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-Converting Enzyme Cleaves the Macrophage Colony-Stimulating Factor Receptor in Macrophages Undergoing Activation1
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Dipartimento di Patologia e Oncologia Sperimentali, Università di Firenze, Florence, Italy; and
Immunex, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The treatment of NIH-3T3 murine fibroblasts, ectopically expressing
M-CSFR, with the PKC activator tetradecanoylphorbol myristate acetate
(TPA) was shown to determine the release into culture medium of a
portion of M-CSFR that maintains its ligand-binding ability
(5). This fact, taken together with our findings, led us
to hypothesize that macrophage activators recruit, via intracellular
PLC/PKC-dependent signals, an extracellularly active endoprotease that
cleaves cell surface M-CSFR (4). A PKC-mediated ectodomain
shedding was, in fact, shown for a number of cell surface proteins,
including pro-TNF-
(TNF), pro-TGF-, prostem cell factor, CD14,
CD16, CD43, CD44, and L-selectin, and the receptors for TNF and IL-6
(6, 7, 8). However, as the cleavage sites of these proteins
share only little, if any, sequence similarity, they are believed to be
shed by a number of different proteases. In particular, no information
was available about the identity of the protease responsible for the
down-modulation of M-CSFR in macrophages undergoing activation.
We report in this study a stepwise approach to the identification of M-CSFR-cleaving protease in macrophages undergoing activation. Using a murine macrophage cell line, we found that the enzyme responsible for the down-modulation of M-CSFR by either LPS or TPA is a transmembrane metalloprotease. M-CSFR cleavage was abolished by pretreating cells in vivo with an Ab directed against the extracellular, catalytic domain of pro-TNF-converting enzyme (TACE). Monocytes obtained from TACE-negative mice were unable to down-modulate M-CSFR in response to TPA. These data indicated that TACE-dependent cleavage is the mechanism responsible for M-CSFR down-modulation in activated macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BAC-1.2F5 (BAC) cell line was derived from murine (BALB/c x A.CA)F1 adherent spleen cells immortalized by transfection with replication-deficient SV40 DNA. BAC cells are strictly dependent on M-CSF for survival and proliferation in culture and retain a large number of the phenotypical and functional characters of normal macrophages (9). BAC cells therefore represent a homogeneous population, free of cells usually contaminating primary macrophage preparations and potential source of interfering cytokines, and were used for all previous studies on M-CSFR down-modulation in macrophages (2, 3, 4). BAC cells were cultured in DMEM (EuroClone, Cramlington, Northumberland, U.K.) supplemented with 2 mM glutamine, 10% heat-inactivated FBS (EuroClone; catalogue ECS-0180), and murine rM-CSF (6 ng/ml). M-CSF was bacteria expressed, HIS-TAG conjugated, and affinity purified on Ni2+-NTA-agarose columns (Qiagen, Hilden, Germany). Incubation was conducted at 37°C in 95% air, 5% CO2, water-saturated atmosphere. Culture medium was completely renewed every 2 days. BAC cells were passaged by scraping at subconfluency (80% saturated growth surface, i.e., approximately 5 x 104 cells/cm2), corresponding to a density of 3 x 105 cells/ml.
The Dexter-ras-myc (DRM) cell line derived from long-term
cultures established with bone marrow cells of (C57BL/6 x
129)F1 mice, following immortalization of
nonadherent cells by infection with a
ras/myc-encoding retrovirus. DRM cells are
strictly dependent upon GM-CSF for growth in culture and exhibit a
monocytic cell surface phenotype (10). DRM cells,
homozygous for a TACE mutation
(TACE
Zn/Zn)
which deletes the Zn-binding domain, thereby abolishing
metalloproteinase activity (TACE-negative DRM cells), were obtained
from chimeric mice generated with C57BL/6 cells and 129-derived
TACE-negative cells as above, except for the treatment of long-term
cultures with G418 to select against TACE+/+
cells (10). Wild-type (TACE+/+) and
TACE-negative DRM cells were cultured in RPMI 1640 (EuroClone),
supplemented with 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM
sodium pyruvate, 100 µM 2-ME, 10% FBS, 0.5 ng/ml murine rGM-CSF, and
10 ng/ml murine rIL-3 (PeproTech, Rocky Hill, NJ; catalogue 315-03 and
213-13, respectively) (11). Incubation was conducted as
above. DRM cells are loosely adherent and were passaged by gentle
scraping, after reaching a density of approximately 2 x
106 cells/ml.
Cell stimulation and lysis
Subconfluent BAC cell cultures were incubated for 16 h in DMEM supplemented with 10% FBS, in the absence of M-CSF, to up-regulate M-CSFR expression. Cells were then treated for 60 min with 1 µM TPA (Sigma, St. Louis, MO; catalogue P-8139) or 10 ng/ml LPS (from Escherichia coli; Sigma; catalogue L-4391). In some experiments, cell monolayers were pretreated with an acid buffer (125 mM NaCl, 50 mM glycine-HCl, pH 3) or with a neutral buffer (same composition, pH 7), washed three times with PBS, and then stimulated for 60 min with 1 µM TPA in FBS-free DMEM. At the end of treatments, cell monolayers were washed three times with PBS at 0–2°C, and cells were removed by scraping in PBS (1 ml/plate), transferred into Eppendorf tubes, centrifuged, and lysed in 50 µl/tube of an ice-cold buffer (pH 7.1) containing 10 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, 5 mM disodium EDTA, 1 mM PMSF (Merck, Darmstadt, Germany; catalogue 7349), 10 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (Sigma; catalogue T-4376), 0.1 U/ml aprotinin (Sigma; catalogue A-1153), and 4 µg/ml pepstatin-A (Sigma; catalogue P-4265). Insoluble material was removed by centrifugation (18,000 x g, 30 min, 4°C), and the protein concentration of supernatants was determined by the Bradford method (12).
Inhibitors used in vivo
1,10-Phenanthroline, 5 mM (Sigma; catalogue P-9375); IC-3, TNF
protease inhibitor, 200 µM (Immunex, Seattle, WA); BB3103, 10 µM
(chosen as a representative of a group including BB2116, BB2275, and
BB2284; British Biotech, Oxford, U.K.); EDTA, 10 mM (Sigma; catalogue
E-1644); aprotinin, 0.1 U/ml; PMSF, 1 mM; calpain inhibitor-I, 20 µM
(Boehringer Mannheim, Mannheim, Germany; catalogue 1086 090);
pepstatin-A, 6 µM; 2-macroglobulin, 7.5
mg/ml (Boehringer Mannheim; catalogue 602 442); dec-RVKR-cmk, 50 µM
(Alexis, Läufelfingen, Switzerland; catalogue 260-022-M001);
phalloidin, 10 µM (Sigma; catalogue P-2141);
phosphoinositide-specific PLC (PI-PLC), 100 µmol/ml (Boehringer
Mannheim; catalogue 1 143 069).
Antibodies
Anti-murine M-CSFR rabbit polyclonal Ab (anti-M-CSFR), raised against the bacteria-expressed, affinity-purified extracellular domain of murine M-CSFR (NH2-terminal aa 1–311), was kindly provided by Dr. Manuela Baccarini, Institut für Mikrobiologie und Genetik, Wiener Biozentrum (Vienna, Austria). Anti-TACE mouse mAb Tc3-7.49 (anti-TACE), raised against the catalytic domain of rTACE, was kindly provided by Dr. Marcia Moss, GlaxoWellcome (Durham, NC) (13). Anti-TNF mouse mAb (anti-TNF; PeproTech; catalogue 500-P64), which was effective in inhibiting TNF activity, was assessed by an HLA induction expression assay in murine melanoma cells (14).
Determination of soluble M-CSFR release
Subconfluent BAC cell cultures were incubated for 16 h in M-CSF-free medium (see above), washed three times with sterile PBS, and then treated for 30 min with 1 µM TPA in DMEM. Culture medium (about 3 ml/dish) was recovered, and 5 mM EDTA, 1 mM PMSF, and 10 µg/ml TPCK were added before centrifugation to remove cells and dialysis through a 10-kDa cutoff membrane in 1 L of Tris-HCl, 500 µM (pH 7.4; two changes of buffer in 24 h). Dialyzed medium was lyophilized and then dissolved in 50 µl of cell lysis buffer (see above), and the protein concentration was determined by the Bradford method. Samples were then subjected to SDS-PAGE and electroblotting, as described above.
Determination of M-CSFR and TACE expression by immunoblotting
Volumes of cell lysate or culture medium containing equal amounts of protein were processed for SDS-PAGE by adding appropriate volumes of a 4-fold concentrated Laemmli buffer and 2-ME solution (100 mM final concentration) and boiling for 7 min. Proteins were separated in 7.6% polyacrylamide, 5-cm-long, 0.75-mm-thick minigels (200 V, 60 min) in a buffer (pH 8.1–8.4) containing 25 mM Tris, 192 mM glycine, 0.5% SDS, and then transferred onto nitrocellulose membranes by electroblotting (100 V, 90 min) in a buffer (pH 8.1–8.4) containing 25 mM Tris, 192 mM glycine, and 10% methanol.
To estimate M-CSFR expression, nitrocellulose membranes were incubated (3 h at room temperature (RT)) in PBS containing 0.1% Tween 20 (TPBS), with the addition of 5% BSA (Sigma; catalogue A-3059) to saturate unspecific protein binding sites. Membranes were then washed in TPBS, incubated (6 h at RT) in a 1/500 dilution of anti-M-CSFR in TPBS containing 5% BSA, washed in TPBS again, and incubated (1 h at 4°C) in TPBS containing 2% BSA and a 1/5000 dilution of a HRP-conjugated anti-rabbit IgG Ab (Sigma; catalogue A-6154). After a final wash in TPBS, membranes were incubated (1 min at RT) in a chemiluminescent reagent (ECL protein detection system; Amersham International, Little Chalfont, U.K.), and HRP-coated protein bands were visualized on Hyperfilm-ECL (Amersham) after a 2- to 5-min exposure. To estimate TACE expression, nitrocellulose membranes were blocked as above, washed in PBS, incubated (overnight at 4°C) in a 1/5 dilution of anti-TACE in PBS containing 5% BSA, washed in PBS again, and incubated (1 h at 4°C) in PBS containing 5% BSA and a 1/1000 dilution of an HRP-conjugated anti-mouse IgG Ab (Sigma; catalogue A-4416). After a final wash in PBS, proteins were revealed by ECL as above.
Determination of M-CSFR expression by flow cytometry
Cells were treated with biotinylated human M-CSF, followed by streptavidin-conjugated PE (PharMingen, San Diego, CA; catalogue 13025D), and processed as described (15). Cells were counted with a FACScan (Becton Dickinson, Mountain View, CA). M-CSF-untreated/PE-treated cells were used for zeroing the analyzer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that a short treatment
of BAC macrophages with TPA resulted in the disappearance from cell
lysates of M-CSFR, as detected by immunoblotting with M-CSFR, raised
against the extracellular portion of murine M-CSFR (Fig. 1A). This was paralleled by the release into culture medium
of a soluble 90- to 100-kDa M-CSFR detectable using the same Ab (Fig. 1B). Thus, TPA activated an endoprotease cleaving M-CSFR in
macrophages, as expected (5).
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2-macroglobulin (all protease
families), PMSF and aprotinin (serinoproteases), calpain-inhibitor-I
(cysteinoproteases), pepstatin-A (aspartatoproteases), and the cation
chelators EDTA and 1,10-phenanthroline (metalloproteases). Cation
chelators prevented TPA or LPS from down-modulating M-CSFR (Fig. 2, A and B). This
effect was abolished in the presence of zinc chloride at concentrations
saturating ion-chelating capacity (Fig. 2B). All other
families of inhibitors were ineffective (not shown). No protein band
other than M-CSFR, cross-reacting with anti-M-CSFR antiserum, was
altered by the treatment with TPA or LPS. The effects of competitive
inhibitors of metalloproteases such as IC-3 and BB3103
(16, 17, 18) were then tested. These compounds also completely
inhibited the TPA- or LPS-induced down-modulation of M-CSFR (Fig. 2, C and D), an effect abolished by an extensive
wash of cells before the treatment with the activator (not shown),
which indicates that the inhibition was reversible. None of
metalloprotease inhibitors used had any effect on basal M-CSFR
expression or was toxic for cells (not shown). These results indicated
that the M-CSFR-cleaving protease is a metalloprotease.
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A) or LPS (not shown) in the
absence of serum. To rule out the possibility that the M-CSFR-cleaving
protease was an endogenous, soluble protease rapidly secreted following
the acid wash and upon activation, cells were treated with phalloidin,
an inhibitor of exocytosis (19), before and immediately
after the acid wash and then throughout the stimulation with TPA.
Phalloidin did not interfere with the down-modulation of M-CSFR,
indicating that the M-CSFR-cleaving protease is not a rapidly secreted,
soluble protein (Fig. 3A). On the other hand, the treatment
of macrophages with PI-PLC, before and throughout the stimulation with
TPA, did not prevent the down-modulation of M-CSFR, indicating that the
M-CSFR-cleaving protease is not a GPI-anchored protein (Fig. 3B). No effect on M-CSFR expression was determined by
phalloidin or PI-PLC alone (not shown). The above results, taken
together, indicated that the M-CSFR-cleaving protease is a
transmembrane metalloprotease.
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shows that in cells pretreated with
dec-RVKR-cmk, the effect of TPA was inhibited, indicating that the
M-CSFR-cleaving protease requires PACE-dependent processing for its
activation. These results confirmed that the M-CSFR-cleaving protease
is a transmembrane metalloprotease.
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A). When cells were
pretreated in vivo with anti-TACE, the down-modulation of M-CSFR
induced by TPA was inhibited (Fig. 5B). The treatment with
anti-TACE alone had no effect on M-CSFR expression, nor did the
treatment with an unrelated, control Ab interfere with the TPA-induced
down-modulation of M-CSFR (not shown). These data indicated that TACE
is involved in M-CSFR cleavage.
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).
Flow cytometry using biotinylated M-CSF to label M-CSFR showed that the
treatment with TPA markedly reduced the percentage of
M-CSF/M-CSFR-positive, wild-type DRM cells. On the contrary, in
TACE-negative cells, TPA was completely ineffective, indicating that
the TPA-dependent down-modulation of M-CSFR requires TACE expression.
To further characterize the importance of this requirement, we
addressed the question of whether the involvement of TACE in M-CSFR
down-modulation may be indirect, being mediated by TACE-dependent
release of TNF from cell surface, a possibility suggested by the
previous observation that exogenous TNF rapidly reduces M-CSFR
expression (27, 28). The pretreatment of cells with
anti-TNF at dilutions (10 µg/ml) preliminarily assayed to block
TNF activity in vivo (not shown) did not interfere with the
down-modulation of M-CSFR induced by either TPA or LPS (not shown),
indicating that the TACE-dependent cleavage of M-CSFR is not mediated
by autocrine TNF.
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shows that under basal conditions (i.e., without TPA treatment),
wild-type DRM monocytes exhibit a higher variability and lower average
values of M-CSFR fluorescence intensity than TACE-negative cells. This
feature suggested that in the absence of macrophage activators, low
levels of M-CSFR shedding are driven by constitutive TACE activity.
This hypothesis was tested by treating BAC macrophages, in the absence
of macrophage activators, with BB3103 or IC-3 at the same
concentrations used above. Fig. 7 shows
that the inhibitors enhanced basal M-CSFR expression (by 2.15-fold, on
the average, as determined by band densitometry of immunoblots). This
enhancement is apparently due to the block of TACE activity, and of the
related basal M-CSFR shedding shown in Fig. 1B
(left lane). Taken together, the data shown in Figs. 1, 6, and 7 demonstrated an either constitutive or macrophage
activator-inducible TACE-dependent shedding of M-CSFR in mononuclear
phagocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The latter result
ruled out the possibility that chelators were toxic for cells or
aspecifically effective due to depletion of intracellular calcium, and
led to add M-CSFR to the group of receptors that, following TPA
stimulation, are cleaved by a metalloprotease sensitive to hydroxamate
inhibitors. The group includes the receptors for TNF (p55–60 and
p75–80), IL-6, IL-2, nerve growth factor (low affinity), and
thyroid-stimulating hormone (8, 29, 30, 31). These receptors
are cleaved at an extracellular site, a fact in keeping with the
release of soluble M-CSFR in culture medium (Fig. 1B). The
sensitivity of M-CSFR shedding to metalloprotease inhibitors, however,
could not be considered a demonstration of the extracellular nature of
the enzyme catalytic activity, as all the inhibitors we used are cell
diffusible. For the same reason, nothing more could be inferred from
the removal of inhibition by an extensive cell wash following the
treatment with the inhibitor (Fig. 3A). We were, on the
other hand, able to surely exclude that the M-CSFR-cleaving protease is
a soluble membrane-associated or a GPI-anchored protein (Fig. 3B) and, therefore, to indirectly conclude that the enzyme
is a transmembrane protein. This conclusion is supported by the
ineffectiveness of 1,10-phenanthroline in the presence of zinc chloride
(Fig. 2B) and by the results of Fig. 1B, since
transmembrane metalloproteases are preferentially zinc metalloproteases
(26) and their catalytic domain is extracellular.
To confirm that the M-CSFR-cleaving protease was a transmembrane
metalloprotease, we tried to inhibit M-CSFR down-modulation by
interfering with the expression of this type of proteases. While
enzymatically active, soluble matrix metalloproteases (MMP) are
usually generated via the extracellular processing of proenzymes,
transmembrane metalloproteases are cleaved to their active form
intracellularly, within the secretory pathway, by furin-type proteases
of the PACE family (24, 25). The furin-recognition motif
(Arg-X-Lys/Arg-Arg), necessary for processing by PACE
(26), is present in all transmembrane metalloproteases,
but not in MMP, the only exception being stromelysin-3/MMP-11
(24, 26). As the latter enzyme is, like any other cell
surface-associated soluble protein, sensitive to the acid wash of cells
(Fig. 3A), the effectiveness of dec-RVKR-cmk in preventing
M-CSFR down-modulation (Fig. 4) indicated that the M-CSFR-cleaving
protease belongs to either the MT-MMP or the ADAM family of
transmembrane metalloproteases.
The facts that the enzymes of the MT-MMP family exhibit a very short
cytoplasmic domain, unlikely target of intracellular activatory signals
triggered by macrophage activators, and that only one member of this
family (MT4-MMP) is expressed in leukocytes (32), directed
our attention to the ADAM proteases. The main result obtained using
anti-TACE, reactive with the catalytic domain of TACE, was the
inhibition of the TPA-induced cleavage of M-CSFR by pretreating cells
with the Ab in vivo, apparently due to the block of TACE catalytic
activity (Fig. 5B). These experiments demonstrated that TACE
is responsible for M-CSFR cleavage and definitively confirmed that the
catalytic site of the enzyme, as well as the cleavage site of M-CSFR,
is extracellular. An even stronger indication of the role of TACE in
M-CSFR cleavage was obtained by using TACE-negative monocytes, in which
TPA was completely ineffective in down-modulating M-CSFR expression
(Fig. 6). An interesting side-result of these experiments was the
discovery of a sizeable TACE-dependent shedding of M-CSFR in the
absence of macrophage activators (Figs. 1B, 6, and 7),
pointing to a constitutive activity of TACE in controlling M-CSFR
density on cell surface. A soluble M-CSFR fragment, including the
ligand-binding domain, is likely to behave as a decoy receptor and to
interfere with the M-CSF concentration active on cells
(33). The physiological role of a constitutive M-CSFR
shedding from macrophage surface is worthy of further
investigation.
A question we needed to address was whether TACE is directly or
indirectly responsible for M-CSFR down-modulation. The fact that TNF
had been shown to down-modulate M-CSFR (27, 28) suggested
the possibility that an M-CSFR-cleaving protease different from TACE
was activated following a TACE-dependent release of TNF. This
possibility resulted very unlikely, as an in vivo treatment with TNF
was completely ineffective in preventing M-CSFR shedding. On the other
hand, it is worth pointing out that, on the basis of the results of
Figs. 2–4, TNF, to determine M-CSFR cleavage, would need to activate
in any case a transmembrane metalloprotease, or a cascade including at
least one enzyme of this type. Thus, the most straightforward
conclusion we could derive from all above is that TACE is the protease
cleaving M-CSFR in mononuclear phagocytes undergoing activation. As for
the down-modulation of M-CSFR by TNF, its mechanism has not been
elucidated yet. Our results are compatible with the hypothesis that TNF
actually activate (yet being not the only activator of) TACE, which
would, in parallel, cause direct M-CSFR shedding and establish a
positive feedback of TNF release. In conclusion, on the basis of all
above, M-CSFR can be added to the list of TACE substrates, such as
-amyloid protein precursor and members of the epidermal growth
factor receptor family (34, 35, 36).
The conclusion that TACE is responsible for M-CSFR down-modulation was
reached by inducing receptor shedding with TPA. Part of the results
presented (those of Figs. 2 and 3), however, were obtained with either
TPA or LPS, suggesting that the effects of the two compounds, as far as
M-CSFR shedding is concerned, are largely overlapping. Indeed, all the
physiological macrophage activators tested to date, including LPS,
IL-2, and IL-4, trigger M-CSFR down-modulation via the PLC
PKC
pathway (2, 3, 4), while TPA interacts with this process
downstream, directly activating PKC. Further work is necessary to
elucidate how the activation of PKC is linked to that of TACE.
The identification of the protease responsible for M-CSFR shedding in mononuclear phagocytes undergoing activation extends the characterization of M-CSFR down-modulation vs down-regulation. M-CSF-dependent down-regulation is based on the internalization of ligand-receptor complexes in peripheral endosomes and their targeting to esoproteases of the lysosomal system (37), whereas the trans/down-modulation induced by macrophage activators is operated by endoproteases active on cell surface (5). Down-regulation, differently from down-modulation, requires tyrosine kinase-active M-CSFR and is PKC independent (5). Down-regulation involves a progressive decrease of M-CSFR levels in individual cells, synchronous for all cells within a population. On the other hand, down-modulation is driven via an all-or-nothing response of individual cells, progressively extended to the entire population. Once a signal threshold is reached in an individual cell, the response is triggered and all receptors from cell surface are rapidly removed (38). Our results indicated that this signal is represented by TACE activation.
The question of the physiological meaning of the rapid M-CSFR
down-modulation induced by macrophage activators requires further
investigation. We know that this down-modulation occurs in any sort of
mononuclear phagocytes, including monocyte or macrophage cell lines and
peritoneal or bone marrow-derived primary macrophages (2 ,
this study), is triggered by different types of macrophage activators
(2, 3, 4), and it regulates macrophage function by blocking
some nonproliferative effects of M-CSF (4). M-CSF has been
actually shown to inhibit the induction by IFN-
of class II MHC
molecules, thereby suppressing macrophage functions in the immune
response (39). On the other hand, macrophages and
dendritic cells are capable of converting into one another until the
late stages of their respective maturation processes, enabling
tissue-resident macrophages to convert to dendritic cells upon
appropriate signals (40). It is interesting to note in
this work that these signals are mainly represented by IL-4 and GM-CSF,
both able to down-modulate M-CSFR (4, 41). We
(4) and others (38) hypothesized that the
induction of a fully activated (in particular, the APC) phenotype in
mononuclear phagocytes requires the rapid block of M-CSF-dependent
signals. Investigations on this hypothesis will take advantage of the
findings reported in this study, as they will enable to restore, by
preventing M-CSFR down-modulation, the sensitivity of macrophages to
M-CSF while they respond to the activators.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Persio Dello Sbarba, Dipartimento di Patologia e Oncologia Sperimentali, viale G. B. Morgagni 50, 50134 Firenze, Italy.
3 Abbreviations used in this paper: PLC, phospholipase C; ADAM, a disintegrin and a metalloprotease; DRM, Dexter-ras-myc; MMP, matrix metalloprotease; MT-MMP, membrane-type MMP; PACE, paired basic amino acid-cleaving enzyme; PI-PLC, phosphoinositide-specific PLC; PKC, protein kinase C; RT, room temperature; IC-3, TNF protease inhibitor; TACE, pro-TNF-converting enzyme; anti-TACE, anti-TACE mouse mAb Tc3-7.49; TPA, tetradecanoylphorbol myristate acetate; TPBS, PBS containing 0.1% Tween 20; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; anti-M-CSFR. anti-murine M-CSFR rabbit polyclonal Ab; anti-TNF, anti-TNF mouse mAb.
Received for publication August 7, 2000. Accepted for publication November 2, 2000.
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 S. Sather, K. D. Kenyon, J. B. Lefkowitz, X. Liang, B. C. Varnum, P. M. Henson, and D. K. Graham A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation Blood, February 1, 2007; 109(3): 1026 - 1033. [Abstract] [Full Text] [PDF]
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 J Plumb, S McQuaid, A K Cross, J Surr, G Haddock, R A. Bunning, and M N Woodroofe Upregulation of ADAM-17 expression in active lesions in multiple sclerosis Multiple Sclerosis, August 1, 2006; 12(4): 375 - 385. [Abstract] [PDF]
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 M. Mahmoodi, S. Sahebjam, D. Smookler, R. Khokha, and J. S. Mort Lack of Tissue Inhibitor of Metalloproteinases-3 Results in an Enhanced Inflammatory Response in Antigen-Induced Arthritis Am. J. Pathol., June 1, 2005; 166(6): 1733 - 1740. [Abstract] [Full Text] [PDF]
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 C. A. Buckley, F. N. Rouhani, M. Kaler, B. Adamik, F. I. Hawari, and S. J. Levine Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1132 - L1138. [Abstract] [Full Text] [PDF]
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S. Fabre-Lafay, S. Garrido-Urbani, N. Reymond, A. Goncalves, P. Dubreuil, and M. Lopez Nectin-4, a New Serological Breast Cancer Marker, Is a Substrate for Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE)/ADAM-17 J. Biol. Chem., May 20, 2005; 280(20): 19543 - 19550. [Abstract] [Full Text] [PDF]
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 A. J. Singh, R. D. Meyer, H. Band, and N. Rahimi The Carboxyl Terminus of VEGFR-2 Is Required for PKC-mediated Down-Regulation Mol. Biol. Cell, April 1, 2005; 16(4): 2106 - 2118. [Abstract] [Full Text] [PDF]
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S. J. Levine Mechanisms of Soluble Cytokine Receptor Generation J. Immunol., November 1, 2004; 173(9): 5343 - 5348. [Abstract] [Full Text] [PDF]
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X. Li and H. Fan Loss of Ectodomain Shedding Due to Mutations in the Metalloprotease and Cysteine-rich/Disintegrin Domains of the Tumor Necrosis Factor-{alpha} Converting Enzyme (TACE) J. Biol. Chem., June 25, 2004; 279(26): 27365 - 27375. [Abstract] [Full Text] [PDF]
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D. V. Bax, A. J. Messent, J. Tart, M. van Hoang, J. Kott, R. A. Maciewicz, and M. J. Humphries Integrin {alpha}5{beta}1 and ADAM-17 Interact in Vitro and Co-localize in Migrating HeLa Cells J. Biol. Chem., May 21, 2004; 279(21): 22377 - 22386. [Abstract] [Full Text] [PDF]
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 K. Wilhelmsen and P. van der Geer Phorbol 12-Myristate 13-Acetate-Induced Release of the Colony-Stimulating Factor 1 Receptor Cytoplasmic Domain into the Cytosol Involves Two Separate Cleavage Events Mol. Cell. Biol., January 1, 2004; 24(1): 454 - 464. [Abstract] [Full Text] [PDF]
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