THE JOURNAL OF BIOLOGICAL CHEMISTRY
45255–45268, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulated Cell Surface Pro-EGF Ectodomain Shedding Is a
Zinc Metalloprotease-dependent Process*
Received for publication, July 17, 2003, and in revised form, August 21, 2003
Published, JBC Papers in Press, August 28, 2003, DOI 10.1074/jbc.M307745200
Sylvain M. Le Gall‡, Rodolphe Auger, Catherine Dreux, and Philippe Mauduit§
From the UMR 8619, Institut de Biochimie Biophysique Moléculaire et Cellulaire, Université Paris XI,
91405 Orsay Cedex, France
Epidermal growth factor receptor (EGFR) ligands are
synthesized as type I membrane protein precursors ex-
posed at the cell surface. Shedding of the ectodomain of
these proteins is the way cells regulate the equilibrium
between cell-associated and diffusible forms of these
growth factors. Whereas the regulated shedding of
transforming growth factor-␣, HB-EGF, and amphiregu-
lin precursors have been clearly established, regulation
of full-length pro-EGF shedding has not been clearly
demonstrated. Here, using both wild-type and M2 mu-
tant CHO-K1 as well as HeLa cell lines transiently trans-
fected with epitope-tagged rat pro-EGF expression plas-
mid, we demonstrate that these cells synthesize EGF as
a high molecular weight membrane-associated precur-
sor glycoprotein expressed at the cell surface. All cell
lines are able to release the entire ectodomain of pro-
EGF in the extracellular medium following juxtamem-
brane cleavage of the precursor once it is present at the
cell surface. More significantly we clearly established
that CHO-M2 and HeLa cells only constitutively release
low levels of pro-EGF. This shedding is a regulated phe-
nomenon in wild-type CHO cells where it can be induced
by different agents such as phorbol 12-myristate 13-ac-
etate (PMA), pervanadate, and serum but not by calcium
ionophores. Using specific inhibitors as well as protein
kinase C (PKC) depletion, PMA stimulation was shown
to be completely dependent on PKC activation whereas
pervanadate and serum stimulation were not. Regulated
ectodomain shedding involves the activity of a zinc met-
alloprotease as determined by inhibition with phenan-
trolin and TAPI-2 and by the results obtained with the
CHO-M2 shedding defective mutant cell line. Compari-
son of the ability of CHO and HeLa cell lines to shed
pro-EGF and pro-TNF-␣ upon stimulation greatly sug-
gests that TACE (ADAM 17) may not be the ectoprotease
involved in the secretion of pro-EGF ectodomain and
that this protease, which remains to be identified, shows
a restricted cellular expression pattern.
Epidermal growth factor (EGF)1 is a small (about 6 kDa)
diffusible polypeptide first discovered and purified from rodent
* 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.
‡ Supported by a Doctoral contract from the Ministère de la jeunesse,
de l’éducation et de la recherche.
§ To whom correspondence should be addressed. Tel.: 33-1-69158033;
Fax: 33-1-69853715; E-mail: philippe.mauduit@bbmpc.u-psud.fr.
1
The abbreviations used are: EGF, epidermal growth factor; EGFR,
EGF receptor; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal
calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albu-
min; HA, hemagglutinin; RIA, radioimmunoassay; WT, wild type; PMA,
This paper is available on line at http://www.jbc.org
This is an Open Access article under the CC BY license.
Vol. 278, No. 46, Issue of November 14, pp.
Printed in U.S.A.
submaxillary glands by Cohen (1) based on its ability to stim-
ulate epithelial cell growth. This growth factor is now known to
be part of a family of growth factors that includes transforming
growth factor-␣ (TGF-␣) (2), heparin binding EGF-like growth
factor (HB-EGF) (3), amphiregulin (AR) (4), betacellulin (5),
and epiregulin (6). These growth factors share the ability to
bind the same receptor, i.e. the EGF receptor (EGFR) and
activate its intrinsic tyrosine kinase activity that induces cel-
lular differentiation and mitogenic effects (7, 8). Most growth
factors are produced as soluble inactive precursors that are
activated by proteolysis in vesicular compartments of the se-
cretory pathway before being released in the extracellular me-
dium. Both cDNA and protein structure analysis clearly indi-
cate that EGFR ligands are also derived from precursor
molecules (9). However, they are not synthesized as soluble but
as nondiffusible, glycosylated membrane-associated precursors
exposed at the cell surface.
In the case of EGF, the cDNA analysis from human, mouse,
and rat species predicted very large precursor molecules (re-
spectively 1207, 1217, and 1133 amino acids) (10 –13) with an
apparent molecular mass ranging from 150 to 185 kDa for the
mature glycosylated precursors. These precursor molecules are
predicted to be type I membrane proteins. In the case of rat
pro-EGF, the 1035 N-terminal amino acids constitute the ex-
tracellular domain that includes the EGF sequence in the jux-
tamembrane region and eight additional “EGF-like” modules, a
22-amino acid transmembrane domain, and a short C-terminal
intracellular part of 76 amino acids (13).
The biological importance of EGFR ligands underscores the
interest in the study of the enzymatic processing leading to
growth factor bioavailability (14, 15). The presence of EGF in
external biological fluids such as saliva, urine, milk, and tears
(16 –22) indicates that EGF secretion is mainly an exocrine
secretory process. However, the cellular events leading from
the high molecular weight precursor molecule to secretion of
lower molecular weight soluble EGF into the external fluid are
currently not clearly identified.
Recent evidence suggests that proteolytic cleavage of cell
surface proteins, i.e. ectodomain shedding, is an important
mechanism whereby cells regulate the repertoire of proteins
expressed on their surface, shifting molecular species from
their membrane-bound forms to soluble ones with the potential
modification of their biological properties (9, 23, 24). Several
types of membrane proteins such as receptor ligands (among
them EGFR ligands), receptors, cell-adhesion molecules, and
ectoenzymes undergo ectodomain shedding (9, 23–26). With the
important exception of the ␤-secretase activity (an aspartyl-
protease) that sheds the prodomain of the ␤-amyloid precursor
protein (␤-APP) from the cell surface, cell surface proteolysis
phorbol 12-myristate 13-acetate; CHO, chinese hamster ovary; PKC,
protein kinase C; TGF-␣, transforming growth factor-␣.
45255
45256 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
appears to be mediated by members of the metzincin super-
family of zinc-dependent proteases that include the matrix
metalloproteinases (MMPs) and ADAMs (for A Disintegrin And
Metalloproteinase) (24, 26 –28). The involvement of proteases
such as ADAM 9 (MDC9/meltrin-␥) and more importantly
ADAM 17 (TACE) in the regulation of EGFR ligand shedding
as been proposed for TGF-␣ (24, 25, 28) HB-EGF (29, 30), and
amphiregulin (29, 31). Unfortunately, the information concern-
ing pro-EGF maturation and secretion does appear much
more controversial.
In rodents submaxillary glands, the precursor molecule is
not detectable and only the mature and diffusible 6 kDa form of
EGF accumulates in the secretory granules of the regulated
exocrine secretory pathway (12, 32, 33). In this case, the release
of EGF in the biological fluid (saliva) involves exocytosis, i.e.
fusion of the secretory granule membrane with the apical cel-
lular membrane. This phenomenon is highly regulated and
may be achieved at least through the activation of adrenergic
receptors (34, 35).
In other tissues studied so far such as kidney (12, 36 –39),
mammary (40), and lacrimal glands (41), the fully mature 6
kDa EGF was undetectable, and EGF accumulated as its high
molecular weight precursor associated with the apical mem-
brane of epithelial cells. The presence of EGF-containing pep-
tides of varying molecular masses (from 6 to 160 kDa) in urine
(37, 42, 43) and milk (22) of different species indicates that
EGF can be released (shed) from its membrane-anchored pre-
cursor. This occurs through proteolytic cleavage in the jux-
tamembrane region. The full-length precursor was the major
substrate for shedding, leading to the initial release of the
entire ectodomain. In order to lead to the ultimate, mature
6-kDa form of the molecule, the ectodomain needs to be further
maturated in the extracellular medium. It has already been
shown that, in vitro, mature EGF could be released from its
precursor through the action of different type of proteases;
acidic protease such as pepsin (36) or serine-proteases such as
trypsin (22, 39, 41) and plasmin (44). Moreover, membrane
fractions of both kidney (45, 46) and mammary gland (47)
contain a membrane-bound proteolytic activity that colocalized
with pro-EGF and released soluble EGF from its membrane-
associated precursor. Based on its sensitivity to a set of inhib-
itors, it was shown to belong to the serine-protease family. This
experimental evidence is in conflict with results obtained in
two independent studies performed with cultured cells. In
these cells transfected either with a full-length (48) or trun-
cated (49) human pro-EGF cDNA, EGF release was sensitive to
metalloprotease inhibitors (batimastat and BB-2116) and cat-
ion chelators (EDTA and EGTA) and insensitive to non-metal-
loprotease inhibitors (48). Another important feature concern-
ing EGFR ligand shedding is its potential regulation. It is now
clearly established that secretion of TGF-␣, HB-EGF, and am-
phiregulin are regulated phenomena that can be triggered by
PMA (through PKC activation), calcium ionophore (calcium
influx), serum or pervanadate (tyrosine phosphatase inhibi-
tion) (9, 23, 25, 31, 50 –53). Concerning EGF, its regulated
shedding from cell surface is not so clearly established. Demp-
sey et al. (48) working with MDCK cells transfected with the
full-length human pro-EGF cDNA failed to stimulate EGF
secretion with serum or PMA whereas Dong and Wiley (49)
working with mammary cells (HB2) transfected with a prodo-
main-truncated human pro-EGF cDNA also failed to stimulate
secretion with PMA but observed a stimulation of EGF release
with both calcium ionophore and a tyrosine phosphatase
inhibitor.
From these studies, it appears that important discrepancies
exist in the conclusions that can be drawn from the studies on
pro-EGF shedding compared with other EGFR ligands. In or-
der to provide insights into pro-EGF shedding properties, we
decided to study pro-EGF secretion in CHO-K1 (both wild type
and M2 mutant) and HeLa cell lines transiently transfected
with a full-length rat pro-EGF cDNA. The CHO-K1 cellular
model has been used previously to establish the mechanism of
regulation of the secretion of other EGFR ligands (TGF-␣,
HB-EGF), and HeLa cells have also been used to study the
shedding of various proteins including HB-EGF (54, 55). In the
present study, we have been able to demonstrate that once
pro-EGF is exposed at the cell surface, it can be shed to release
its entire ectodomain in the extracellular medium. In CHO-K1,
this ectodomain shedding can be stimulated by PMA, serum, or
pervanadate. PMA acts through a PKC-dependent pathway
whereas serum and pervanadate act through PKC-independ-
ent pathway(s). The use of specific protease inhibitors as well
as of the CHO-M2 mutant cell line that displays impaired
constitutive and regulated pro-EGF ectodomain shedding dem-
onstrated that shedding was achieved through the activation of
a zinc-dependent metalloprotease activity. More important is
the fact that, whereas the zinc-dependent metalloprotease
ADAM 17 (TACE) has an important role in the regulation of
EGFR ligands shedding such as TGF-␣, HB-EGF, and amphi-
regulin (24, 25, 28, 29, 30, 31), our results suggest that TACE
may not be relevant in the shedding of pro-EGF since pro-EGF
shedding was not regulated in HeLa cells, which are known to
possess high amounts of TACE.
MATERIALS AND METHODS
Chemicals—Peroxidase-conjugated goat anti-mouse IgG, trypsin
(10000 BAEE units/mg), extravidin-peroxidase, soybean trypsin inhib-
itor (SBTI, 1 mg inhibits 10000 units trypsin), aprotinin, pepstatin A,
E-64, phenantrolin, bisindolylmaleimide I (BIM I), EDTA, tunicamycin,
protein molecular weight markers, DMEM without methionine and
cysteine, A23187, ionomycin, PMA, ampicillin, LB broth, LB agar, and
protein G-Sepharose were obtained from Sigma Chemical Co. QIAquick
Gel Extraction and PCR purification kits were obtained from Qiagen.
BCA protein assay kit and Sulfo-NHS-LC-Biotin were from Perbio.
Triton X-100 was obtained from MERCK. N- and O-deglycosylation as
well as Quantum Prep Plasmid purification kits were from BioRad
Laboratories. Desoxynucleotide trisphosphates, agarose, and DNA size
markers were obtained from Amersham Biosciences. Upstream and
downstream oligonucleotide primers were synthesized by MWG-bio-
tech. pTargeTTM mammalian expression vector system, TNT-coupled
reticulocyte lysate systems and JM-109 or DH5␣-competent cells were
from Promega. ExpandTM Reverse Transcriptase and ExpandTM High
Fidelity PCR System were from Roche Applied Science. 125I-Na (100
␮Ci/ml, 3.7 MBq/ml) and Renaissance® Western blot Chemilumines-
cence Reagent Plus were purchased from PerkinElmer Life Sciences.
TRAN35S-Label was from ICN. Ro-31-8220 (BIM IX; methanesulfon-
ate), polyclonal anti-human TACE (AB1) antibody, TAPI-2 and TIMP1
and 3 were from Calbiochem. Phosphate-buffered saline (PBS), NUT.
MIX.F12(HAM) Glutamax (HAM-F12), fetal calf serum (FCS),
OptiMEM, Dulbecco’s modified Eagle’s medium (DMEM) Glutamax,
fungizone, penicillin-streptomycin solution, random primers,
PcDNA3.1/Myc-His mammalian expression vector and monoclonal anti-
Myc antibody were from Invitrogen. Exgen 500 was from Euromedex.
Monoclonal anti-HA antibody was from Covance. Polyclonal rabbit anti-
human TNF-␣ antibody was from Promocell and TNF-␣ from Prepro-
Tech Inc.
Rat Pro-EGF cDNA Cloning and Construction of Epitope-tagged Pro-
EGF—Total RNAs from Sprague Dawley rats were prepared as de-
scribed previously (56). Kidney RNAs were first subjected to reverse
transcription using random priming and ExpandTM Reverse Tran-
scriptase as described by the manufacturer’s protocol. PCR amplifica-
tion of the cDNA was performed according to rat pro-EGF cDNA se-
quence as published by Saggi et al. (Ref. 13, GenBankTM accession
number: M63585). The 5⬘ sense primer, complementary to nucleotides
350 –368, carries a HindIII restriction site whereas 3⬘ antisense primer,
complementary to nucleotides 3881–3900, carries an EcoRI restriction
site. Amplification was performed using the ExpandTM high fidelity
PCR system (Roche Applied Science). After an initial denaturation for 2
min at 94 °C, cDNA was first subjected to 10 cycles of amplification with
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
the following conditions: denaturation for 15 s at 94 °C, annealing for
30 s at 55 °C, and extension for 2 min 30 s at 68 °C then followed by 25
cycles in the same conditions but with extension time incremented of 5 s
for each cycle. A 3550 bp cDNA was obtained, which was purified,
A-tailed, inserted into pTargeTTM mammalian expression vector as
described by the manufacturer’s protocols (Promega) and finally cloned
into JM-109 bacterial cells leading to the pT12 clone. The sequence of
clone pT12 was confirmed by both restriction analysis and sequencing
using selected primers. Its ability to drive the synthesis of prepro-EGF
protein of high molecular weight was further confirmed by in vitro
transcription using TNT®-coupled reticulocyte lysate systems (Pro-
mega). To introduce epitope tags into the cloned rat prepro-EGF cDNA
sequence, the entire coding sequence (HindIII-EcoRI fragment) was
first excised and subcloned into PcDNA3.1/Myc-His mammalian ex-
pression vector (Invitrogen). The prepro-EGF/Myc-His construct was
generated by suppressing the stop codon using a PCR-based strategy.
An antisense oligonucleotides complementary to nucleotides 3768 –
3787 and carrying an ApaI restriction site was used, allowing the
in-frame ligation of the prepro-EGF cDNA with both c-Myc and His6
epitopes sequences at the 3⬘-end of the cDNA. This cDNA was further
modified by inserting the HA1 epitope (YPYDVPDYA) of influenza
virus hemagglutinin (57) between amino acids Ser975 and Asn976 of rat
prepro-EGF using PCR approach as described by Arribas and Massagué
(51) for the insertion of the HA epitope into the rat TGF-␣ sequence.
This final modification supplies the pcD12-HA plasmid, which was
confirmed by DNA sequencing.
PCR-based strategy was also used to generate a cDNA allowing the
synthesis of a HA-tagged soluble form of EGF, which lacked most of its
prodomain. Briefly, a stop codon was introduced in the extracellular
domain of pro-EGF-HA following the Lys1024 codon (i.e. the C-terminal
end of the mature urinary EGF sequence) and the prodomain deleted
between Leu32 and Val972 to generate the ⌬pro-⌬TM-EGF expression
plasmid. This expression plasmid allows the synthesis of a secreted
soluble form of rat pro-EGF that conserved the signal peptide and the
first ten amino acids of the EGF prodomain (Met1 to Pro31) as well as
the arginine residue (Arg973) that preceded the mature EGF sequence
(Asn974 to Lys1024) but lacks the downstream sequences encoding the
juxtamembrane, transmembrane, and intracellular domains.
Cell Culture and Transfection—The CHO-K1 and CHO-M2 cells
were cultured in monolayers in HAM-F12 medium whereas HeLa cells
were cultured in DMEM. Both medium were supplemented with 10%
FCS, 100 ␮g/ml streptomycin, 100 units/ml penicillin, and 1.25 ␮g/ml
amphothericin B (fungizone) at 37 °C under 5% CO2 and used within
ten passages. Cell monolayers were suspended by treatment with 5 mM
EDTA in PBS (CHO) or trypsin (HeLa) and usually plated in 6-well
plates (750,000 cells/plate) in culture medium the day before
transfection.
CHO or HeLa cells (50 –70% confluence) were transiently transfected
with 2.5 ␮g of the appropriate expression plasmid containing either
native (pT12), HA-tagged pro-EGF (PcD12-HA), or HA-tagged soluble
pro-EGF cDNA (⌬pro-⌬TM-EGF) or else human pro-TNF-␣ cDNA in
PcDNA 3 (a kind gift from Dr. F. Peiretti) using Exgen 500 reagent as
described by the manufacturer. Following transfection, cells were
switched in HAM-F12 or DMEM supplemented with 10% FCS and
antibiotics. Transfected cells were further incubated for 48 h to allow for
pro-EGF or pro-TNF-␣ expression and analyzed either directly or after
additional incubations in varying conditions. At the end of these differ-
ent types of experiments, cells and conditioned media were collected.
Conditioned medium was cleared of contaminating cells by centrifuga-
tion (1000 ⫻ g/5 min). Cells monolayers were washed twice with PBS
and lysed for 30 min in detergent-containing buffer (50 mM sodium
phosphate, pH 7.4 supplemented with 150 mM NaCl, 5 mM EDTA, and
1% Triton X-100). Cell lysates were cleared from insoluble material by
centrifugation at 15,000 ⫻ g for 20 min. Both incubation media and cell
lysates were used for EGF or TNF-␣ content analysis as described
below.
EGF and TNF-␣ Radioimmunoassay—Rat EGF or human TNF-␣
was labeled with carrier free 125I-Na to a specific activity of 150 –200
␮Ci/␮g using the chloramine-T method. RIA analysis for human TNF-␣
contents was performed exactly as previously described for rat EGF
(41). The IgG fraction of the rabbit polyclonal anti-rat EGF antibody
(rEGF2, 1/5000) or of the anti-human TNF-␣ antibody (1/1000) were
used as immunoprecipitating antibodies. Immunoreactive rat EGF
(irEGF) in both cells and conditioned media and human TNF-␣ in
conditioned media were quantified 48 h after cells have been tran-
siently transfected. As discussed previously (41), in order to adequately
quantify EGF amounts, aliquots from both conditioned media and cell
lysates were subjected to trypsin hydrolysis in order to release low
45257
molecular mass fully immunoreactive mature EGF from putative pre-
cursor molecules.
Metabolic Labeling of Cellular Proteins—Plated CHO (both wild type
and M2 mutant) cells grown in 6-well plates as described above were
transiently transfected with the pcD12-HA expression plasmid. 48 h
later, monolayers were washed once with PBS and then preincubated in
serum-free, methionine/cysteine-free DMEM (Met-Cys-free DMEM)
supplemented with 1% lipid-free BSA for 30 min at 37 °C under 5%
CO2. Cells were then labeled in the same medium containing 250
␮Ci/ml for 2 h of TRAN35S Label at 37 °C under 5% CO2. Labeling was
stopped by washing the cells three times with PBS. Cells were then
further incubated in the chase medium (complete DMEM containing
either 1% BSA or 10% FCS) containing the appropriate drugs and for
the time specified in the figure legends.
Biotinylation of Cell Surface Proteins—CHO (both wild type and M2
mutant) or HeLa cells were grown on 6-well plates for 48 h after
transient transfection with the appropriate expression plasmid. Prior to
biotinylation, cells were washed three times with ice-cold PBS contain-
ing 1 mM CaCl2 and 1 mM MgCl2 (PBS⫹), then biotinylated in the
presence of 1 mg/ml sulfo-NHS-LC-Biotin in the same buffer for 30 min
on ice. Biotinylation was stopped by incubating cells for 10 min at 4 °C
in the presence of 100 mM glycine in PBS⫹ and washed once with
PBS⫹. Following biotinylation, cells were incubated at 37 °C with the
indicated chase medium (complete DMEM containing either 1% BSA or
10% FCS) containing the appropriate drugs and for the time specified in
the figure legends.
Immunoprecipitation, Western Blotting Analysis, and Autoradiogra-
phy—All immunoprecipitation protocols were performed at 4 °C, unless
otherwise stated. After incubating cells in the different conditions spec-
ified in the legends of the figures, conditioned media and cell lysates
were prepared as described above. Equal relative amounts of cell ly-
sates and conditioned media were incubated overnight with either
anti-rat EGF (rEGF2, 1/200), anti-HA (HA-11, 1/500) or anti-Myc (1/
500) antibodies. In order to precipitate immune complexes, 40 ␮l of a
50% slurry of protein A (rEGF2) or protein G-Sepharose (HA-11 or Myc)
were added for 2 h under constant shaking. Protein A- or G-Sepharose
was pelleted by centrifugation and immune complexes washed three
times with high salt lysis buffer (phosphate 50 mM, NaCl 300 mM, pH
7.4; EDTA 5 mM, Triton X-100 1%).
Immunoprecipitated incubation medium or cell lysates were solubi-
lized in SDS containing electrophoresis sample buffer and separated by
SDS-PAGE on a 7.5% Tris-HCl or 16.5% Tris-Tricine slab gel. Following
electrophoresis, proteins from non-labeled, 35S-labeled or biotinylated
samples were electrotransferred overnight to optitran BA-S 85 re-
inforced nitrocellulose membrane (0.45 ␮m, Schleicher & Schuell). For
detection of 35S-radioactivity, membranes were air-dried and exposed to
photosensitive storage phosphorimaging plates in a STORM 840 instru-
ment (Molecular Dynamics). Quantification of the radioactivity of the
bands in each lane was performed using ImageQuant software.
For non-labeled and biotinylated samples, membranes were blocked
with 5% nonfat milk in TBS containing 0.2% Tween 20 (TBST) for 1 h
at 37 °C. Blots of biotinylated samples were directly probed with horse-
radish peroxidase-conjugated streptavidin (1/2500 in 0.5% milk con-
taining TBST) for 1 h at room temperature. To reveal specific HA or
Myc epitope-containing proteins, blots were immunostained with mono-
clonal antibodies (anti-HA or anti-Myc, 1/5000 in 0.5% milk containing
TBST) for 3 h at room temperature and probed with 1/10000 dilution of
goat anti-mouse IgG antibody linked to horseradish peroxidase as de-
scribed previously (56). Blots were probed in Renaissance® Western
blot Chemiluminescence Reagent Plus according to manufacturer’s rec-
ommendations and visualized by exposure to Amersham Biosciences
Hyperfilm-ECL.
Immunodetection of TACE (ADAM 17) in cell lysates from CHO and
HeLa cells was performed following 7.5% slab gel electophoresis. West-
ern blot analysis was performed with the rabbit polyclonal anti-TACE
antibody (1/2000) exactly as described above.
RESULTS
In an attempt to gain insight into the mechanisms involved
in the fate of rat EGF secretion, we first decided to clone the
entire rat pro-EGF cDNA. To facilitate analysis of membrane-
anchored pro-EGF shedding, the rat EGF cDNA was modified
in order to introduce epitope tags in both the ectodomain (HA
epitope) and the cytoplasmic tail (Myc epitope) of the molecule.
The HA tag was introduced downstream of the prodomain,
between Ser975 and Asn976, i.e. the second and third amino
45258 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
TABLE I
RIA analysis of EGF synthesis and secretion
by transfected CHO-K1 cells
CHO-K1 cells were grown in culture medium (HAM F12 supple-
mented with 10% FCS) to about 70% confluency in 90-mm dishes.
Plated cells were either not transfected (WT) or transiently transfected
with unmodified (pT12) or epitope-tagged (pcD12-HA) prepro-EGF-con-
taining expression plasmids. Following transfection, cells were further
incubated in 5 ml of culture medium at 37 °C under 5% CO2. 48 h later,
conditioned media were harvested and cell lysates prepared. Aliquots of
both cell lysates and conditioned media were subjected or not to trypsin
hydrolysis before quantification of rat EGF by RIA as described previ-
ously (41). Total amounts of immunoreactive rat EGF in cell lysates and
conditioned media are reported as well as the percentage of secreted
EGF for each experimental condition.
Amount of EGF in % of EGF in
conditioned
Conditioned
medium Cell lysates medium
pmol
CHO WT ⫺ Trypsin NDa ND
⫹ Trypsin ND ND
CHO pT12 ⫺ Trypsin 2.3 2.6 46.9
⫹ Trypsin 6.8 9.5 41.5
CHO pcD12-HA ⫺ Trypsin 4 3.4 54.4
⫹ Trypsin 14 16.3 46.1
a
Not detected.
acids of mature EGF. The Myc and His6 tags were C-terminal
extension of the precursor molecule (see Fig. 1A for the sche-
matic representation of the epitope-tagged rat pro-EGF con-
struct). Both unmodified (pT12) and epitope-tagged (pcD12-
HA) expression plasmids encoding the EGF precursor as well
as the derived expression plasmid encoding the prodomain-
truncated HA-tagged soluble EGF (⌬pro-⌬TM-EGF) were used
to transiently transfect established cultured cells.
RIA Analysis of the Synthesis and Secretion of Rat EGF
Immunoreactive Materials in Transiently Transfected CHO-K1
Cells—We first wanted to determine if a heterologous protein,
which contains the EGF epitope, was expressed in CHO-K1
cells transfected either with the pT12 or PcD12-HA plasmids.
EGF contents under different experimental conditions were
first determined by RIA. As reported in Table I, EGF was
undetectable in both lysate and conditioned medium of non-
transfected cells both before and after trypsin treatment. In
contrast, important and similar amounts of immunoreactive
EGF could be detected in both lysate and conditioned medium
following transfection of cells with either the untagged pro-
EGF (pT12) or the pro-EGF-HA/Myc-tagged encoding plasmid
(pcD12-HA). In the experiments realized with the EGF-precur-
sor encoding plasmids, EGF immunoreactivity was increased
by 3– 4.8-fold following trypsin treatment. These results indi-
cate that CHO cells do not naturally synthesize nor secrete
detectable levels of EGF or that EGF synthesized by cells from
Chinese hamster cannot be detected by our antibody, which is
directed against rat EGF (41). The presence of immunoreactive
EGF in both cell lysates and conditioned media indicates that
cells transfected with either one of the two plasmids secreted
EGF materials. Cells transfected with the two different pro-
EGF plasmids did not only synthesize similar amounts of EGF
but also secreted the same percentage of EGF (about 50% of
total). This points out that the cellular behavior of the encoded
proteins toward synthesis and secretion is very similar and
suggests that epitope tagging of pro-EGF does not have any
significant effect. Moreover, as stated in a previous study (41),
the increase in EGF immunoreactivity following trypsin treat-
ment may be an indication that EGF is synthesized and se-
creted as high molecular weight EGF-containing precursor
proteins.
Western Blot Analysis of Expression and Secretion of Rat
pro-EGF in Transiently Transfected CHO-K1 Cells—Our initial
attempt to detect EGF-containing proteins by Western blot
analysis using rEGF2 antibody failed because this antibody
was not able to recognize the denatured and reduced forms of
EGF (data not shown). That is why we decided to introduce
epitope tags in both extracellular and intracellular parts of the
precursor protein (see Fig. 1A for the schematic structure of
EGF constructs). Western blot analysis of anti-EGF immuno-
precipitates from pcD12-HA-transfected CHO cells show that
pro-EGF is expressed as three high molecular weight species
containing both HA and Myc epitopes (Fig. 1B, Cell), and we
did not observe any protein lacking one of the two epitopes. Size
analysis showed a major species of 176 kDa and two minor ones
of 136 and 111 kDa. Three high molecular weight immunore-
active proteins, one major species of 165 kDa and two minor
species of 121 and 99 kDa were observed in the anti-EGF
immunoprecipitates from conditioned medium of pcD12 HA-
transfected cells (Fig. 1B, Medium). These proteins only con-
tained the HA epitope and not the Myc epitope. Performing
electrophoresis with 16.5% Tris-Tricine acrylamide slab gels
did not allow us to detect any other lower molecular weight
immunoreactive species, especially mature 6 kDa EGF, either
in cell lysates or in the conditioned medium (data not shown).
As could be predicted, no EGF-containing protein could be
detected either with anti-HA or anti-Myc antibodies in both cell
lysate and conditioned medium from pT12-transfected CHO
cells.
The presence of both extracellular (HA) and intracellular
(Myc) epitopes in anti-EGF immunoprecipitates from cell ly-
sates suggests that these species represent membrane-an-
chored forms of pro-EGF. The 176-kDa species would be the
full-length pro-EGF whereas the two others may represent
species shortened in the prodomain of the molecule. The loss of
the intracellular C-terminal Myc tag in pro-EGF species from
the conditioned medium as well as the shift in molecular
weight led us to hypothesize that these proteins are derived
from the membrane-anchored pro-EGF through proteolytic
cleavage in the juxtamembrane domain, i.e. between EGF and
the transmembrane domain. Moreover, the similar shift in
molecular mass (respectively 11, 15, and 12 kDa) suggests that
the different soluble pro-EGF species; 165, 121, and 99 kDa are
respectively derived from the 176, 136, and 111 cellular species
through proteolytic cleavage in the juxtamembrane domain.
As shown on Fig. 1C (upper panel), labeling of cell surface
proteins with sulfo-NHS-LC-biotin demonstrated that all three
different membrane-anchored pro-EGF species detected in Fig.
1B were exposed at the cell surface. In pcD12-HA transfected
cells, biotinylated proteins of 176, 136, and 111 kDa could be
immunoprecipitated with the anti-EGF, anti-HA, or the anti-
Myc antibodies. These pro-EGF species were also observed in
anti-EGF immunoprecipitates from pT12-transfected cells. In-
cubation of biotinylated cells for 4 h in culture medium supple-
mented with 10% serum resulted in the release of biotinylated
pro-EGF species of 165, 121, and 99 kDa (Fig. 1C, lower panel).
According to the results shown in Fig. 1B, these biotinylated
proteins were immunoprecipitated with both anti-EGF and
anti-HA antibodies but not with the anti-Myc antibody. All
three soluble species were also observed in anti-EGF immuno-
precipitates from pT12-transfected cells. Since all three species
(176, 136, and 111 kDa) of cellular pro-EGF could be biotiny-
lated at the cell surface, this greatly suggests that the lower
molecular mass species (136 and 111 kDa) may not represent
some non-glycosylated precursor forms of the 176 kDa species.
This hypothesis was confirmed by performing short pulse la-
beling experiments (5 min) of newly synthesized proteins with
35
S-methionine/cysteine followed by chase incubation (up to
3 h). Following 35S labeling, the radioactive 176 kDa anti-HA
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding 45259

FIG. 1. Epitope-tagged expression and shedding of pro-EGF in CHO-K1 cells. A, structure of epitope-tagged prepro-EGF constructs used
in this study. Location of HA, Myc, and His6 epitopes are as indicated. S, signal peptide; EGF, mature epidermal growth factor; TM, transmem-
brane domain; Cyt, cytoplasmic domain. B, characterization of the epitope-tagged EGF-containing proteins in cell lysate and conditioned medium.
CHO cells were transiently transfected with either unmodified prepro-EGF (pT12) or epitope-tagged prepro-EGF (pcD12-HA) containing expres-
sion plasmids and further incubated in conditioning medium (HAM F12 supplemented with 10% FCS). 48 h later, lysates and media were
harvested for immunoprecipitation (IP) with anti-rat EGF antibody. Immunoprecipitated EGF species were analyzed by Western blot (WB) using
the indicated antibodies as described under “Materials and Methods.” Apparent molecular masses of the different EGF species in lysate (°, 176 kDa;
°°, 136 kDa; °°°, 111 kDa) and incubation medium (*, 165 kDa; **, 121 kDa; ***, 99 kDa) were deduced from comigration with known standards.
C, characterization of cell surface expression and shedding of rat pro-EGF. CHO cells were transiently transfected with pT12 or pcD12-HA
expression plasmids. 48 h later, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin as described under ‘‘Materials and Methods’’ and
further incubated for 4 h in conditioning medium as in B. Cell lysates and incubation media were harvested and immunoprecipitation (IP)
performed with the indicated antibody. Immunoprecipitated proteins were separated by SDS-PAGE, blotted to nitrocellulose and biotinylated
proteins revealed with peroxidase-conjugated streptavidin. D, expression and secretion of the HA-tagged prodomain-truncated soluble EGF. CHO
cells were transiently transfected with the ⌬pro-⌬TM-EGF expression plasmid. 48 h later, cells were washed and further incubated and processed
as described above (C). Anti-EGF immunoprecipitates were analyzed by electrophoresis on 16.5% Tris-Tricine slab gels, blotted to nitrocellulose
and analyzed by Western blot (WB) using the anti-HA (HA-11) antibody as described under ‘‘Materials and Methods.’’ Apparent molecular masses
of the different EGF species reported on the right side of the figures were deduced from comigration with known standards (left side).

immunoprecipitated protein is instantaneously and highly de-
tectable in the cell lysate. At any time tested, this major pro-
EGF showed the same high molecular weight. Lower molecular
mass proteins of 136 and 111 kDa were only faintly detected
later (data not shown), a result that argues against their role as
precursor forms of the 176-kDa species.
Taken altogether, these results indicate that pT12- and
pcD12-HA-transfected CHO cells contain one major (176 kDa)
and two minor (136 and 111 kDa) pro-EGF species that are
membrane-anchored proteins exposed at the cell surface. Once
exposed at the cell surface, these pro-EGF species may undergo
proteolytic cleavage in their juxtamembrane domain in order to
release soluble pro-EGF of 165, 121, and 99 kDa. The protein
pattern observed with CHO cells transfected with the epitope-
45260 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
tagged pro-EGF encoding pcD12-HA plasmid is clearly not a
consequence of epitope tagging of the molecule. As a matter of
fact, the same pattern of proteins was observed in both cell
lysate and conditioned medium following biotinylation of un-
tagged pro-EGF synthesized by pT12-transfected CHO cells.
Since the calculated size of the full-length epitope-tagged
membrane-anchored EGF precursor, 128 kDa, as determined
from cDNA sequence, is far smaller than the estimated size
found by SDS-PAGE (176 kDa) we suggested that this protein
might be glycosylated. This was confirmed following treatment
of both secreted and cellular pro-EGF with different glycosi-
dases (N-glycosidase, O-glycosidase, and N-acetyl-neuramini-
dase) or incubation of cells with tunicamycin (an inhibitor of
endogenous glycosylation). These experiments show that both
soluble (165, 121, and 99 kDa) as well as membrane-anchored
pro-EGF (176, 136, and 111 kDa) were N-glycosylated proteins
(data not shown). However, the shift in molecular mass follow-
ing deglycosylation (15–20 kDa) was not sufficient to explain
the discrepancy between the one calculated from the peptide
backbone and the one measured on SDS-PAGE. Size analysis of
the in vitro transcription/translation product from the
pcD12-HA expression plasmid yielded only one product, whose
estimated molecular mass of 154 –159 kDa is very close to the
estimated size (160 kDa) of the deglycosylated 176 kDa mem-
brane-anchored pro-EGF. These results greatly favor the hy-
pothesis that an unusual electrophoretic behavior of the EGF
precursor molecules may explain the overestimation of both
membrane-anchored and soluble high molecular weight pro-
EGF found in transfected CHO cells but also in other cell types
such as MDCK and HB2 cells.
Fig. 1D show the results obtained with CHO cells transiently
transfected with the HA-tagged prodomain-truncated soluble
EGF expression plasmid (⌬pro-⌬TM-EGF). As can be shown,
following an incubation of 4 h in serum-containing medium,
anti-EGF immunoprecipitates from both cell lysate and condi-
tioned medium were shown to contain an anti-HA immunore-
active protein of 8.4 kDa. This molecular mass is greater than
the one that could be predicted for fully mature HA-tagged
EGF (6.9 kDa) but perfectly matches the one predicted for an
expressed protein that lacks the signal peptide but was not
cleaved at the level of its residual prodomain. The identical
molecular masses that are observed for both cellular and se-
creted EGF indicated that the last one is not derived from the
first one through proteolytic cleavage. Thus, as observed with
the full-length pro-EGF-encoding plasmids (Fig. 1, A–C), se-
creted EGF does not appear to be processed in its ectodomain
and particularly at the Arg973 level that would have led to the
fully mature HA-tagged soluble 6.9-kDa EGF species as ob-
served in rat urine (58). The way by which the 8.4 kDa soluble
EGF is secreted appeared to be completely different from the
one involved for full-length pro-EGF and must follow the se-
cretory pathway for soluble proteins that do not involve cell
surface shedding.
Shedding of Rat Membrane-anchored pro-EGF Is Regulated
in CHO-K1 Cells—As shown in Fig. 2 (upper panel), pcD12-HA-
transfected 35S-labeled CHO cells show an increased release of
all three species of pro-EGF upon incubation in the presence of
PMA, pervanadate (V2O7), or serum (FCS). No stimulation
of pro-EGF release was detected when cells were incubated
with either Me2SO (vehicle), the calcium ionophores A23187
(see Fig. 2) or ionomycin (data not shown). Following quantifi-
cation by phosphorimaging, the amount of each [35S]pro-EGF
species was expressed relative to the unstimulated condition
(medium). As seen in Fig. 2 (lower panel), the stimulatory
agents tested increased the release of the three pro-EGF to the
same extent. This indicates that any of the soluble pro-EGF can
FIG. 2. Induction of pro-EGF shedding by PMA, pervanadate,
and FCS. CHO cells were transiently transfected with pcD12-HA ex-
pression plasmid. 48 h later, cells were labeled for 2 h with TRAN35S
label. At the end of the labeling, cells were extensively washed three
times with PBS and chased at 37 °C under 5% CO2 for 2 h in DMEM
supplemented with 1% BSA and containing either no addition (medi-
um) or one of the following agents: 0.1% Me2SO (DMSO), 1 ␮M calcium
ionophore (A23187), 1 ␮M phorbol ester (PMA), 100 ␮M Pervanadate
(V2O7) or 10% serum (FCS). Conditioned media were immunoprecipi-
tated with the anti-HA antibody as described under ‘‘Material and
Methods.’’ Immunoprecipitated proteins were further separated by
SDS-PAGE, blotted to nitrocellulose, and visualized by phosphorimag-
ing using a STORM 840 instrument (upper panel). Radioactivity asso-
ciated with each secreted pro-EGF (165, 121, 99 kDa as indicated on the
figure) was quantified for each experimental condition (lower panel),
and results are expressed as the percentage of the amount of that
35
S-labeled pro-EGF present in the control medium.
be used to account for the properties of pro-EGF secretion.
Thus, in the following experiments only results concerning the
major pro-EGF kilodalton species (pro-EGF165) will be reported
and quantified.
We have demonstrated here that in contrast to previously
published results (48), the release of soluble rat pro-EGF may
be regulated at least in one cell type, the CHO-K1 cells. How-
ever, with that kind of experiment, the increased release of
soluble forms of pro-EGF may be due to the stimulation of
intracellular protein traffic in the secretory pathway and/or an
increase in the shedding rate of the membrane precursor. In
order to test for an increased cleavage of the cell surface pro-
EGF, we analyzed pro-EGF secretion upon PMA stimulation
following both 35S labeling and cell surface protein biotinyla-
tion. As shown in Fig. 3, compared with control experiments,
PMA induced a time-dependent increase of both 35S-labeled
(upper panel) and biotinylated pro-EGF165 (middle panel) in
the conditioned medium, suggesting that the HA-tagged pro-
teins secreted came from a previously labeled intrinsic cell
surface protein, i.e. the membrane-anchored pro-EGF. Quanti-
fication of the kinetics of 35S-labeled pro-EGF165 release indi-
cates that the effect of PMA was linear for up to 2 h (lower
panel). Stimulation rapidly decreases thereafter probably as a
result of both desensitization of the PKC-dependent pathway
and down-regulation of the PKC activity. From these results, it
is obvious that the rise in pro-EGF detected in the medium
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
FIG. 3. Kinetic of stimulation of cell surface pro-EGF secre-
tion. CHO cells were transiently transfected with pcD12-HA expres-
sion plasmid. 48 h later, cells were labeled for 2 h with TRAN35S Label.
At the end of the labeling, cells were extensively washed three times
with PBS and cell surface proteins biotinylated using sulfo-NHS-LC-
biotin as described under ‘‘Materials and Methods.’’ Labeled cells were
chased for up to 180 min, in the absence or presence of 1 ␮M PMA as
indicated on the figure. At each time point, conditioned media were
immunoprecipitated with the anti-HA antibody and immunoprecipi-
tated proteins separated by SDS-PAGE and blotted to nitrocellulose.
35
S-labeled proteins were visualized by phosphorimaging using a
STORM 840 instrument (upper panel) and biotinylated proteins visu-
alized by ECL⫹ using peroxidase-conjugated streptavidin (middle
panel) as described under ‘‘Materials and Methods.’’ Radioactivity as-
sociated with 165 kDa [35S]pro-EGF was quantified for each time point
(lower panel), and results are expressed as the percentage of the
amount of 35S-labeled pro-EGF165 present in the incubation medium
upon 180 min of stimulation with PMA.
upon PMA stimulation is due to the stimulation of membrane-
anchored precursor shedding.
This conclusion was further confirmed by the experiments
performed with CHO cells transiently transfected with ⌬pro-
⌬TM-EGF expression plasmid. RIA analysis of EGF content in
both conditioned media and cell lysates show us that cells
transfected with ⌬pro-⌬TM-EGF or pcD12-HA synthesized
equal amounts of immunoreactive EGF (data not shown), how-
ever the rate of EGF secretion by cells expressing the truncated
8.4 kDa soluble form of EGF (11.2 ⫾ 0.89% per h) was two
times that of cells expressing the full-length membrane-an-
chored EGF precursor (4.9 ⫾ 0.35% per h), suggesting that both
EGF species do not follow the same pathway to be secreted into
the extracellular medium. Moreover, compared with pro-EGF-
transfected cells, incubation in the presence of PMA, serum, or
pervanadate showed that secretion of the soluble form of EGF
could not be up-regulated whereas the one resulting from ex-
pression of the full-length membrane-anchored pro-EGF could
be (Fig. 4). This result, and the fact that no molecular weight
shift between the cellular and secreted HA-EGF was detected
(8.4 kDa, Fig. 1D) indicate that a general stimulation of intra-
cellular protein trafficking does not account for the stimulation
of pro-EGF ectodomain secretion and reinforces the hypothesis
that the rise in pro-EGF165 detected in the medium upon stim-
45261
FIG. 4. Regulated EGF secretion in ⌬pro-⌬TM-EGF versus
pcD12-HA transfected cells. CHO cells were transiently transfected
with either HA-tagged full-length pro-EGF (pcD12-HA) or with the
HA-tagged prodomain truncated soluble EGF (⌬pro-⌬TM-EGF) expres-
sion plasmid as indicated on the figure. 48 h later, cells were extensively
washed three times with PBS and chased at 37 °C under 5% CO2 for 2 h
in DMEM supplemented with 1% BSA and containing or not (medium)
one of the following agents: 1 ␮M phorbol ester (PMA), 100 ␮M pervana-
date (V2O7), or 10% serum (FCS). Immunoreactive EGF in both condi-
tioned medium and cell lysates was quantified by RIA following trypsin
treatment of the different samples as described under ‘‘Materials and
Methods.’’ EGF secretion in the incubation medium was first calculated
as a percentage of total immunoreactive EGF in both medium and cell
lysate then normalized to percentage of EGF secretion in the medium
alone. Results are the average ⫾ S.E. of triplicate experiments.
ulation is due to the stimulation of membrane-anchored pre-
cursor shedding.
Role of PKC in the Regulation of Membrane-anchored pro-
EGF Shedding—Since PKC activation is often linked to protein
secretion and was clearly involved in the PMA-dependent reg-
ulation of EGFR ligand shedding, we have tested its role in the
control of rat pro-EGF secretion. Implication of phorbol ester
activable PKC in pro-EGF secretion was first demonstrated by
the use of specific PKC inhibitors such as Ro 31-8220 or BIM I.
As can be seen on Fig. 5A, preincubation of CHO cells in the
presence of PKC inhibitors either partially (Ro 31-8220) or
completely (BIM I) abolished a subsequent stimulation of pro-
EGF165 secretion by PMA. In the same conditions, basal secre-
tion as well as pervanadate and serum stimulation was unaf-
fected (data not shown). The involvement of PKC in the
stimulatory effect of PMA was further confirmed following its
down-regulation prior to cells stimulation. PcD12-HA-trans-
fected cells were first incubated with 1 ␮M PMA for 18 h prior
to 35S labeling. As shown in Fig. 5B, down-regulation of PKC
completely abolished the stimulation of pro-EGF165 secretion
by PMA. This inhibition is not the consequence of a general and
nonspecific inhibitory effect of the treatment since pervanadate
stimulation was only slightly affected and serum-induced se-
cretion was not. Moreover, PMA pretreatment did not show any
inhibitory effect on the incorporation of 35S-amino acids into
cellular pro-EGF, i.e. pro-EGF synthesis (data not shown).
Taken together, these results and those obtained with PKC
inhibitors, clearly indicate that the stimulatory effect of PMA is
completely dependent on PKC activities whereas both pervana-
date and serum stimulation are not.
Pro-EGF Ectodomain Shedding in CHO-K1 Cells Is Associ-
ated with a Zinc Metalloprotease Activity—The following exper-
iments were performed in order to determine the type of pro-
tease activity involved in membrane-anchored pro-EGF
45262 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding

FIG. 5. Role of PKC in the regulation of pro-EGF shedding. A, effect of PKC inhibitors on PMA induced pro-EGF shedding. CHO cells were
transiently transfected with pcD12-HA expression plasmid. 48 h later, cells were labeled for 2 h with TRAN35S Label. At the end of the labeling,
cells were extensively washed three times with PBS and chased for 30 min in DMEM supplemented with 1% BSA in the absence or presence of
PKC inhibitors (5 ␮M BIM or 5 ␮M RO 31– 8220) before addition or not of PMA (1 ␮M final concentration) for an additional 2 h. B, effect of PKC
‘‘down-regulation’’ on the regulation of pro-EGF shedding. CHO cells were transiently transfected with pcD12-HA expression plasmid. 30 h later,
cells were washed and further incubated for 18 h in the absence or presence of 1 ␮M PMA in order to down-regulate PKCs. Cells were then labeled
for 2 h with TRAN35S Label and extensively washed three times with PBS. Following labeling, cells were chased for 2 h at 37 °C under 5% CO2
in DMEM supplemented with 1% BSA in the absence (medium) or presence of different agents: 1 ␮M PMA; 100 ␮M V2O7; 0,1% Me2SO (DMSO);
10% FCS, or 1 ␮M A23187 as mentioned on the figure. Following chase incubations, conditioned media were immunoprecipitated with anti-HA
antibody. Immunoprecipitated proteins were further separated by SDS-PAGE, and blotted to nitrocellulose. 35S-labeled proteins were visualized
by phosphorimaging using a STORM 840 instrument (upper panels A and B). Radioactivity associated with 165 kDa [35S]pro-EGF was quantified
for each condition, and results are expressed as the percentage of the amount of 35S-labeled pro-EGF165 present in the incubation medium alone.

shedding. Therefore, we tested the effects of different protease
inhibitors on pro-EGF165 secretion from PcD12-HA transfected
CHO-K1 cells. The inhibitory effect was assayed both on PMA-
stimulated and -unstimulated 35S-labeled cells. As shown in
Fig. 6A, none of the protease inhibitors tested had any signif-
icant inhibitory effect on the unstimulated secretion. However,
the PMA-stimulated [35S]pro-EGF165 secretion was signifi-
cantly reduced by the metalloprotease inhibitor phenantrolin, a
zinc chelator molecule, and this inhibition can be overcome by
the addition of an excess of zinc in the incubation medium (data
not shown). Neither aprotinin nor SBTI (two serine protease
inhibitors), nor pepstatin (an aspartyl protease inhibitor), nor
E64 (a cysteine protease inhibitor) had any significant inhibi-
tory effect either on basal or stimulated secretion. EGTA and
EDTA, two commonly used metalloprotease inhibitors, which
preferentially chelate Mg2⫹ and Ca2⫹, were also inefficient
(data not shown).
These results indicate that the activity of the protease in-
volved in pro-EGF cleavage is greatly dependent upon the
presence of some divalent cations and suggest that it might be
a Zn2⫹ metalloprotease. This hypothesis was further supported
by the use of TAPI-2, a hydroxamate acid-based derivative,
which inhibits zinc-dependent metalloprotease activities by
binding to the catalytic site of the enzyme. As shown in Fig. 6B,
TAPI-2 inhibited both unstimulated and PMA-stimulated
[35S]pro-EGF165 secretion at concentrations below 1 ␮M. Half-
maximal inhibition of the PMA stimulation was achieved for a
concentration of 0.1 ␮M and complete inhibition reached at a
concentration of 3 ␮M. The inhibitory effect of TAPI-2 was not
restricted to the PMA stimulation since a 2 ␮M concentration
completely inhibited the serum- and pervanadate-stimulated
[35S]pro-EGF165 secretion (data not shown). Tissue inhibitors
of matrix metalloprotease (TIMP 1 and 3) were also tested at
concentrations up to 50 nM but failed to prevent the PMA
stimulation of immunoreactive EGF secretion (data not
shown).
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
FIG. 6. Effect of different protease inhibitors on pro-EGF shed-
ding. CHO cells were transiently transfected with pcD12-HA expres-
sion plasmid. 48 h later, cells were labeled for 2 h with TRAN35S Label,
extensively washed three times with PBS and chased at 37 °C under 5%
CO2 in DMEM supplemented with 1% BSA. A, effect of type-specific
proteases inhibitors on pro-EGF shedding. Chase was first performed
for 30 min in the absence (medium) or presence of different proteases
inhibitors (10 ␮M aprotinin, 20 ␮M SBTI, 100 ␮M pepstatin, 100 ␮M E64,
and 1 mM phenantrolin) before the addition (lower panel) or not (upper
panel) of PMA (1 ␮M final concentration) for 2 h. B, effect of the specific
metalloprotease inhibitor TAPI-2 on PMA induced pro-EGF secretion.
Labeled cells were first chased for 30 min in the presence or not of
increasing concentrations of TAPI-2 before stimulation (right part) or
not (left part) with PMA (1 ␮M final concentration) for 2 h. Following
chase incubations, conditioned media were immunoprecipitated with
anti-HA antibody. Immunoprecipitated proteins were further separated
by SDS-PAGE, and blotted to nitrocellulose. 35S-labeled proteins were
visualized by phosphorimaging using a STORM 840 instrument (panel
A and upper panel B). Radioactivity associated with 165 kDa [35S]pro-
EGF as reported in upper panel B was further quantified for each
conditions, and results are expressed as the percentage of the amount of
35
S-labeled pro-EGF165 present in the incubation medium upon stimu-
lation with PMA alone (lower panel B).
The metalloprotease-dependent ectodomain shedding of pro-
EGF was further confirmed by studying pro-EGF secretion in a
CHO cell line (CHO-M2) defective in the metalloprotease-de-
pendent shedding of many cell surface proteins (25, 28, 51).
45263
FIG. 7. Expression and regulation of pro-EGF secretion in
CHO-M2. Wild-type CHO (CHO-wt) and mutant CHO (CHO-M2) cells
were transiently transfected with the pcD12-HA expression plasmid.
48 h later, cells were processed for cell surface proteins biotinylation
using sulfo-NHS-LC-biotin (part A) or labeled for 2 h with TRAN35S
Label (part B) as described under ‘‘Materials and Methods.’’ A, biotiny-
lated cells were further incubated for 4 h in conditioning medium as in
Fig. 1B. Cell lysates and incubation media were harvested and immu-
noprecipitated with the anti-HA antibody (HA-11) as described under
‘‘Materials and Methods.’’ Immunoprecipitated proteins were separated
by SDS-PAGE on 7.5% slab gels, blotted to nitrocellulose and biotiny-
lated proteins revealed with peroxidase-conjugated streptavidin. B, at
the end of the labeling, cells were extensively washed three times with
PBS and chased at 37 °C under 5% CO2 for 2 h in DMEM supplemented
with 1% BSA and in the presence or not of different agents as indicated
below the figure. Conditioned media were harvested and immunopre-
cipitated with the anti-HA antibody (HA-11) as described under ‘‘Ma-
terial and Methods.’’ Immune complex were further separated by SDS-
PAGE on a 7.5% slab gel, blotted to nitrocellulose and visualized by
phosphorimaging using a STORM 840 instrument. Radioactivity asso-
ciated with the pro-EGF165 species was quantified for each experimen-
tal condition, expressed as arbitrary units, and are the average ⫾ S.E.
of triplicate experiments.
Wild-type CHO (wt) and mutant (M2) cells were first tran-
siently transfected with the pcD12-HA expression plasmid, cell
surface biotinylated, and analyzed for their ability to release
pro-EGF upon incubation in a serum-containing medium. Bi-
otinylated HA-tagged proteins in both cell lysate and incuba-
tion medium were further analyzed as described in Fig. 1. As
can be seen on Fig. 7A, the amount of soluble biotinylated
pro-EGF165 released by the M2 cells was greatly reduced com-
pared with the wild-type cells. This reduction was associated to
a concomitant accumulation of pro-EGF at the cell surface as
shown by the increase of the biotinylated pro-EGF176 species in
the cell lysate from CHO-M2 compared with CHO-wt. Since we
verified by RIA analysis that both cell lines synthesized equal
amounts of pro-EGF (data not shown), this result clearly dem-
onstrates that the mutant CHO cell line was also defective in
cell surface pro-EGF shedding.
45264 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
CHO-wt and CHO-M2 cell were further compared for their
ability to up-regulate pro-EGF secretion upon stimulation.
PcD12-HA- transfected 35S-labeled CHO-wt and CHO-M2 were
incubated in the presence of PMA, serum or pervanadate, and
pro-EGF secretion analyzed as described in Fig. 2. Results
reported in Fig. 7B showed that these agents were unable to
increase pro-EGF shedding from the cell surface of CHO-M2
cells and demonstrate that both constitutive and regulated
pro-EGF secretion was completely impaired in the CHO-M2
cell line compared with CHO-wt.
Taken as a whole, the results obtained with protease inhib-
itors as well as CHO-M2 clearly established that, at least in
CHO cells, both constitutive and regulated EGF secretion in-
volve one or more zinc-dependent metalloprotease(s) activities.
This or these metalloprotease activities shed the ectodomain of
pro-EGF from the membrane-anchored pro-EGF once exposed
at the cell surface.
Lack of Regulated pro-EGF Shedding in HeLa Cells—The
zinc metalloprotease TACE (ADAM 17) is now known to be
involved in shedding of many different cell surface proteins and
among them three members of the EGF growth factor family,
i.e. TGF-␣ (24, 25, 28, 29), HB-EGF (29, 30), and amphiregulin
(29, 31). So, in order to determine its possible involvement in
the shedding of pro-EGF, we have used the established HeLa
cell line, which is well known to possess high amounts of TACE
(Ref. 59 and Fig. 8A) and was able to cleave one of the sub-
strates of TACE, the ␤-amyloid precursor protein (␤APP) (60).
Using an anti-TACE antibody directed against the cytoplas-
mic domain of the protease, we have first verified the presence
of the protease in both CHO-K1 and HeLa cell lines (Fig. 8A).
As can be shown, HeLa cells contained high amount of both the
inactive high molecular weight proenzyme and of 2 mature
forms with lower masses. In contrast, when equal amounts of
cellular proteins from our CHO-K1 cells were analyzed, they
appeared to contain only low amounts of mature enzymes.
Like CHO-K1 cells, HeLa cells transiently transfected with
the pcD12-HA expression plasmid synthesize EGF as its high
molecular weight precursor. This precursor is membrane-an-
chored and is expressed at the cell surface as shown by bioti-
nylation experiments. However, compared with CHO cells, the
presence of HA-tagged pro-EGF in conditioned medium follow-
ing incubation in the presence of serum was only barely detect- FIG. 8. Regulation of EGF and TNF-␣ secretion in CHO and
able (data not shown). This result was confirmed when immu- HeLa cell lines. A, cell lysates from both CHO and HeLa cell lines
were prepared as described under ‘‘Materials and Methods.’’ Equal
noreactive EGF secretion was measured by RIA. HeLa cells
amount of cellular proteins were separated by SDS-PAGE on 7.5% slab
synthesized two to three times less EGF than CHO cells and gel and analyzed by Western blot using an anti-human TACE antibody.
with a basal EGF secretion rate, which was also 2⫻ lower (2.2% Arrows on the right show the position of the unprocessed full-length
per hour compared with 4.9% for CHO cells). In contrast to TACE precursor (proTACE) as well as of the mature and active TACE
species (TACE). B, HeLa (upper part) and CHO (lower part) cells were
CHO, HeLa cells have EGF receptors (EGFR) expressed at the
transiently transfected with either HA-tagged full-length pro-EGF
cell surface (61). As reported for TGF-␣ secretion by MDCK (pcD12-HA, left side) or with the human pro-TNF-␣ (right side) expres-
cells (62), we tested the possibility that secreted EGF could be sion plasmids. 48 h later, cells were extensively washed three times
lowered by a rapid consumption through receptor binding and with PBS and chased at 37 °C under 5% CO2 for 30 min for cells to
measure TNF-␣ secretion or for 2 h to measure EGF secretion. Chase
internalization. However, this is clearly not the explanation in
incubation was performed in DMEM supplemented with 1% BSA and
HeLa since none of the manipulations intended to inhibit this containing either no addition (medium) or one of the following agents: 1
effect had any increasing effect on the amount of secreted EGF. ␮M phorbol ester (PMA), 100 ␮M pervanadate (V2O7) or 10% serum
The conditions tested were the following: incubation in the (FCS). Immunoreactive TNF-␣ in conditioned medium as well as EGF
in both conditioned medium, and cell lysates were quantified by RIA as
presence of an excess of human EGF or of an anti-EGFR anti-
described under ‘‘Materials and Methods.’’ TNF-␣ secretion was ex-
body (mAb 225) that blocked the binding of secreted EGF to the pressed as percentages relative to the amount of immunoreactive
EGFR as well as blockade of receptor kinase activity by TNF-␣ in conditioned medium of unstimulated cells (medium). EGF
AG1478 that inhibited EGFR internalization (data not shown). secretion in the incubation medium was first calculated as a percentage
of total immunoreactive EGF in both medium and cell lysate then
More importantly, in HeLa cells, EGF secretion did not show normalized to percentage of EGF secretion in medium alone. Results
any strong up-regulation upon incubation in the presence of are the average ⫾ S.E. of triplicate experiments.
either PMA, serum, or pervanadate (Fig. 8B, upper left) under
conditions that strongly increase EGF secretion in CHO cells regulated shedding of cell surface proteins. To test this hypoth-
(Fig. 8B, lower left). Only a small but significant increase could esis, HeLa and CHO cells were transiently transfected with a
be observed upon PMA stimulation. One simple explanation pro-TNF-␣-containing expression plasmid, and secretion of
may be that our HeLa cell line may be unable to achieve TNF-␣ in both cell lines was analyzed by RIA. Results obtained
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
allowed us to rule out this hypothesis since they clearly showed
that in HeLa cells, shedding of pro-TNF-␣ was enhanced by the
different treatments: PMA, FCS, or pervanadate (Fig. 8B, up-
per right). Moreover, both basal and PMA-stimulated shedding
could be inhibited by the zinc metalloprotease inhibitor TAPI-2
(data not shown). In contrast, CHO cells show only a small
increase in pro-TNF-␣ shedding upon stimulation with PMA
and serum (⬃140% of basal) when compared with the effect
obtained on pro-EGF shedding in the same cell line (200 –500%
of basal) or to the release of TNF-␣ from HeLa cells (200 – 400%
of basal).
Taken together, these results greatly favor the hypothesis
that HeLa cells may not have the enzymatic machinery (ecto-
protease) necessary to shed the EGF precursor. The ADAM 17
protease (TACE) was first identified as a protease that specif-
ically cleaves full-length pro-TNF-␣ (63, 64). The fact that
HeLa cells, which possess a high amount of TACE, did not
efficiently shed pro-EGF, whereas these cells were able to
cleave pro-TNF-␣, questioned the role of TACE in the process-
ing of pro-EGF. This conclusion is further corroborated by the
ability of CHO cells that contain low amounts of TACE to
efficiently shed pro-EGF while being unable to shed pro-TNF-␣
from the cell surface. Thus, while both pro-TNF-␣ and pro-EGF
shedding are achieved by zinc-dependent metalloproteases,
pro-TNF-␣ shedding appears to be correlated to the amount of
TACE, while this correlation does not apply to pro-EGF
shedding.
DISCUSSION
In the course of a recent study (41) we have developed an
efficient polyclonal antibody (rEGF2) directed against the na-
tive form of rat EGF. This antibody allowed us to demonstrate
the presence of immunoreactive EGF in the exocrine rat lacri-
mal gland. In this tissue EGF was shown to be stored as its
high molecular weight membrane-anchored precursor as al-
ready shown in kidney (12, 36, 37, 39). Immunoreactive rat
EGF is also present in corresponding secretory fluids, i.e. tears
and urine (18 –20, 37, 42, 43). However, the nature of the
regulatory pathways as well as the proteases involved in the
shedding of membrane-anchored pro-EGF that may account for
the release of soluble EGF are very poorly understood. The
reasons may be that there was neither simple physiological
model nor any immortalized cell line available to study the
endogenous synthesis and secretion of EGF.
Since the tight regulation of shedding of EGFR ligands such
as TGF-␣ (50, 53, 65) and HB-EGF (66) have been previously
demonstrated in the CHO cell line, we were then interested in
determining the possible regulation of rat pro-EGF secretion in
this cellular model. In this way, we first cloned rat prepro-EGF
cDNA and introduced epitope tags in both the extracellular and
intracellular domain of the molecule. This was done in order to
conveniently follow the different parts of the precursor mole-
cule and also because our anti-rat EGF antibody does not
recognize the denatured and reduced form of EGF, precluding
any analysis by SDS-PAGE. RIA analysis of cell lysate and
conditioned media indicates that CHO cells transfected with
pro-EGF-containing expression plasmids efficiently synthesize
and secrete EGF immunoreactive molecules.
Cellular pro-EGF was found to be expressed as three differ-
ent species that are all present at the cell surface. These pro-
teins that contain EGF as well as the intracellular and extra-
cellular epitopes, certainly represent the membrane-anchored
pro-EGF. The size of the major pro-EGF species (176 kDa) is
very similar to the one of the human pro-EGF expressed in
MDCK (48) and in NIH 3T3 (67) cell lines (respectively, 185
and 170 kDa) and may be the full size rat pro-EGF. The lower
and minor molecular mass species (136 and 121 kDa) are
45265
probably derived from the major 176 kDa full-length precursor
through cleavage in the N-terminal distal part of the prodo-
main. They have not been observed with NIH 3T3 and MDCK
cells transfected with the human pro-EGF (48, 67) but we
previously reported a similar maturation of the membrane-
associated pro-EGF in rat kidney (41). Nevertheless, from the
present experiments, this limited prodomain release does not
seems to have any consequences on EGF secretion since all
species appeared to be equally sensitive to juxtamembrane
cleavage.
According to studies on MDCK (48) and NIH 3T3 (67) cells,
we did not observe any proteolytic maturation of pro-EGF that
would lead to the release of the entire EGF prodomain. These
observations are quite different from those reported for the
maturation process of other EGFR ligands such as TGF-␣ (50)
and human HB-EGF (68) where the precursor prodomain ap-
pears to be cleaved very rapidly once it has reached the cell
surface. However, our results are very similar to the ones
obtained with mouse HB-EGF (68) and amphiregulin (31),
where the full-length precursor preferentially accumulates at
the cell surface. This may reflect some specific and intrinsic
properties of the EGF prodomain that would be independent
both from the origin of EGF and of the expressing cells. It is
often thought that membrane-anchored EGFR ligands are able
to interact with the EGF receptor (EGFR) located on an adja-
cent cell, thus acting as a juxtacrine growth factor (9, 71). This
property of EGFR ligands has been clearly demonstrated for
both TGF-␣ (69 –71) and human HB-EGF (3, 68), whose mem-
brane-anchored precursors are known to be rapidly subjected
to prodomain release (50, 66). In contrast, the juxtacrine activ-
ity of the membrane-anchored mouse HB-EGF precursor is
greatly reduced because of a decreased maturation of its prodo-
main (68). The physiological significance of the N-terminal
processing still remains unknown. However, prodomain re-
lease leads to the exposure of the growth factor moiety to the
cellular environment, thus potentially facilitating its interac-
tion with the EGFR on an adjacent cell. Under these condi-
tions, the observation that both human (48, 67) and rat mem-
brane-anchored pro-EGF (this study) do not undergo any
significant cleavage of their prodomain led us to question
whether it would act as a juxtacrine growth factor. The hypoth-
esis that the EGF precursor may have juxtacrine potency has
not been deduced from co-culture experiments but was inferred
from the ability of both soluble (secreted) pro-EGF (43, 67) and
detergent-solubilized membrane pro-EGF (36) to bind and ac-
tivate the EGF receptor. However, under these two conditions
EGF is freed from the environmental constraint (steric hin-
drance) of the membrane. This would be sufficient to allow a
low affinity interaction between the EGF moiety and the EGFR
(high molecular mass pro-EGF is less biologically active than
mature 6 kDa EGF) but it does not necessarily imply that the
native membrane-anchored pro-EGF has this property. To
more definitely answer this question, we think that the results
that could be obtained from co-culture experiments with CHO
cells expressing or not the membrane-anchored rat EGF pre-
cursor and EGFR-expressing cells will be valuable.
We did not observe any precursor form to the 176-kDa rat
pro-EGF species, and in contrast to the results obtained in
MDCK cells, cell surface biotinylation experiments led to the
efficient labeling of the 176-kDa pro-EGF (as well as the 136-
and 111-kDa species). This favors the hypothesis that they
represent mature pro-EGF species that have reached the cell
surface after trafficking through the cell. Thus, in CHO as in
NIH 3T3 cells, intracellular post-translational maturations
and trafficking of EGF must be much more rapid than in
45266 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
MDCK, leading to an increased steady-state level of the mature
precursor at the cell surface.
As found previously with NIH 3T3 and MDCK cells, pro-
EGF-expressing CHO cells release high molecular mass EGF
into the extracellular medium. Secreted pro-EGF species (one
major 165 kDa and minor 121 and 99 kDa species) are shorter
in size and appear to directly derive from their membrane
counterpart (respectively, the major 176 kDa and minor 136
and 111 kDa form). Because of the great similarity in the
molecular size of cellular and secreted pro-EGF in NIH 3T3,
Mroczkowsky et al. (67) suggested that soluble pro-EGF might
be the consequence of an inefficient stop-transfer sequence.
Under these conditions, the non-inserted precursor may repre-
sent the secreted form. However, we can rule out this hypoth-
esis since, at least in CHO cells, secreted pro-EGF lacks the
intracellular Myc epitope indicating that it has been cleaved at
its C-terminal domain. These shorter soluble species do not
appear to be synthesized in this state since they were not
observed in cell lysates. Nevertheless, we cannot exclude that
intracellular processing of the C-terminal domain might lead to
pro-EGF species immediately released into the extracellular
medium. However, the fact that following cell surface biotiny-
lation, pro-EGF released into the extracellular medium is bi-
otinylated does not favor such an interpretation. To our knowl-
edge, this is the first direct evidence that part if not all of the
secreted pro-EGF arise from the proteolytic cleavage of mem-
brane-anchored pro-EGF exposed at the cell surface. The de-
termination of the exact cleavage site was not in the scope of
this study. However, the size decrease between the different
cellular and soluble pro-EGF species was comprised between
11 and 15 kDa. This is very close to the predicted value (13–14
kDa) when making the hypothesis that cleavage of cellular
pro-EGF occurred in the extracellular juxtamembrane domain.
Thus, our experimental results, if they do not directly demon-
strate the juxtamembrane cleavage of membrane-anchored
pro-EGF, significantly favors this hypothesis.
The regulated ectodomain shedding of EGFR ligands such as
TGF-␣, HB-EGF, and amphiregulin is now clearly established.
It can be triggered by different agents such as PMA (through
PKC activation), calcium ionophore (calcium influx), serum, or
pervanadate (tyrosine phosphatase inhibition) (9, 23, 25, 31,
50 –53) and inhibited by zinc-dependent metalloprotease inhib-
itors (24, 25, 28 –31). Concerning EGF, its regulated shedding
from cell surface is not so clearly established and experimental
results are sparse and controversial. To our knowledge, only
two studies have addressed this question. In an extensive study
performed with MDCK cell line transfected with a full-length
human pro-EGF cDNA-encoding plasmid, the authors failed to
observe any stimulation of pro-EGF ectodomain cleavage and
particularly upon phorbol ester (PMA) or serum treatments
(48). However, in this cellular model, the constitutive basolat-
eral and apical secretion of pro-EGF is partially sensible to the
metalloprotease inhibitor batimastat. In a second study per-
formed with a human mammary gland cell line (HB2) trans-
fected with a truncated human pro-EGF cDNA construct lack-
ing most of the prodomain region, EGF secretion was found to
be stimulated by a calcium ionophore (A23187) and a tyrosine
phosphatase inhibitor (phenylarsine oxide) but not by phorbol
esters (49). The metalloprotease inhibitor BB-2116 inhibited
the calcium ionophore-stimulated EGF release.
With regard to the above results, our finding that full-length
rat pro-EGF ectodomain shedding was regulated in CHO-K1
was surprising. As for other EGFR ligands, PMA, serum, and a
tyrosine phosphatase inhibitor stimulated shedding. However,
whereas CHO cells expressing TGF-␣ showed an increased
shedding upon calcium ionophore treatment (A23187, see Ref.
50), we did not find any stimulatory effect using two different
calcium ionophores, namely A23187 and ionomycin. Addition-
ally, we found that, as well as for TGF-␣, both constitutive and
regulated pro-EGF ectodomain shedding was sensitive to zinc-
dependent metalloprotease inhibitors (phenantrolin and TAPI-
2). Moreover, the PMA-stimulated pro-EGF ectodomain shed-
ding was very sensitive to inhibition by the metalloprotease
inhibitor TAPI-2. The IC50 value found in our study (0.1 ␮M)
was 10 –100 times less than those currently reported for the
inhibition of membrane protein shedding (23, 25). The key role
played by a metalloprotease in both constitutive and regulated
pro-EGF shedding was also deduced from secretory experi-
ments performed with the mutant CHO-M2 cell line. CHO-M2
were initially selected for their defect in TGF-␣ shedding but
were subsequently shown to be defective in the metalloprotease-
dependent shedding of many cell surface proteins (25, 28, 51).
A defect that can now be extended to EGF since both constitu-
tive and regulated pro-EGF secretion was completely impaired
in the CHO-M2 cell line compared with CHO-wt.
We do not have any clear explanation for the inability of
MDCK cells to secrete pro-EGF in a regulated manner (48).
However, since intracellular maturation and/or traffic ap-
peared to be limiting steps in the course of EGF secretion, an
explanation could be that a too low amount of steady state level
in cell surface pro-EGF would preclude any detection of cell
surface precursor cleavage regulation. In addition, the consti-
tutive metalloprotease activity of the basolateral membrane
that prevents accumulation of pro-EGF in this cellular domain
may be already too high to be further increased. At last, the
possibility that the regulated metalloprotease that cleaves pro-
EGF in CHO cells may not be present or correctly located
nearby its substrate in MDCK cells cannot be ruled out. This
hypothesis may also be applied to HB2 cells since they ap-
peared to have different possibility to regulate pro-EGF shed-
ding (49). As was stated by the authors, based on a differential
sensitivity of proHB-EGF and pro-EGF shedding to PMA and
calcium ionophore, different metalloproteases may be involved
in the cleavage of EGF versus HB-EGF. Our results demon-
strate that CHO cells cleave pro-EGF in a PMA but not calcium-
dependent manner whereas HB2 cells exhibit the opposite reg-
ulation. As stated above, the difference between the two cell
lines may come from the involvement of different proteases.
This discrepancy may not necessarily imply different protease
populations in the two cell lines, but could be the result of
differential recruitment of specific proteases because of the
different type of substrates involved in the shedding event. The
deletion of the prodomain would allow the interaction of the
substrate with one type of metalloprotease that can be acti-
vated by the calcium-dependent pathway whereas the full-
length substrate would interact with a metalloprotease acti-
vated through the PMA/PKC pathway. So, in addition to the
role that can be played by the stalk region, comparison of the
results of both studies suggests that the prodomain of pro-EGF
may contribute to the specificity of the interaction between the
substrate and the protease(s).
An increasing amount of experimental evidence suggests
that the ADAM protease TACE may be the sheddase of TGF-␣
(24, 25, 28), HB-EGF (29), and amphiregulin (29, 31). However,
the recent demonstration of the, in vitro, inability for purified
TACE to cleave synthetic peptide presenting the cleavages
sites of human pro-EGF (both N- and C-terminal) (29) would
argue against a role for TACE as the pro-EGF sheddase and
would favor the possibility that different metalloproteases
could be involved in the release of EGF versus TGF-␣. Our
demonstration of the inability of HeLa cells to shed EGF from
the cell surface in a regulated manner, whereas this cell line
 Metalloprotease-dependent Regulated Pro-EGF Ectodomain Shedding
appears to contain high amounts of TACE and secrete TNF-␣
also argue against a role for TACE in regulated pro-EGF se-
cretion. This conclusion was reinforced by the observation that
wild-type CHO cells we used in this study do efficiently regu-
late pro-EGF ectodomain shedding whereas they contain low
amounts of TACE and are not able to regulate TNF-␣ secretion,
and that TIMP-3, which is known to be a potent inhibitor of
TACE (72), failed to inhibit pro-EGF secretion. Borroto et al.
(73) recently showed that the defect in ectodomain shedding of
the mutant CHO-M2 cells was the consequence of a defective
removal of the prodomain that keeps TACE in an inactive form
in the early secretory pathway. Thus, our results obtained with
this mutant CHO cells are in favor of a pro-EGF shedding
controlled by TACE. However, despite their control experi-
ments that show a normal processing for some other metallo-
proteases involved in activated ectodomain shedding, it is pos-
sible that the M2 cells are not only deficient in TACE activity
but also in an other unsuspected ADAM or zinc metallopro-
tease-related activity, which will be responsible for the con-
trolled shedding of pro-EGF. We tried to obtain more direct
evidences about the involvement of TACE in pro-EGF shedding
using fibroblasts expressing an inactive form of TACE (Tace
⫺/⫺ cells). Unfortunately, we failed to obtain any expression of
pro-EGF in this cell line despite the fact that we tested many
different transfection protocols and were able to transfect a
GFP-containing expression plasmid.
As was demonstrated previously, shedding of TGF-␣, HB-
EGF, and amphiregulin precursors are regulated phenomena
that can be triggered by PMA (through PKC activation), cal-
cium ionophore (calcium influx), serum, or pervanadate (tyro-
sine phosphatase inhibition) (9, 23, 25, 31, 50 –53). We have
been able to clearly demonstrate that pro-EGF shedding is also
increased upon PMA, serum, and pervanadate treatment.
However, comparison of the effect of specific PKC inhibitors
and PKC depletion on the different ways of stimulation indi-
cates that different intracellular pathways may be involved. In
fact, the PMA stimulation is completely dependent on the ac-
tivation of some PKC activities whereas both serum and per-
vanadate stimulation are not. The fact that a specific PKC can
be involved in the PMA stimulation of pro-EGF shedding can-
not be evidenced here but a study on proHB-EGF suggests that
PKC-␦ was the target for the phorbol ester-induced proHB-
EGF shedding (30). It has been recently suggested that the
MAP kinase signaling cascade could be involved in the regula-
tion of the ectodomain shedding of both TGF-␣ (53) and HB-
EGF (66). Our preliminary results suggest that it may be also
involved in the regulation of pro-EGF shedding activated by
PMA, serum, and pervanadate. Since both PKC-dependent and
PKC-independent pathways can activate the MAP kinase path-
way, MAP kinases such as ERK1/2 may be the target of these
different types of stimulation. The recent discovery that ERK
was able to phosphorylate the cytoplasmic tail of TACE at a
specific site strongly suggests that it may modulate TACE
activity through serine phosphorylation (74), and potentially
act in the same way to regulate other metalloproteases.
In this study, we have clearly demonstrated that as other
members of the EGFR ligand family, when expressed in CHO
cells, EGF is synthesized as a membrane-anchored precursor
present at the cell surface. The entire ectodomain of the pre-
cursor can be shed from the cell through a proteolytic cleavage
that involved a zinc-dependent metalloprotease activity that
appears not to be TACE but can be activated by both PKC-de-
pendent and -independent pathways. However, these experi-
mental results obtained with cultured cells, are in conflict with
those obtained with membrane fractions of both kidney (45, 46)
and mammary gland (47). Indeed, these preparations contain a
45267
membrane bound proteolytic activity that colocalizes with pro-
EGF. However, this activity, which is able to release soluble
EGF from its membrane-associated precursor, belongs to the
serine protease family and not to the metalloprotease one. In
order to resolve such a discrepancy, the establishment of a
more physiologically relevant cells line from organ cells, which
naturally secrete EGF appears necessary and is now under
investigation.
Acknowledgments—We thank Dr. F. Peiretti from INSERM (EPI
99 –36), Marseille, France for providing us with the human TNF-␣
expression plasmid, Dr. Helene Jammes, from INRA Jouy en Josas,
France for providing us with the CHO-K1 cell line, Dr. K. Benihoud
from CNRS Orsay, France for the HeLa cell line, and Dr. J. Arribas
from Barcelona, Spain for the mutant CHO-M2 cell line. We particu-
larly thank Jocelyne Dujancourt for expert technical assistance.
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