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INTRODUCTION |
The transferrin receptor
(TfR)1 is a type II
transmembrane protein that mediates uptake of iron by binding the iron
carrier protein transferrin (Tf). Following internalization of the
complex, iron is released in the acidic endosomes and the TfR·Tf
complex recycles back to the cell surface where apotransferrin is
released at neutral pH. The TfR is composed of two homologous peptide
chains of 760 amino acids linked by two disulfide bonds (Cys-89 and
Cys-98) close to the transmembrane domain. Each polypeptide is divided into a large C-terminal extracellular domain of 672 amino acids, a
transmembrane domain of 21 amino acids, and an N-terminal cytoplasmic domain of 67 amino acids. The extracellular domain that binds Tf is
kept by a juxtamembrane stalk at 2.9 nm from the plasma membrane (1).
Within the stalk Thr-104 is O-glycosylated (2, 3), but
detailed structure and function of the stalk remain unknown.
Although the appearance of the soluble form of the transferrin receptor
(sTfR) in human serum has been known for quite a long time (4) and its
concentration is accepted as diagnostic marker for erythropoietic
activity (5, 6), little is known about the molecular basis of the
shedding process, in particular the nature of the shedding protease
involved. In diseases accompanied by enhanced erythropoiesis or TfR
expression, like iron deficiency anemia, increased serum sTfR levels
are observed (7-10), whereas lower levels of serum sTfR were
determined in patients with aplastic anemia (8). The sTfR level remains
unchanged in anemia arising from chronic disease, so that it is a
reliable marker to distinguish iron deficiency anemia from the anemia
of chronic inflammation and liver disease (11). It was shown that a
human red blood cell fraction does not have an own TfR-specific
proteolytic activity, whereas a white blood cell fraction does have
(12). The latter was also observed in human primary granulocytes,
activated mononuclear blood cells, HL-60 cells, and K562 cells
(13-16).
A mixture of protease inhibitors consisting of leupeptin, pepstatin,
EDTA, and PMSF was used to block the release of a TfR fragment from
HL-60 membranes (15). Cleavage of purified TfR by HL-60 plasma membrane
fractions could be inhibited by the serine protease inhibitors PMSF and
diisopropyl fluorophosphate at concentrations of 1 mM but not with inhibitors of other classes of proteases. Furthermore, four matrix metalloproteinases (MMP1, MMP2, MMP3, and
MMP9) did not cleave TfR (17). Using sheep exosome-bound TfR as
substrate for a protease present on human granulocytes, release of a
TfR fragment was inhibited by 1 mM PMSF (13). However, the
actual cleavage site was not determined.
The N terminus of sTfR isolated from human serum starts with Leu-101
revealing a cleavage site C-terminal of Arg-100 in the stalk region 11 amino acids distal to the plasma membrane (18). Recently we have
identified alternative cleavage sites at Val-108 and Lys-95
within the TfR stalk, which are processed by neutrophil elastase and
cathepsin G, respectively (19). A further cleavage site at Gly-91 was
reported for two N-terminal truncated TfRs lacking either the entire
cytoplasmic domain or the proximal 31 amino acids of the transmembrane
domain. Pulse-chase analysis revealed that this cleavage occurs during
the biosynthetic pathway (20). In contrast, the wild-type TfR that was
cleaved at Arg-100, when transfected in Chinese hamster ovary cells,
was shown to be processed during cycling through an endosomal
compartment, although cleavage was not dependent on acidification of
the endosomes (21).
Elimination of the O-linked carbohydrate at Thr-104 may
enhance the susceptibility of TfR to cleavage (22). Nevertheless, purified sTfR from human serum was sensitive to O-glycanase
and could bind to jacalin lectin, indicating that the sTfR contains an
O-linked oligosaccharide (3). This suggests that the glycan at Thr-104 does not modulate the shedding process of TfR in
vivo.
Several members of the metalloprotease family known as ADAM (for
a disintegrin and
metalloprotease), including tumor necrosis factor 
(TNF)-converting
enzyme (TACE or ADAM-17), ADAM-9, ADAM-10 (KUZ), and
ADAM-19, play an important role in ectodomain shedding (reviewed in
Ref. 23). The ADAM proteins fall within the metzincin superfamily that
is characterized by an extended zinc binding consensus sequence
(HEXXHXXGXXH) and a common tight Met-containing turn and includes the
matrix metalloproteinases (MMPs).
Besides these structural similarities, the proteolytic activities of
most MMPs and several ADAMs could be inhibited by hydroxamate-based
inhibitors and four endogenous tissue
inhibitors of metalloproteinases
(TIMPs). However, the TIMP inhibitor profile of ADAMs is different from
that of MMPs (25-27). TACE was originally found to release TNF (28,
29), but was later shown to be involved in the shedding of a variety of
other membrane proteins (e.g. L-selectin, TNF receptors,
transforming growth factor (Ref. 30)). Like TACE, ADAM-10 releases
TNF (31), participates in the nonamyloidogenic pathway of APP
processing to its soluble form (32), and is required for Notch
signaling (33). ADAM-9 is responsible for the shedding of
heparin-binding epidermal growth factor (EGF)-like growth factor (34)
and ADAM-19 for the release of neuregulin-
1, a member of the EGF
family (35). Besides the ADAMs, other types of proteases are involved
in shedding processes. Thus, the matrix metalloproteinase stromelysin-1
(MMP-3) releases active heparin-binding EGF-like growth factor (36) and
matrilysin (MMP-7) generates active soluble Fas ligand (37) and is
required for TNF release from macrophages (38). In addition,
the membrane type 4 matrix metalloproteinase (MMP-17) and
the serine protease proteinase-3 may participate in TNF
processing (39, 40).
All these data show that one type of protein can be processed by
different proteases or, vice versa, one protease can process several different proteins. Recently the idea emerged that regulating steps may determine which protease processes the target protein. In
general it can be differentiated into (i) regulated shedding mediated
by activating protein kinase C (PKC) by phorbol esters like phorbol
12-N-myristate 13-acetate (PMA) and (ii) constitutive shedding, which is present at a basal level. The stimulation of PKC by
treating cells with PMA leads to a reduction of cell surface expression
of L-selectin and p75 TNF receptor only from wild-type cells but not
TACE inactive cells (30). The PMA-induced shedding of interleukin-6
receptor is also strongly reduced in TACE-deficient fibroblasts,
whereas a basal release could still be detected (41). In addition
disruption of the TACE gene also abolishes regulated -cleavage of
APP in cultured cells, whereas the basal secretion of soluble APP is
unaffected in cells derived from TACE knockout mice (42). Thus, TACE
appears to play a central role in regulating shedding processes in most
cases, although it was reported that constitutive -secretase
cleavage could also be increased in cells transfected with TACE (43).
In contrast to TACE, ADAM-10 is reported to be involved in the
constitutive shedding of APP (32, 44), whereas overexpression of
ADAM-10 enhanced PKC-stimulated -secretase cleavage (32).
In the present study we developed a cell-free assay to detect the
release of sTfR from membrane-bound TfR. Membranes of the shedding
active cell line HL-60 were isolated, and following incubation at
37 °C a TfR fragment of ~80 kDa could be purified that lacked the
cytoplasmic and transmembrane domain. N-terminal sequencing of the
fragment revealed that the TfR is cleaved C-terminal of Arg-100; thus,
this fragment is identical to the major sTfR found in human serum. We
could show that the TfR-shedding protease is an integral membrane
metalloprotease that can be inhibited by specific metalloprotease
inhibitors. In addition to inhibiting release of sTfR from HL-60
membrane preparations, the inhibitors also effectively diminish
constitutive TfR shedding from HL-60 cells.
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EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Mouse mAb OKT9 directed against the
extracellular domain of TfR was prepared from a hybridoma cell line as
previously described (45), and mouse mAb H68.4 directed against the
intracellular domain of TfR was purchased from Zymed Laboratories (San
Francisco, CA). Polyclonal rabbit antibody pAB063 was generated by
immunization with purified human placental TfR (in cooperation with R. Geßner, Charité, Berlin, Germany). Horseradish
peroxidase-labeled anti-mouse and anti-rabbit antibodies were obtained
from Dako A/S (Glostrup, Denmark). MMP inhibitor 1, MMP inhibitor 2, TAPI-2, and recombinant human TIMP-3 were purchased from Calbiochem
(Schwalbach, Germany). Furin convertase inhibitor (FCI;
decanoyl-Arg-Val-Lys-Arg-chloromethylketone), human TIMP-1 (isolated
from stimulated neutrophils), and human TIMP-2 (isolated from
rheumatoid synovial fibroblasts) were obtained from Alexis
Biochemicals (Grünberg, Germany). PefablocSC and n-octylglucoside were from Roche Molecular Biochemicals
(Mannheim, Germany), and octylpolyoxyethylene (8-POE) was from
Bachem Biochemica GmbH (Heidelberg, Germany). Other
inhibitors and reagents were purchased from Sigma. Pervanadate was
generated immediately before use by mixing equivalent volumes of 2 M sodium vanadate (Sigma) and 1 M hydrogen
peroxide (Sigma) to obtain 0.5 M pervanadate. Sodium
vanadate was used in excess to completely eliminate hydrogen peroxide,
because remaining hydrogen peroxide in the pervanadate solution turned
out to be toxic to the leukocytic cells.
Cell Culture--
HL-60, Jurkat, and U937 cells were cultured in
RPMI 1640 medium supplemented with 5% (v/v) fetal calf serum in the
presence of 100 units/ml penicillin, 100 µg/ml streptomycin, and 1×
Glutamax. The cell cultures were maintained at 37 °C in a 5%
CO2 humidified atmosphere.
Isolation of the Soluble Transferrin Receptor from HL-60 Culture
Medium--
The cell culture (3500 ml) was centrifuged (210 × g, 4 °C, 15 min), and the supernatant was passed through
a ferri-transferrin-Sepharose affinity column and eluted under
nondenaturing conditions with 2 M KCl, 1% 8-POE in PBS
(150 mM NaCl, 10 mM phosphate, pH 7.5), as
described recently (46). TfR was detected by Western blotting using
OKT9 mAb directed against the C-terminal extracellular domain of the
TfR. sTfR-containing fractions were dialyzed against 1 mM
phosphate, pH 7.4, 15 mM NaCl and concentrated ~10 times
by lyophilization. For amino acid sequencing, proteins were transferred onto a polyvinylidene difluoride sequencing membrane (Millipore, Bedford, MA) with blotting buffer A (50 mM boric acid/NaOH,
pH 9.0, 10% (v/v) methanol) for 1 h at a constant voltage of 50 V according to the method of Xu and Shively (47). The band of interest
was cut out and sequenced by Edman degradation either commercially
(Peptide Specialty Laboratories GmbH, Heidelberg, Germany) or by using
an Applied Biosystems type 473A automated protein sequencer.
Preparation of Membrane Fractions--
A 300-ml cell suspension
of actively growing HL-60 cells (~1 × 106 cells/ml)
was centrifuged at 130 × g for 15 min and washed once with 30 ml of Dulbecco's PBS (Invitrogen GmbH, Karlsruhe, Germany). All following steps were carried out at 4 °C. The pelleted cells were resuspended in 10 ml of Dulbecco's PBS, homogenized by douncing 15 times in a Dounce homogenizer, and differentially centrifuged at
500 × g for 15 min, followed by 2600 × g for 15 min and finally at 20,000 × g for
30 min. The membrane pellet was washed once in 5 ml of PBS and again
centrifuged at 20,000 × g for 30 min. The microsomal
membrane pellet was resuspended in PBS to a final concentration of 6 mg/ml protein.
Digestion of Membrane-bound Transferrin Receptor--
In order
to detect membrane-associated TfR cleaving activity, 50 µl of HL-60
membrane fraction was incubated for 18 h at 4 °C or 37 °C.
The samples were applied to a 12% SDS-polyacrylamide gel under either
reducing or nonreducing conditions, and TfR fragments were detected by
immunostaining with OKT9 or H68.4 as described below.
sTfR Release Assay--
HL-60 membrane fractions were incubated
in aliquots of 50 µl for 18 h at 4 °C as negative control or
37 °C in the absence or presence of reagents. The samples were
centrifuged at 20,800 × g for 15 min at 4 °C, the
supernatants analyzed by 12% SDS-polyacrylamide gel electrophoresis
under nonreducing conditions, and sTfR detected by immunostaining with
OKT9 as described below. For quantitation of the chemiluminescence
signals, membranes were scanned with a FujiFilm LAS-1000 system and
analyzed with the Image Gauge version 3.2 software.
pH-dependent release was determined by resuspending the
HL-60 membranes in 50 mM BisTris buffer, 150 mM
NaCl (pH 6.0-7.5) or 50 mM Tris buffer, 150 mM
NaCl (pH 8.0-9.5).
Western Blot Analysis--
Samples were prepared according to
Laemmli (48) by boiling for 5 min in sample buffer containing 2% SDS
with or without 2% 2-mercaptoethanol and separated by electrophoresis.
For immunostaining, electrotransfer of proteins to a nitrocellulose
membrane, pore size 0.2 µm (Sartorius AG, Göttingen, Germany),
was performed with blotting buffer B (25 mM Tris, 192 mM glycine, 10% (v/v) ethanol). After blotting the
membrane was blocked with Dulbecco's PBS containing 5% dry skim milk.
TfR was detected either with OKT9 (6 µg/ml) or H68.4 (0.5 µg/ml) in
Dulbecco's PBS containing 0.2% Brij 58 (PBSB) and 1% dry skim milk
and a subsequent incubation with peroxidase-conjugated polyclonal
rabbit anti-mouse antibody (60 µg/ml). Each antibody incubation step
was followed by intensive washes with PBSB. Secondary antibody
detection was carried out by the enhanced chemiluminescence system from
PerkinElmer Life Sciences (Boston, MA).
sTfR-ELISA--
Using a 96-well microplate, each well was coated
with 100 µl of anti-TfR mAb OKT9 (2 µg/ml in Dulbecco's PBS) for
90 min and subsequently blocked with 175 µl of 10% (v/v) fetal calf
serum, 3% (w/v) bovine serum albumin in PBS for 30 min. All incubation steps were carried out under gentle agitation at room temperature. Cell
culture supernatant was centrifuged (20,800 × g,
4 °C, 20 min) to remove cellular debris and 100-µl aliquots
pipetted in triplicate and incubated for 2.5 h. The plate was
washed with PBS containing 0.05% (v/v) Tween 20 (PBST) and bound TfR
detected with 100 µl of anti-TfR polyclonal antibody pAB063 (1:2000
in PBST) and 100 µl of peroxidase-labeled swine anti-rabbit IgG
(1:2000 in PBST). The purification of placental TfR, which served as
internal standards for the quantitation of sTfR, and colorimetric
reaction were performed as previously described (46).
Protein Determination--
Protein concentrations were
determined in duplicate in microplates using the BCA protein assay (no.
23225, Pierce) and the appropriate microscale protocol of the manufacturer.
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RESULTS |
HL-60 Cells Release sTfR Cleaved at Arg-100--
Because the
promyelocytic cell line HL-60 release relatively high amounts of sTfR
(19), we chose this cell line for further investigations. First we
isolated sTfR from the culture cell medium by
ferri-transferrin-Sepharose affinity chromatography and analyzed the
site of cleavage by N-terminal sequencing. The N terminus of the
sequenced sTfR was constituted by LAG(T)ES, revealing the physiological
cleavage site at Arg-100 as described by Shih et al. (18).
Because Thr-104 is reported to be modified by
O-glycosylation (2, 3), only a weak signal was detected for
this amino acid.
Detection of TfR fragments--
HL-60 cells were washed once in
PBS and subsequently lysed in PBS containing 1% Triton X-100. The
lysate was separated by 10% SDS-PAGE under nonreducing conditions and
TfR detected by Western blotting using OKT9 mAb directed against the
extracellular domain or H68.4 mAb directed against the intracellular
domain of the TfR (Fig. 1A).
To determine the TfR shedding activity in membranes of HL-60 cells,
membrane preparations were incubated at 4 or 37 °C for 18 h and
separated by 12% SDS-PAGE under either nonreducing or reducing
conditions. TfR was detected by Western blotting using OKT9 or H68.4
(Fig. 1B). Altogether six TfR fragments were detected and
identified by size and immunological characteristics as follows:
~190-kDa dimer of TfR (TfR·TfR), ~110-kDa dimer of TfR lacking
one extracellular domain (TfR·mfTfR), ~90-kDa monomeric TfR (TfR),
~80-kDa soluble monomeric TfR (sTfR), ~25-kDa dimer of TfR lacking
both extracellular domains (mfTfR·mfTfR), and ~13-kDa monomeric
membrane fragment (mfTfR). The TfR fragments are schematically presented in Fig. 1E. The sTfR was not detected by H68.4,
indicating that it is N-terminally truncated, whereas the fragments
that only consist of small membrane fragments were not detectable by OKT9, revealing that they are C-terminally truncated. The ratios of the
detected fragments varied from preparation to preparation. Nevertheless, in most of the blots derived from nonreducing SDS-PAGE, the amount of TfR dimer exceeded the amounts of monomeric TfR and
TfR·mfTfR. Furthermore, in all cases the levels of fragments containing full-length TfR (TfR·TfR, TfR·mfTfR, and TfR) decreased after incubation at 37 °C, whereas the levels of cleaved fragments (sTfR, mfTfR·mfTfR, and mfTfR) increased. Several wash steps of the
membrane with detergent-free buffer did not reduce the sTfR releasing
activity, indicating that the TfR-shedding protease is tightly bound to
the membrane. Just below the sTfR we detected a faint band that
coappears in some cases with the sTfR, but in much lower concentrations
(see also Figs. 1D, 2A, 5B, and
6D). It remains unclear under which conditions this
alternative sTfR is generated, but in general it appears in a linear
relationship to the major sTfR. The molecular weight of the alternative
sTfR corresponds to the molecular weight of the product of a
TfR-cleaving protease recently identified in our lab as neutrophil
elastase (19).
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Fig. 1.
Six TfR fragments were detected in HL-60
membranes. A, HL-60 cell lysate was separated by 10%
SDS-PAGE under nonreducing conditions and analyzed by Western blotting
with anti-TfR mAb directed against the extracellular domain (OKT9) or
intracellular domain (H68.4). B, a membrane fraction from
HL-60 cells was incubated for 18 h at 4 or 37 °C, separated by
12% SDS-PAGE under either nonreducing or reducing conditions, and
analyzed by Western blotting with OKT9 or H68.4 mAb. The proposed
fragment compositions are indicated. C, incubated HL-60
membranes were centrifuged at 20,800 × g, and the
supernatant separated by 12% SDS-PAGE under nonreducing conditions and
analyzed by Western blotting with OKT9. Only the sTfR could be
detected, indicating the lack of the transmembrane domain.
D, HL-60 membrane fractions were solubilized with indicated
detergents and incubated and analyzed as described above, but using a
7.5% SDS-PAGE. The table shows the relative amounts of sTfR
and alternative sTfR quantified by chemiluminescence imaging on a
FujiFilm LAS-1000 system. E, schematic presentation of the
six TfR fragments detected in HL-60 cell membranes. Dimers are linked
by two disulfide bonds in the stalk region. MW, molecular
weight.
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sTfR Release Assay--
After pelleting the incubated membranes at
20,800 × g, the supernatant was separated by
nonreducing SDS-PAGE and analyzed by Western blotting; OKT9 solely
detected the sTfR (Fig. 1C), whereas H68.4 did not detect
any TfR fragment in the supernatant (data not shown). The apparent
molecular mass of the sTfR did not change under nonreducing conditions,
revealing the lack of intermonomeric disulfide bonds (Fig.
1B). This provides evidence that the sTfR lacks the
transmembrane domain and that the TfR is cleaved within the stalk
region C-terminal of Cys-98. The sTfR-releasing protease is active at
4 °C but the level of cleaved product increased ~5-fold at
37 °C. To confirm the exact site of cleavage, sTfR was purified by
affinity chromatography on ferri-transferrin-Sepharose. N-terminal
sequencing revealed that the fragment released from membranes is indeed
generated by cleavage of the TfR C-terminal of Arg-100, as determined
for sTfR from the culture medium of HL-60 cells. Because the release of
sTfR from isolated HL-60 membranes represents a cell-free system
similar to that in living cells, the quantitation of sTfR using OKT9 is
a valuable tool for examining the shedding process of TfR. Moreover,
the cell-free assay provides a few advantages over a whole cell system.
First, it enables the examination of the sTfR-shedding protease without
effecting synthesis and intracellular trafficking of TfR so that an
observed inhibition can be traced back to a direct inhibition of the
sTfR shedding activity. Second, the enzyme system involved is more
accessible to inhibitors in the sTfR release assay than in whole cell
systems. Finally, the isolated HL-60 membranes may represent a suitable starting material for purifying the TfR-shedding protease.
Solubilization of the protease is an absolute requirement for
purification; however, solubilization of the membranes with various
detergents (Triton X-100, CHAPS, Brij 58, 8-POE, or
n-octylglucoside) almost completely destroyed the sTfR
releasing activity (Fig. 1D). Interestingly the appearance
of the alternative sTfR is not affected as much by solubilization with
Triton X-100, Brij 58, and n-octylglycoside as compared with
the major sTfR. This suggests that the protease that processes the
alternative sTfR may not require membrane localization for TfR cleavage.
Rate of sTfR Release--
Quantitation of the
time-dependent release of sTfR from HL-60 membranes showed
that, within 1 h, ~50% of the total generated sTfR was released
(Fig. 2A). Because the amount
of full-length TfR is not significantly altered over a 24-h period at
37 °C (Fig. 2A, right inset), the
limited sTfR release is not the result of substrate deprivation but
rather of a limited stability of the sTfR releasing activity.
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Fig. 2.
sTfR release assay. Generation of sTfR
(a typical result is shown in Fig. 1C) upon incubation at
37 °C was quantified by chemiluminescence imaging on a FujiFilm
LAS-1000 system. Data represent mean values for at least two
independent experiments with S.E. A, rate of sTfR release at
37 °C over a 24-h period. A representative result is shown
(left inset). Right inset
shows a similar experiment without centrifugation of the incubated
membranes. Samples were separated by SDS-PAGE under nonreducing
conditions and detected by Western blotting using OKT9. B,
pH dependence of sTfR release at 37 °C analyzed and quantified as
described above.
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pH Dependence of sTfR Release--
Incubating the membranes at
different pH showed that cleavage activity was completely abolished at
less than pH 6.5, increased substantially within the pH range from 6.5 to 7.0, reached a maximum at 7.5 and decreased slightly at more basic
pH (Fig. 2B). Maximum activity at neutral pH provides
evidence that the shedding process occurs in a neutral cell compartment
or at the cell surface and indicates the catalytic type of the
protease. Aspartic proteases are generally active at acidic pH,
metalloproteases are restricted to neutral or basic pH, and the serine
and cysteine proteases are active over a more broad pH range (for a
comprehensive review, see Ref. 49).
Integral Membrane Anchoring--
To determine how the protease is
anchored in the membrane, we incubated the membranes under various
conditions resulting in the release of either peripheric membrane
proteins (hypertonic buffer, hypotonic buffer, low or high pH) or
glycosylphosphatidylinositol (GPI)-anchored membrane proteins (by
phosphatidylinositol-specific phospholipase C). Only the preincubation
of membranes at low pH significantly lowered the sTfR releasing
activity of the membranes (Fig. 3). This
is, however, the result of the instability of the protease at low pH
(Fig. 2B), activity of which cannot be restored at neutral
pH, rather than a result of the release of the protease from the
membrane. Thus, it can be concluded that the TfR-shedding protease and
potential cofactors are integral membrane proteins.
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Fig. 3.
sTfR releasing activity is membrane-anchored
by an integral domain. HL-60 membrane fractions were preincubated
under conditions releasing peripheric membrane proteins (hypertonic
buffer (1 M NaCl), hypotonic buffer (0.015 M),
pH 3.3 and 9.5 for 30 min at 4 °C) or in the presence of
GPI-specific phospholipase C to release GPI-anchored membrane proteins
(30 min at 37 °C). Subsequently the membranes were pelleted by
centrifugation at 20,800 × g, washed once, and
incubated at 37 °C for 18 h. The sTfR was quantified using the
sTfR release assay.
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Inhibition of sTfR Release--
To further determine the nature of
the sTfR releasing activity, we tested various protease inhibitors
(summarized in Table I). The protease
inhibitors specific for aspartic, serine, or cysteine proteases showed
no significant inhibitory effect at the concentrations tested, with the
exception of the serine protease inhibitor PefablocSC. This will be
discussed below in context with inhibition by the furin inhibitor FCI.
In contrast, the general metalloprotease inhibitor 1,10-phenanthroline
inhibits cleavage dose-dependently (Fig.
4A). Cleavage could also be
inhibited by the chelating reagents EDTA and EGTA, but only at
concentrations exceeding the concentration of Ca2+ (0.9 mM) and Mg2+ (0.5 mM) cations in
the reaction buffer. Because binding of divalent cations to chelators
may lower the pH of a solution and thus inactivate the sTfR releasing
activity, we tested the pH of the incubation buffer and found no
significant change. We observed that preincubation of the membranes
with 1,10-phenanthroline in the absence of Ca2+ and
Mg2+ destroyed the sTfR releasing activity irreversible,
possibly because of its alkaline earth metal-chelating ability. The
activity, however, remained unaffected in the presence of 0.9 mM Ca2+ and 0.5 mM
Mg2+. Addition of Zn2+ to membranes
preincubated with 1 mM 1,10-phenanthroline, in the presence
of 0.9 mM Ca2+ and 0.5 mM
Mg2+, restored the sTfR releasing activity almost
completely, whereas additional Ca2+ in the same
concentrations did not (Fig. 4B). It could be concluded that
the inhibition by 1,10-phenanthroline is specific because of its
chelating activity toward transition metals. We found that the decrease
in sTfR releasing activity in the presence of high Zn2+
concentrations is caused by an inhibitory effect of transition metals
on sTfR release (Fig. 4C). The fact that metalloproteases are inhibited by millimolar concentrations of Zn2+ has been
shown, and the structural features of the inhibition effect were
determined for carboxypeptidase A (50). Ca2+ reduced sTfR
release only at high concentrations, whereas Mg2+ resulted
in a slight increase in sTfR release at high concentrations. To further
characterize the metalloprotease, we tested more specific metalloprotease inhibitors. An inhibitor developed for the specific inhibition of matrix metalloproteinases (MMP inhibitor 1) showed no
significant effect, whereas MMP inhibitor 2 and an inhibitor that
blocks shedding of TNF (TAPI-2) specifically decreased sTfR release
(Fig. 5, A and B).
MMP inhibitor 2 and TAPI-2 are hydroxamic acid-based inhibitors of
metalloproteases belonging to the MMP and ADAM families (51, 52).
Members of these families of metalloproteases are often activated by
furin-type pro-protein convertases; we therefore tested the furin
inhibitor FCI (Fig. 5A). This inhibitor decreased the amount
of sTfR release, indicating that the sTfR releasing activity is indeed
activated by a furin-type pro-protein convertase. The serine protease
inhibitor PefablocSC, tested within the same concentration range,
reduced sTfR release only at high concentrations (Fig. 5A).
This could be a result of the moderate inhibitory effect of serine
protease inhibitors on furin (53). The most specific inhibitors tested,
the TIMPs, are naturally occurring proteins of ~20-28 kDa, which
bind to metalloproteases of the MMP or ADAM families. The inhibitors
were tested at concentrations up to 1 µM in the sTfR
release assay and 20 nM in HL-60 cell culture. It has been
reported that TIMPs in these concentrations inhibit the shedding of
TRANCE (54), L-selectin (55), and HER-2 (56). No clear inhibitory
effect for TIMP-2 or TIMP-3 could be detected in the sTfR release assay
(Table I), or in HL-60 cell culture (data not shown). TIMP-1 showed a
moderate inhibitory effect; however, this appeared to be
batch-dependent. TIMP-1 and TIMP-2 are supplied in a
detergent-containing buffer (0.05% Brij 35), which appeared to enhance
sTfR release from HL-60 membranes, when added in low concentrations.
Because different batches of TIMP-1 and TIMP-2 seemed to contain
different amounts of Brij 35, the results were not consistent between
batches.
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Fig. 4.
sTfR releasing activity is inhibited by
chelating reagents. Various reagents were added to HL-60
membranes, incubated at 37 °C for 18 h, and cleavage activity
analyzed in the sTfR release assay. A, 1,10-phenanthroline,
EDTA, and EGTA were added in the indicated concentrations.
B, all samples were preincubated with 1 mM
1,10-phenanthroline for 5 min at 4 °C and ZnCl2 and
CaCl2 subsequently added in the indicated concentrations.
C, ZnCl2, CoCl2, CaCl2,
and MgCl2 were added in the indicated concentrations.
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Fig. 5.
Inhibition of sTfR releasing activity by
specific protease inhibitors. HL-60 membranes were incubated in
the presence of four synthetic protease inhibitors and cleavage
activity analyzed in the sTfR release assay. A,
concentration-dependent (0-1 mM range)
inhibition of sTfR release. B, a representative Western blot
showing the effect of 0.1 mM inhibitor.
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TfR Shedding from HL-60, Jurkat, and U937 Cells--
To assess the
level of sTfR released into the cell culture medium, we developed an
ELISA specific for TfR (for details see "Experimental Procedures").
The basal sTfR levels measured correspond well to the sTfR detected by
immunoprecipitation as reported earlier by our group (19). Moreover,
for HL-60 cells we determined the correlation between cell growth and
rate of sTfR release into the cell culture medium (Fig.
6A). The rate of sTfR release
correlated with the growth rate with a maximal release during the log
phase. This indicates that both events are in a close relationship. The common effector of ectodomain shedding, PMA, exhibited no strong effect
on TfR shedding in the leukocytic cell lines HL-60 and Jurkat (Fig.
6B). PMA stimulated sTfR release solely in U937 cells, which
display a very low basal level of sTfR release, to a level equivalent
to the basal level of HL-60 cells. Because PMA induces endocytosis of
TfR, one could argue that internalization withdraws the TfR from the
protease. PMA also did not reduce TfR shedding; thus, it may be
concluded that TfR shedding is mediated independent of PKC-activation
as well as of TfR localization in the cell. The phosphatase inhibitor
pervanadate was the only reagent we found that was suitable to
significantly stimulate TfR shedding. However, this effect was
dependent on the cell line. Pervanadate increased TfR shedding
severalfold in all cell lines examined, with the greatest effect
observed in Jurkat cells (Fig. 6B). The inhibitors shown to
be active in the sTfR release assay and exhibiting no effect on cell
growth (see "Discussion"), namely TAPI-2, MMP inhibitor 2, FCI, and
PefablocSC, were utilized to examine constitutive TfR shedding into the
medium by HL-60 cells. sTfR was quantified using the sTfR-ELISA (Fig.
6C). The cells were harvested in the log phase, fresh
culture medium added, and grown for 20 h in the absence and
presence of the inhibitors (50 µM concentration). In
comparison to the untreated cells, the hydroxamic acid-based inhibitors
TAPI-2 and MMP inhibitor 2 decreased sTfR release by 40-50%, the
furin inhibitor by 60%, whereas the serine protease inhibitor
PefablocSC inhibited sTfR release by only 10%. To prove the
specificity of the sTfR-ELISA for the major sTfR, we precipitated sTfR
from the HL-60 inhibition experiment (Fig. 6C) with
ferri-transferrin-Sepharose, separated the precipitate by SDS-PAGE, and
detected TfR by Western blotting using OKT9 mAb (Fig. 6D).
We found that the levels of precipitated sTfR were in good agreement
with the sTfR levels measured by the sTfR-ELISA. Furthermore, only very
small amounts of alternative sTfR were detectable in the HL-60 culture
medium, confirming that the alternative sTfR did not significantly
contribute to the total amount of released sTfR. Moreover, we compared
the gel mobility of sTfR isolated from HL-60 cell culture medium and from supernatant of incubated HL-60 cell membranes by SDS-PAGE (Fig.
6D). We found that sTfR from both assays revealed the same molecular weight, as expected because N-terminal sequencing revealed that they possessed the same N termini.
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Fig. 6.
TfR shedding in leukocytic cells.
A, HL-60 cells (1 × 105/ml) were cultured
for 100 h and cell counts performed every 12 h. The sTfR in
the cell culture supernatant was determined by sTfR-ELISA. Data were
quantified by using purified human TfR as standard. B,
HL-60, Jurkat, and U937 cells (1 × 106/ml) were
cultured for 5 h in the absence or in the presence of 100 ng/ml
PMA or 10 µM pervanadate and sTfR release determined by
sTfR-ELISA. Data were quantified from four independent experiments
(mean value ± S.E.). C, HL-60 cells (1 × 106/ml) were cultured for 20 h in fresh medium with or
without inhibitors at a final concentration of 50 µM and
sTfR levels quantified using an sTfR-ELISA. Data are expressed as
percentage of control from four independent experiments (mean
value ± S.E.). D, the supernatant (100 µl) of
incubated HL-60 membrane preparations from Fig. 1C and 5 ml
of HL-60 cell culture supernatants from panel C were
precipitated with ferri-transferrin-Sepharose, separated by
7.5% SDS-PAGE under reducing conditions, and TfR detected by Western
blotting using OKT9 mAb.
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DISCUSSION |
In the present study we could show for the first time, in a
microsomal membrane fraction of HL-60 cells, the existence of all
theoretical TfR fragments that are generated during shedding of TfR. As
the question regarding the location of the TfR shedding process is
unresolved, the membrane-bound remnants that remain after shedding of
at least one extracellular domain are of particular interest. In
previous studies a 105-kDa TfR fragment was detected under nonreducing
conditions in cell membranes of K562 and HL-60 cells. This fragment was
proposed to be consistent with the truncation of one extracellular
domain of the transferrin receptor (57). A small TfR fragment, lacking
both extracellular domains, could not be observed in the cell
membranes, but was detected in the exosomes obtained from cell culture
supernatant (57). An 18-kDa peptide was also detected in sheep exosomes
but not in plasma membranes of human red blood cells, indicating that
the sTfR originating from maturing red blood cells may be cleaved by a
leukocyte protease after the release of exosomes containing full-length
TfR (13). In contrast, in HL-60 cell membrane preparations, we were
able to detect small TfR fragments, levels of which increased after incubation at 37 °C. Furthermore, we could also show the existence of TfR fragments of the same sizes in HL-60 cell lysates, as detected in membrane preparations. This provides strong evidence that TfR shedding occurs at the cell membrane independent of exosome formation.
The 80-kDa TfR fragment could only be detected by an antibody directed
against the extracellular domain of TfR, the molecular mass remained
unchanged under nonreducing and reducing conditions, and it is the only
fragment that could be detected in the supernatant of incubated
membranes. All these properties are consistent with the identity of
sTfR, which could be finally proven by N-terminal sequencing. The
observation that sTfR derived from HL-60 cell culture, as well as from
digested HL-60 membranes, could be purified on Tf-Sepharose illustrates
that sTfR retains its Tf binding capacity.
Because sTfR levels increased after incubation at 37 °C, the HL-60
membrane preparations contain a protease that is responsible for TfR
shedding. This protease is membrane-bound because membrane fractions
washed free of soluble or loosely associated membrane proteins are
competent for cleavage. Furthermore, the protease is an integral
membrane protein as activity remains membrane-bound under conditions
that release peripheric membrane proteins or GPI-anchored membrane
proteins. The limited stability of the sTfR releasing activity may be
explained by proteolytic degradation or by specific shedding of the
sTfR-shedding protease itself, such as described for MT5-MMP (58) or
furin (59, 60). This conclusion would imply that the secreted soluble
TfR-shedding protease can no longer cleave the membrane-bound TfR. This
assumption is supported by our observation that solubilization of the
membrane fraction almost completely destroyed sTfR releasing activity, revealing that the TfR-shedding protease and/or its substrate need to
be anchored in the membrane. Similarly it has been shown for
-secretase and angiotensin-converting enzyme secretase that the
intact membrane is required to specifically shed APP and
angiotensin-converting enzyme (61, 62).
Our studies show that the sTfR releasing activity is caused by a
metalloprotease that is membrane-anchored. The sensitivity of the sTfR
releasing activity to hydroxamic acid-based inhibitors supplies further
evidence for the characterization of the TfR-shedding protease. The
hydroxamate group complexes the catalytic zinc and forms two hydrogen
bonds to a Glu and an Ala amino acid residue that are located in the
catalytic domain. Selectivity can be obtained by varying those groups
that fit the S1' and S2' pocket of the target protease. MMP inhibitor 2 has been shown to be a potent inhibitor of MMP-1, MMP-3, MMP-7, and
MMP-9 (51), but other related proteases may be inhibited too. TAPI-2
was originally developed to block TNF release from cells (63). Later
it was shown that TAPI-2 is also effective in preventing the cleavage of the 80-kDa TNF receptor (64), the transmembrane glycoprotein receptor HER2 (56), and the constitutive as well as PMA-activated shedding of transforming growth factor , L-selectin, interleukin-6 receptor, and APP (65). Furthermore, TAPI-2 was reported to inhibit the
apoptotic induced shedding of the E-cadherin ectodomain in epithelial
cells (66). Because TACE and ADAM-10 have been shown to be involved in
TNF release, these enzymes appear to be the main mediators of the
above-mentioned shedding processes. However, the TIMP inhibitor profile
observed in our study argue against an involvement of TACE or ADAM-10
in TfR shedding because TACE is inhibited only by TIMP-3 (25) and
ADAM-10 by TIMP-1 and to a lesser extent by TIMP-3 (26). Thus, other
proteolytic active ADAMs, which have not been tested to date in their
sensitivity to TAPI-2 and TIMPs, are likely to contribute to TfR shedding.
Proteases of the MMP and ADAM families are synthesized as inactive
precursors, in which the N-terminal prodomain blocks the catalytic site
by coordinating the Zn2+ by a conserved unpaired Cys
residue. Proteolytic cleavage between the prodomain and catalytic
domain enables a "cysteine switch" mechanism that leads to the
active form of the enzyme (67). The membrane type forms of the MMPs and
most proteolytic active ADAMs (e.g. ADAM-9 (68), ADAM-10
(69), ADAM-12 (70), ADAM-15 (71), TACE (28), and ADAM-19 (72)) share
the feature that they contain the furin recognition sequence
R-X-(K/R)-R between pro- and catalytic domain, which permits
their activation by furin-like pro-protein convertases during their
intracellular trafficking through the secretory pathway. Our
observation that a furin inhibitor reduced the sTfR releasing activity
in cell membranes as well as sTfR release in HL-60 cells suggests that
the TfR-shedding protease is activated by a furin-like pro-protein
convertase. In particular, the processing of ADAM-10 to its active form
by furin-type pro-protein convertases, furin and proprotein convertase 7 (PC7), was examined in detail (73). The authors could inhibit the
maturation of ADAM-10 to its active form in HEK cells by treatment with
FCI.
We observed that the intensity of TfR shedding correlates with cell
growth. This is in agreement with the fact that changes in human serum
sTfR levels are usually observed in diseases accompanied with altered
cell proliferation (7-10). Thus, a decrease in TfR shedding from
living cells can be traced back to either direct inhibition of the
shedding process or diminished cell growth. The latter was confirmed by
the observation that treatment of HL-60 cells with several kinase
inhibitors or apoptosis-inducing reagents that reduce cell
proliferation resulted in a decreased release of sTfR (data not shown).
As protease inhibitors may reduce cell growth, inhibitors like
1,10-phenanthroline that are toxic to cells and lead to an arrest in
cell growth must be excluded from interpretation. All inhibitors shown
in Fig. 6 neither inhibited proliferation nor showed any detectable
toxic effect at the concentrations tested. Our observation that
pervanadate stimulates TfR shedding in leukocytic cell lines has also
been reported for other shedding processes. The cleavage of HER2 has
been shown to be enhanced by pervanadate, but not by PKC activators.
This process is efficiently inhibited by TIMP-1 but not TIMP-2 (56).
The tumor necrosis factor family member TRANCE is shed by at least two
proteolytic activities, which are both distinct from TACE. One
proteolytic activity could be induced by pervanadate and is sensitive
to TIMP-2 but not TIMP-1 (54).
In summary, we found that the protease(s) that releases sTfR is a
transmembrane metalloprotease and its activation is probably controlled
by furin or furin-like proteases, which process transmembrane proteases
to their active form. The cleavage of TfR could be inhibited by
hydroxamic acid-based inhibitors that have been shown to be specific
for metalloproteases of the MMP and ADAM families. All synthetic
inhibitors that were active in the sTfR release assay also
significantly reduced the basal release of sTfR from HL-60 cells. The
low constitutive TfR shedding in some cell lines could be enhanced by
pervanadate but not by the commonly used ectodomain shedding effector
PMA. The above results, especially the inhibition of sTfR release by
TAPI-2 and weakly by TIMP-1, but not by TIMP-2 and TIMP-3, suggest that
the TfR-shedding protease(s) is a member of the ADAMs family.
Protease Inhibitor-2*
,
TOP
ABSTRACT
INTRODUCTION
