanced by pervanadate but not by phorbol ester implies that in addition to activating a pathway targeted by phorbol ester, pervanadate also activates shedding events not amenable to phorbol ester stimulation. Furthermore, the distinct TIMP inhibition profiles of the TRANCE and HER2 sheddases (HER2 shedding is inhibited by TIMP-1, but not TIMP-2 (68)) suggest that at least two distinct proteases must be activated in response to pervanadate and not PMA.
Finally, given the clear differences in the inducible shedding of
TRANCE and TNF, both of which are TNF family members, we decided to
evaluate which part of the substrates are necessary to determine
specific targeting by one or the other sheddase. Previous studies have
addressed this question for several type I integral membrane proteins
that are shed, including TGF
, APP (54), and erbB-4 (24). In all
three cases, the juxtamembrane region is sufficient for stimulated
shedding. Our results demonstrate that this is also the case for the
type II transmembrane proteins TRANCE and TNF
. For both proteins,
the juxtamembrane region is necessary and sufficient to govern their
characteristic shedding behavior. Interestingly, the juxtamembrane
sequence of TRANCE is significantly longer than that of TNF
, and
TRANCE is cleaved at least 66 amino acids from the membrane, whereas
the predominant TNF
cleavage site is 23 amino acids from the
predicted transmembrane domain. For TNF
, the distance from the
membrane has been suggested to play a critical role in determining
cleavage site selection (55). Similarly, when the
juxtamembrane region of TRANCE is shortened, the
pervanadate-responsive cleavage is lost. However, constitutive shedding
remains intact. Thus, the length of the juxtamembrane stalk region as
well as specific sequences within this domain may be determinants in
regulating the shedding of TRANCE and TNF
.
In summary, biochemical and pharmacological studies have revealed
different endogenous TRANCE and TNF shedding activities in the same
cell type. In CHO cells and embryonic fibroblasts, a constitutive and
an inducible metalloprotease, the latter most likely an MT-MMP, have
emerged as TRANCE sheddases. Both of these sheddases have properties
that clearly distinguish them from the TNF
convertase. Criteria such
as those applied here toward TRANCE shedding should also be useful in
defining the properties of relevant sheddases for other substrates of
interest. These could then be compared with the properties of the
expanding list of potential sheddases. Because TRANCE is shed by at
least one metalloprotease in all cells analyzed in this study, we
predict that ectodomain shedding is likely to occur in cells and
tissues where TRANCE is active as a signaling molecule. If so, shedding
is likely to have a role in regulating TRANCE function, either by
activating or inactivating it or by producing a soluble growth factor
that can function in a paracrine or autocrine manner.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. G. Murphy for donating
recombinant TIMP-1 and TIMP-2 and the full-length murine TACE cDNA
clone, Dr. J. D. Becherer for providing BB-94, Dr. M. Milla for a
human TNF cDNA clone, and Drs. Y.-W. Choi, B. Wong, and J. Arron
for their generous gift of the FLAG-tagged TRANCE cDNA and TR-Fc,
and Drs. M. Roderfeld and H. Tschesche for providing the MT1-MMP
cDNA clone. Mice and murine embryonic fibroblasts carrying various
gene disruptions were kindly provided by Drs. P. Reddy, J. Peschon and
R. Black (TACE-deficient cells), D. J. Pan (KUZ-deficient cells),
A. Fujisawa-Sehara (ADAM 12-deficient mice), H. Zhou (ADAM 19-deficient
cells), and G. Weskamp (ADAM 9/12/15 and ADAM 19-deficient cells). We
are indebted to L. Lacomis and Drs. H. Erdjument-Bromage and P. Tempst for N-terminal microsequencing, and to B. Lee for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from Glaxo Wellcome (to C. P. B.), by National Institutes of Health Grant RO1 GM58668 (to C. P. B.), by Memorial Sloan-Kettering Cancer Center Support Grant NCI-P30-CA-08748, by the Samuel and May Rudin Foundation, and by the DeWitt Wallace Fund.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.
¶ Supported in part by a National Institutes of Health Medical Scientist Training Program Training Grant 5T32GM07739-17 and the Louis and Rachel Rudin Family Foundation.
Present address: Johns Hopkins University School of Medicine,
725 North Wolfe St., PCTB Rm. 714, Baltimore, MD 21205.
** 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@ski.mskcc.org.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010741200
2 G. Weskamp, L. Lum, and C. Blobel, manuscript in preparation.
3 G. Weskamp, A. Fujisawa-Sehara, and C. P. Blobel, manuscript in preparation.
4 H. Zhou, G. Weskamp, B. Skarnes, and C. P. Blobel, manuscript in preparation.
5 J. Schlöndorff, L. Lum, and C. P. Blobel, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
APP, amyloid precursor protein;
MMP, matrix metalloproteinase;
MT-MMP, membrane type matrix metalloproteinase;
PMA, phorbol-12
myristate 13-acetate;
PV, pervanadate;
TACE, tumor necrosis factor convertase;
TIMP, tissue inhibitor of matrix metalloproteinase;
CHO, Chinese hamster ovary;
MAP, mitogen-activated protein;
DME, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
KUZ, kuzbanian..
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