From the
Laboratori de Recerca Oncològica,
Servei d'Oncologia Mèdica, Hospital Universitari Vall d'Hebron, Psg.
Vall d'Hebron 119-129, Barcelona 08035, Spain,
Instituto de Microbiología
Bioquímica and Centro de Investigación del Cáncer,
Consejo Superior de Investigaciones Científicas-Universidad de
Salamanca, 37007 Salamanca, Spain, and
Cellular Biochemistry and Biophysics
Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,
New York, New York 10021
Received for publication, February 17, 2003 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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Experiments with specific inhibitors indicate that practically all of the
shedding events analyzed to date are mediated by zinc-dependent
metalloproteases that belong the metzincin family
(3). In addition,
overexpression of certain metzincins or their dominant-negative forms point to
the participation of these metalloproteases in ectodomain shedding (for
example see Refs. 4 and
5). A definitive confirmation
of the role of a member of the metalloprotease disintegrins (also known as
ADAMs or MDCs), a subfamily of the metzincins, in ectodomain shedding came
with the development of mice genetically deficient in the tumor necrosis
factor--converting enzyme
(TACE1/ADAM17)
(6). The characterization of
cell lines derived from TACE knock-out mice (TACE/ cells)
confirmed the involvement of this metalloprotease disintegrin in the shedding
of a surprisingly large group of cell surface proteins (reviewed in Ref.
1).
Metzincins are synthesized as zymogens containing a prodomain with an odd cysteine, which coordinates the zinc present in the catalytic site inhibiting the metalloprotease activity (7). Certain metzincins contain a stretch of basic amino acids between the prodomain and the metalloprotease domain and are activated in the secretory pathway by proteolytic removal of the former. It has been frequently assumed that serine proteases of the proprotein convertase family that are highly specific for basic amino acid-based motifs and, typically, act in the trans-Golgi network (8) cleave the prodomain of these metzincins. The proprotein convertase family is composed by seven members: four of them (furin, PACE4, PC6, and PC7) are expressed in a broad range of tissues, and two of them (PC1 and PC2) are expressed in neuroendocrine tissues, whereas the expression of PC4 is restricted in spermatogenic cells (Ref. 9 and references therein).
The number of zinc-dependent metalloproteases involved and the mechanisms
that regulate ectodomain shedding remain largely undetermined. To address
these issues, several years ago we isolated two independent mutant cell lines
defective in the shedding of the transmembrane growth factor proTGF-
(M1 and M2 cells) (10,
11). Characterization of these
mutant cells showed that they are also defective in the shedding of a variety
of proteins such as the transmembrane proheparin-binding epidermal growth
factor-like growth factor (proHB-EGF)
(12),
APP
(10), the cell adhesion
molecule L-selectin, the
subunit of the receptor for
interleukin-6 (11), and the
cytokines proTNF-
and fractalkine
(13,
14). The shedding of these
molecules is also defective in TACE/ fibroblasts
(6,
12,
13,
1518).
Nonetheless, somatic cell fusions between mutant cells and TACE/
fibroblasts recover the wild type phenotype, suggesting that the mutation
affects a component different from TACE but necessary for its activity and
perhaps that of other metalloproteases
(13). Therefore, the
characterization of these cell lines may reveal novel aspects of the
regulation of ectodomain shedding.
In this report, we show that the processing of TACE is impaired in M1 and
M2 cells. An analysis of cells defective in the activity of furin and
experiments with 1-Antitrypsin Portland
(
1-PDX), an inhibitor specific for certain proprotein
convertases, indicates that in normal cells TACE is processed by furin and
other proprotein convertases. However, the mutant cells possess a normal
repertoire of proprotein convertases, which are not likely a target of
mutations. An analysis of the subcellular location of TACE in these mutant
cells reveals a defective intracellular trafficking that leads to the
accumulation of the metalloprotease in the early secretory pathway and
explains the phenotype observed. Other metzincins processed by furin-like
convertases and putatively involved in ectodomain shedding such as ADAM10,
ADAM9, or MT1-MMP as well as ADAMTS1 are correctly processed in the mutant
cells, indicating that the defect is highly specific for TACE. In summary,
characterization of shedding defective somatic cell mutants reveals the
existence of a mechanism that specifically mediates the intracellular
trafficking and in turn the proteolytic activation of TACE by proprotein
convertases.
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MATERIALS AND METHODS |
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cDNAs, Cell Lines, Transfections, and Viral InfectionsThe
cDNAs encoding furin, PC7, PC6B/Flag, and 1-PDX were
provided by Dr. Gary Thomas, and the cDNAs encoding PACE4 and ADAM10 were
gifts from Dr. Joseph F. Sucic and Dr. Paul Glynn, respectively. The cDNA
encoding Notch tagged at the C terminus with the Myc epitope and Myc-tagged
TACE have been previously described
(13). To facilitate detection,
HA epitopes were introduced right after the propeptide cleavage site of PC7
(after Ser-142 of human PC7) and at the beginning of the cysteine-rich domain
of human PACE4 (after Pro-697), respectively, using standard techniques. LoVo
and CaCo cells were provided by Drs. Alain Israel and Senén
Vilaró, respectively. Transient transfections were performed using the
DEAE-dextran method. The efficiencies of transfection were routinely
controlled and ranged between 30 and 40%. CHO cells permanently transfected
with
1-PDX were obtained by co-transfecting
1-PDX subcloned into the pcDNA3.1 Z(+) vector (Invitrogen)
and the selectable plasmid pREP-4 (Invitrogen) at a DNA ratio of 10:1 using
the calcium phosphate precipitate method as previously described
(10). Transfectants were
selected in 600 µg/ml hygromycin and subcloned. The levels of expression of
1-PDX were assessed by Western blot using anti-human
1-antitrypsin antibodies.
LoVo cells were infected with retrovirus as follows: 293T cells were plated at 2 x 106 cells/60 mm dish. Five minutes prior to transfection, 25 µM chloroquine was added to each plate. The transfection solution was DNA (2.5 µg of pMDG-VSV, 5 µg of pNGUL-MLV-gag-pol, 3 µg of the retroviral vector pLZR-IRES-GFP bovine-containing furin, 61 µl of 2,5 M CaCl2, and 500 µl of double-distilled water. The medium was replaced with 3 ml of fresh virus-collecting medium 24 h later, and the retrovirus were recovered 1 day thereafter. Cells were then infected with the viral supernatants containing 6 µg/ml Polybrene.
Western BlottingApproximately 106 parental HeLa,
CHO, M1, M2, Caco, or LoVo cells were lysed in PBS containing 1% Nonidet P-40,
10 mM 1,10-phenanthroline, 5 mM EDTA, and 20
µM BB-94 (lysis buffer), and insoluble material was removed by
centrifugation. The resulting supernatants were incubated with 25 µl of
concanavalin A (ConA)Sepharose (Amersham Biosciences) for 1 h at 4 °C to
concentrate glycoproteins. ConA-Sepharose beads were washed twice with lysis
buffer, and the final beads were treated with or without endoglycosidase H
(Endo H) or N-glycosidase F as detailed below, resuspended in sample
buffer, and boiled. Samples were then electrophoresed in 7% polyacrylamide
gels under reducing conditions and subjected to Western blotting analysis
using 1/500 of the anti-prodomain, anti-cytoplasmic domain antibodies directed
against TACE, anti-human 1-antitrypsin or anti-ADAM10,
1/1000 of anti-furin or anti-MT1-MMP, or 1/2.500 of anti-ADAM-TS1 as
indicated. Signals were detected with SuperSignal West Dura Extended Duration
substrate (Pierce). As indicated, films were digitized and quantified using
MacBAS software (Fuji film).
Metabolic Labeling and ImmunoprecipitationTo analyze TACE
processing, 2.106 CHO or M2 cells were metabolically labeled
with 1 mCi/ml [35S]Translabel (Biolink 2000, Arlington Heights, IL)
for 1 h in methionine- and cysteine-free medium at 37 °C and chased for
variable periods of time in complete medium. Cells were then lysed in lysis
buffer as described above, and the cell lysates were immunoprecipitated with 2
µl of antibodies directed against the cytoplasmic tail of TACE. Immune
complexes were collected with protein A-Sepharose, washed three times with
washing buffer (PBS containing 0.1% Triton X-100 and 0.1% SDS) and diluted
1:50 with lysis buffer and incubated with ConASepharose to remove
non-glycosylated background proteins as previously described
(19). The beads were washed,
resuspended in loading buffer, and analyzed on 7% SDS-polyacrylamide gels
under reducing conditions. Gels were exposed to x-ray films or quantified
using a BAS-1800 instrument and software (Fuji film).
To analyze the processing of Myc/Notch by furin, CHO or M2 cells were transfected with Myc/Notch in the presence or absence of furin. Transfected cells were metabolically labeled as above and chased for 1 h in complete medium. Cells were then lysed in lysis buffer, and the cell lysates were immunoprecipitated with 1/100 of anti-Myc antibodies. Immune complexes were collected with protein G-Sepharose, washed, and analyzed on 6% SDS-polyacrylamide gels under reducing conditions. Gels were analyzed as above.
Deglycosylation StudiesFor Endo H (Roche Applied Science) and N-glycosidase F (Roche Applied Science) treatments, washed ConA-binding proteins were resuspended in 40 µl of a buffer containing 0.15 M sodium citrate, pH 5.5, 1 mM phenylmethylsulfonyl fluoride, and 0.25% SDS or in buffer containing 20 mM sodium phosphate, pH 8.3, 10 mM EDTA, and 1% Nonidet P-40, respectively. ConA beads were then boiled for 2 min, allowed to cool down and treated with 2 milliunits of Endo H or 1 unit of recombinant N-glycosidase F for 1216 h at 37 °C, resuspended in SDS sample buffer, and analyzed by Western blot as described above.
Reverse Transcription-PCR and Sequencing of TACE from Wild Type and M2 CellsDouble-stranded cDNAs from CHO cells, M1 and M2 were synthesized using the Amersham Biosciences kit and instructions of the manufacturer. To sequence TACE, different fragments were amplified using aliquots of the cDNA from parental CHO cells or M2 cells as template and the oligonucleotides shown in Supplementary Table I. The PCR fragments were purified from agarose gels and sequenced using BigDyeTM Terminator Cycle Sequencing Ready Reaction DNA sequencing kit from Amersham Biosciences.
Reverse transcription-PCR analysis of parental CHO cells and M1 and M2 mutants was performed using the Amersham Biosciences kit and instructions of the manufacturer. Aliquots of cDNA were used as template for 20, 25, or 30 cycles of PCR, and the products of the reaction were separated on agarose gels and analyzed with a Fluor S device and software (Bio-Rad). The oligonucleotides used to amplify PACE4 and PC6 fragments have been previously described (20). The rest of oligonucleotides used were as follows: furin, 5'-GTG GTC TCC ATC CTG and GCC ATC ATC CTC AGG-3'; PC7, 5'-AAC GGC TTC AAT GAC TG-3' and 5'-ATC CTT GCT GCT GCT-3'; and actin, 5'-CCT GAC CGA GCG TGG CTA C-3' and 5'-GAA GCA TTT GCG GTG GAC G-3'.
Cell Surface BiotinylationFor biotinylation of cell surface TACE, ADAM10, and MT1-MMP, wild type or M2 cells were washed three times with PBS buffer at 4 °C and incubated in the same buffer containing 1 mg/ml sulfo-NHS-LC-biotin (Pierce) for 30 min at 4 °C. Excess biotinylating reagent was quenched by washing with 10 mM glycine in PBS three times. Cells were lysed as described above, and cell lysates were incubated with neutravidin-agarose (Pierce). Washed beads were electrophoresed and analyzed by Western blot with antibodies against the cytoplasmic domain of TACE, polyclonal antibodies against ADAM10, or monoclonal antibodies against MT1-MMP.
Confocal MicroscopyCells grown in coverslips were fixed for 30 min in 2% paraformaldehyde in PBS for 20 min. Cells then were permeabilized with 0.1% saponin in PBS for 30 min. Permeabilized cells were incubated for 45 min in PBS with 1% bovine serum albumin containing 1/100 of IgG purified from polyclonal antibodies against the cytoplasmic tail of TACE, anti-KDEL monoclonal antibodies, or FITC-labeled antibodies against the Golgi marker gm130. After incubation with primary antibodies, cells were washed with PBS and incubated for 45 min with Texas Red-conjugated anti-rabbit antibodies, FITC-conjugated anti-rabbit antibodies, or Texas Red-conjugated anti-mouse antibodies at a 1/100 dilution. Coverslips were washed with PBS, mounted, and viewed on a Leica TCS 4D laser scanning confocal microscope (Leica Lasertechnik). As a control, M2 cells transiently transfected with Myc/TACE were immunostained with anti-Myc and the polyclonal antibody against the cytoplasmic tail of TACE. The extensive colocalization indicates that the staining observed with the polyclonal antibody against TACE is specific (Supplementary Fig. 3A). To further assess its specificity, the antibody was preincubated with 100 µg/ml TACE cytoplasmic tail-glutathione S-transferase fusion protein produced using standard techniques. As expected, no signal was detected in CHO cells in the presence of competing protein (Supplementary Fig. 3B)
To specifically stain cell surface proHA/TGF-, cells were shifted to
4 °C and incubated with 1/100 of anti-HA in PBS containing 1% bovine serum
albumin for 1 h. Cells were fixed, permeabilized, and treated with antibodies
against the cytoplasmic tail of TACE, FITC-conjugated anti-rabbit, or Texas
Red-conjugated anti-mouse antibodies as above.
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RESULTS |
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To further characterize the mutant cell lines, we analyzed the species of
TACE expressed by them using specific antibodies. In agreement with previous
results (19), antibodies
directed against the cytoplasmic domain of TACE stain two proteins in HeLa and
CHO cells (Fig. 1, A and
B), whereas antibodies against the prodomain of TACE only
stain the slowest migrating protein (Fig.
1A). These bands were identified as full-length and
processed TACE devoid of prodomain, respectively
(Fig. 1A). In M1 and
M2 cells, the only apparent TACE species is the full-length form, suggesting
that these cells have a defect in the processing of TACE or, alternatively,
that the processed form of TACE is specifically and rapidly degraded in the
mutant cells. To distinguish between these possibilities, we performed
pulse-chase experiments. Because M1 and M2 cells belong to the same
complementation group (11) and
show a similar absence of processed TACE, pulse-chase and most subsequent
experiments were performed only with M2 cells. The full-length form of TACE is
long-lived in M2 cells (Fig.
1C, t
20 h) compared with that in
wild type cells (Fig.
1C, t
< 8 h, and data not shown). In
these cells, full-length TACE is processed to the form that lacks the
prodomain undetectable in M2 cells (Fig.
1C). These results strongly suggest that M2 cells are
unable to process the prodomain of TACE. Unexpectedly, at long periods of
chase (24 h) the electrophoretic migration of processed TACE in wild type
cells is slightly slower than that at the 12-h time point
(Fig. 1C, wild type
(WT), compare lanes 12 and 24 h). This slight
difference in migration cannot be appreciated in HeLa, CaCo, or LoVo cells
(data not shown) and provides an explanation for the migration of the
processed form of TACE from CHO cells as a broad band in Western blots (for
example see Fig.
1B).
TACE Is Processed by Proprotein ConvertasesBecause the
prodomain is connected with the metalloprotease domain by a typical furin
cleavage site, it has been widely assumed that TACE is activated by furin-like
proprotein convertase(s) in the secretory pathway
(17,
19,
21). To test this assumption,
we analyzed TACE species in cells devoid of furin activity and in cells
overexpressing the 1-PDX, a protein-based inhibitor of
proprotein convertases highly specific for furin and PC6B
(22).
The human colon carcinoma LoVo cell line has been used to characterize the processing of numerous proteins, because it expresses inactive forms of furin (23). As judged by Western blot analysis using anti-TACE antibodies, LoVo cells express the full-length as well as low levels of the processed form (compared with those expressed by CaCo cells (Fig. 2A), indicating that convertases different from furin can process the prodomain of TACE (Fig. 2A). The electrophoretic migration of full-length TACE from LoVo cells is different from that observed using a variety of cell lines such as CaCo, HeLa, or CHO cells (Fig. 2A and data not shown). Because proteins are differentially glycosylated in different cell lines and glycosylation frequently alters the electrophoretic migration, we de-glycosylated samples from the different cell lines and analyzed the electrophoretic migration of TACE in the resulting preparations. Treatment of samples with N-glycosidase F confirmed that the differences in the apparent molecular weight of TACE from LoVo and Caco cells were because of differential glycosylation. To determine the possible ability of furin to process TACE, we infected LoVo cells with retrovirus that included a vector coding for furin and analyzed the processing of the metalloprotease. As shown in Fig. 2B, overexpression of furin increased the levels of processed TACE, indicating that the lack of this convertase accounts for the low levels of TACE processing observed in these cells (Fig. 2A, compare the levels of processed TACE in LoVo and CaCo cells)
To confirm these results, we showed that 1-PDX inhibits
the processing of TACE, although even in cells with a high level of
overexpression of this inhibitor, some residual processing can be detected
(Fig. 2C).
Collectively, these results indicate that TACE is processed by furin in normal
cells. In the absence of furin, alternative convertases can remove the
prodomain of TACE, albeit less efficiently.
Proprotein Convertases in Wild Type and M2 CellsBecause furin and probably other proprotein convertases participate in the removal of the prodomain of TACE, one or more of these processing enzymes could be inactive in the mutant cells. To investigate this possibility, we first determined the set of proprotein convertases expressed by wild type and mutant cells by performing semi-quantitative PCR with oligonucleotides specific for the four convertases widely expressed (24). The result of this analysis showed no major differences in the levels of furin, PACE4, and PC7 in wild type and mutant cells (Fig. 3A). Although it has been previously found that PC6B is expressed in CHO cells (24), we were unable to detect this convertase using two different sets of primers (data not shown).
We next analyzed whether the convertases expressed by M2 cells are the target of the mutation that prevents the processing of TACE by analyzing the effect of the overexpression of wild type convertases on the mutant phenotype. None of the convertases tested had any detectable effect on the processing of TACE in wild type or M2 cells (Fig. 3B), indicating that convertases are not rate-limiting in the processing of TACE in wild type cells and that they are not the defective component in M2 cells. To rule out the possible interference of the epitope tags with the proteolytic activity of PC7, PC6B and PACE4, parallel experiments were performed with non-tagged versions of the convertases and the results were indistinguishable from those shown in Fig. 3B (data not shown). As a positive control, we showed that overexpression of furin in wild type and M2 cells leads to an enhanced processing of Notch, a previously described substrate of this convertase (25), as judged by the quantification of full-length Notch and one of the products of the processing reaction, the C-terminal fragment (Notch TM) (Fig. 3C and see also supplementary Fig. 1). It should be noted that in contrast to the processing of TACE, the processing of overexpressed Notch is limited by the amount of furin because it can be enhanced by furin overexpression. These results confirm that proprotein convertases are not likely the mutated component in the shedding-defective mutant cell lines.
Subcellular Location of TACE in M2 CellsProprotein convertases are synthesized as zymogens containing a prodomain. Upon exit from the ER, the prodomain is autocatalytically removed and the convertases reach the trans-Golgi system as active endoproteases (24). To test whether TACE progresses correctly through the secretory pathway in M2 cells and is, therefore, accessible to proprotein convertases, we analyzed the subcellular location of endogenous TACE in mutant cells and compared it with that in control wild type cells.
In agreement with previous results and results shown here (Figs.
2 and
4)
(19), full-length TACE is
sensitive to Endo H treatment in wild type cells, indicating that this form
resides largely in the early secretory pathway and does not reach the medial
Golgi. Processed TACE is resistant to Endo H
(Fig. 4A) and can be
specifically detected by cell surface biotinylation
(Fig. 4B), indicating
that this form has transversed the medial Golgi and reaches the plasma
membrane. Consistently, immunofluorescence analysis on wild type cells with
anti-TACE antibodies show intracellular staining that partially colocalizes
with anti-KDEL and anti-gm130 (that bind to ER and cis-Golgi markers,
respectively) (Fig. 4, C and
D) and cell surface staining that partially colocalizes
with cell surface proHA/TGF- (Fig.
4E). In M2 cells, full-length TACE is entirely sensitive
to Endo H treatment (Fig.
4A), indicating that it remains in the early secretory
pathway. In agreement with this conclusion, TACE is not displayed at the cell
surface (Fig. 4B).
Furthermore, although the intracellular immunostaining pattern of TACE in M2
cells is similar to that in wild type cells, perhaps with a lower level of
colocalization with anti-gm130 (Fig.
4D), no cell surface immunostaining can be detected in M2
cells (Fig. 4E).
Collectively, these results indicate that the trafficking of TACE is impaired
in M2 cells and that the metalloprotease remains largely in the ER and does
not reach the trans-Golgi where the processing of TACE by proprotein
convertases takes place.
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The Processing of Different Metalloproteases Is Normal in Shedding-defective MutantsM1 and M2 cells show a gross defect in the metalloprotease-dependent shedding of plasma membrane proteins (10, 11); however, since the defect in these cell lines has remained elusive, it has not been possible to determine whether the affected component controls one, few, or many metalloproteases. To investigate this point, we analyzed the transport to the cell surface and processing of several metalloproteases, some of them putatively involved in ectodomain shedding, in M2 cells. Specific antibodies identified endogenous ADAM10 and MT1-MMP in CHO cells (Fig. 5). The processing of these metalloproteases is normal in M2 cells as judged by Western blot (Fig. 5). Moreover, biotinylation experiments showed that the processed forms of these metalloproteases is readily detected on the cell surface of M2 cells and accumulates to levels similar to those observed in wild type cells (Fig. 5), further supporting a normal trafficking of these proteases in M2 cells.
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Although ADAM9 is widely expressed (26), we were unable to detect it in CHO cells with polyclonal antibodies against the mouse protein; therefore, we analyzed the processing of transiently transfected mouse ADAM9 and found it indistinguishable in wild type and M2 cells (Fig. 5). Furthermore, the processing of transfected ADAM-TS1 to the previously described p87- and p65-soluble fragments (27) was also similar in both cell types (Fig. 5). Because it has been suggested that the metalloproteases analyzed are processed by furin-like convertases (2730), these results confirm that convertase activity is normal in mutant cells. Collectively, the evidences shown here unveil the existence of a highly specific mechanism defective in M1 and M2 cells that mediates the intracellular trafficking and therefore the processing of TACE. Ultimately, this mechanism controls the shedding of many proteins through the control of the activity of TACE.
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DISCUSSION |
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Several years ago, we undertook a genetic approach to characterize the
shedding machinery that acts on proTGF-. First, we isolated chemically
induced somatic cell mutants (M1 and M2 cells) defective in the activated
shedding of proTGF-
, and subsequently, we found that these mutant cell
lines have a gross defect in the shedding of a variety of proteins, including
many substrates whose shedding is also defective in TACE/ cells
(1014).
However, the wild type phenotype of somatic cell fusions between M2 and
TACE/ cells indicated that the defect in M2 cells affects a
component other than TACE
(13). TACE transfected into M2
cells does not rescue the wild type phenotype, indicating that the component
defective in these mutant cells is different from TACE
(13). Furthermore, no
mutations can be detected in TACE from M2 cells (this report). Therefore, the
defect in M2 cells does not affect TACE; thus, the characterization of this
defect provides an opportunity to study mechanisms that control the activity
of TACE.
Because of the current availability of polyclonal antibodies that allow the
detection of endogenous TACE in CHO cells, we found that the processing of the
metalloprotease is completely blocked in M1 and M2 cells. This result
apparently contradicts a previous one, because a C-terminal tagged version of
TACE was judged to be processed marginally but to the same extent in wild type
and mutant cells (13).
However, several lines of evidence strongly suggest that the fate of
transfected TACE is not a good indicator of that of the endogenous protein and
that the apparent "processing" previously observed with
transfected TACE is artifactual. Pulse-chase experiments show that prodomain
processing affects almost all of the detectable TACE labeled after the pulse
(this report and Ref. 19),
whereas it affects only to 10% transfected TACE
(13). In addition, the
electrophoretic migration of the marginal amount of TACE processed in
transient transfections is different from that of the endogenous processed
protein (data not shown). Currently, the basis for the artifactual processing
of TACE in transient transfections is unknown. Presumably, they could be
related to the saturation of a component needed for the processing of the
metalloprotease. We are currently investigating this possibility.
An analysis of the processing of TACE in LoVo cells and in cells
overexpressing 1-PDX indicates that the processing of TACE
is carried out by furin and other convertases. Because
1-PDX
is specific for furin and PC6B
(22) and CHO cells express
undetectable levels of the latter convertase, the clear inhibition of TACE
processing in CHO overexpressing the serpin-based inhibitor cells strongly
suggests that furin is the main convertase acting on TACE. However, two main
evidences indicate that convertases are not the target of the mutations in M1
and M2 cells. Transfection of mutant cells with furin does not rescue the wild
type phenotype, and the processing of several metalloproteases known to be
substrates of furin
(2730)
is normal in mutant cells.
Although the components of the machinery responsible for the intracellular
trafficking of TACE await to be identified, the results presented here show
that the factor mutated in M1 and M2 cells is specific for TACE and is not
necessary for the trafficking of other metzincins. This conclusion is
particularly significant when considering the specificity of metalloprotease
disintegrins. It has been proposed that ADAM10 and ADAM9 play a role in the
shedding of APP (4,
33) and that ADAM9 is involved
in the shedding of proHB-EGF
(34). However, the processing
of these metalloprotease disintegrins is normal in M2 cells, indicating that
at least in CHO cells, the activated
-secretase activity that sheds the
ectodomain of
APP and that of proHB-EGF is identical to TACE. Although
the phenotype of TACE/ and ADAM9/ cells obtained
from knockout mice (that show defective and normal shedding of proHBEGF and
APP, respectively) indicates that this is also true in mouse embryonic
fibroblasts (12,
13,
15,
35), it may well be possible
that different metalloprotease disintegrins act on different cell lines. In
this scenario, TACE would be the main endogenous shedding activity in CHO
cells and mouse embryonic fibroblasts, whereas ADAM10 and ADAM9 could be
responsible for the shedding of
APP in HEK 293 and in transiently
transfected COS cells, respectively, and ADAM9 would shed proHB-EGF in Vero
cells (34).
In summary, as expected, the characterization of mutant cell lines
initially isolated for lack of proTGF- shedding has allowed us to
detect the existence of a mechanism that specifically controls the activity of
TACE through the regulation of its intracellular trafficking. Obviously, the
identification of the components that allows the export of TACE from the ER
and therefore its proteolytic activation will be the next step in
understanding this mechanism of control.
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FOOTNOTES |
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* This work was supported by grants from the Spanish Comisión
Interministerial de Ciencia y Tecnología (SAF2000-0203),
Fundació La Marató de TV3, Fondo de Investigación
Sanitaria (PI021003) and the EMBO Young Investigator Program (to J. A.), and
predoctoral fellowships from the Fundació "la Caixa" and
from the Fundació per a la Recerca i Docència dels Hospitals
Vall d'Hebron (to M. B.-P. and S. R.P., respectively). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org)
contains Table 1 and Figs. 1,
2,
3.
Both authors contributed equally to this work.
¶ Recipient of a postdoctoral fellowship from the Fundació per a la
Recerca i Docència dels Hospitals Vall d'Hebron.
|| Recipient of fellowships from the Instituto de Salud Carlos III and the
Spanish Ministry of Education, respectively.
** Present Address: Unitat de Biologia Molecular i Cellular, Institut
Municipal d'Investigació Mèdica, Aiguader 80, 08003 Barcelona,
Spain.
¶¶ To whom correspondence should be addressed: Laboratori de Recerca Oncològica, Hospital Vall d'Hebron Psg., Vall d'Hebron 119-129, Barcelona 08035, Spain. Tel./Fax: 34-93-274-6026; E-mail: jarribas{at}hg.vhebron.es.
1 The abbreviations used are: TACE, tumor necrosis factor--converting
enzyme; TGF, transforming growth factor; HB, heparin binding; EGF, epidermal
growth factor-like growth factor; TNF, tumor necrosis factor;
1-PDX,
1-Antitrypsin Portland; FITC,
fluorescein isothiocyanate; HA, hemagglutinin; CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline; Endo H, endoglycosidase H; ER, endoplasmic
reticulum; gm, Golgi marker; ADAM, a disintegrin and metalloprotease; MT-MMP,
membrane-type matrix metalloprotease; PC, proprotein convertase; PACE, paired
basic amino acid-converting enzyme;
-APP,
-amyloid precursor
protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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