* Departments of Medicine and Cell Biology, Vanderbilt University School of Medicine and Veterans Affairs Medical Center,
Nashville, Tennessee 37232-2279; and Department of Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa
920-02, Japan
EGF precursor (proEGF) is a member of the
family of membrane-anchored EGF-like growth factors
that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor- precursor (proTGF
), which is
sorted basolaterally in Madin-Darby canine kidney
(MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878-16889), we now demonstrate
that human proEGF overexpressed in MDCK cells is
found predominantly at the apical membrane domain
under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical
and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD
EGF form into the medium. Unlike the fate of TGF
in
MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR,
and is not processed to the mature 6-kD peptide. We
show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface
than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically
inhibited the release of soluble 170-kD EGF into the
apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of
185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that
preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel
mechanism to achieve a polarized distribution of cell
surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and
TGF
in polarized epithelial cells offer a new conceptual framework to consider the actions of these
polypeptide growth factors.
EGF is the prototypical member of the EGF-like family of growth factors that display high-affinity binding for the EGF receptor (EGFR).1 Other mammalian EGF-like ligands include transforming growth factor- Structural and functional characteristics of the EGF
precursor (proEGF) distinguish it from other EGF-like
growth factors. First, human proEGF is synthesized as a
very large membrane-anchored precursor of 1,207 amino
acid residues, whereas the other, smaller EGF-like growth
factor precursors range in length from 160 to 252 amino
acid residues (3, 17, 32). Second, it is the only EGF-like
growth factor that contains multiple EGF-like repeats. Nine EGF-like repeats are found in the extracellular domain of proEGF with the soluble, mature 6-kD EGF derived from the most distal EGF repeat, which is positioned
near the transmembrane domain (3, 17, 32). Third, proEGF
has a very restricted expression pattern in vivo compared
to the other, more widely expressed EGF-like growth factors (3, 17, 32). Fourth, a polarized distribution of proEGF
has been demonstrated in the kidney, where it is expressed
exclusively on the luminal surface of epithelial cells in the
distal convoluted tubule (5, 48, 51). Finally, in various
epithelial cell types, differential processing of proEGF has
been demonstrated to release different soluble forms of
EGF. In the salivary gland, mature 6-kD EGF is secreted
after intracytoplasmic proteolytic cleavage by an arginine esterase-like activity (13, 14, 28), whereas in vitro studies using NIH 3T3 cells stably transfected with proEGF have
demonstrated that proEGF is proteolytically cleaved to
release a high-molecular mass 160-kD form (39, 40). Recent in vivo studies indicate that the predominant EGF
species released from most epithelial cells is the high-molecular mass 160-kD EGF, which is found at high concentrations in urine and milk (30, 38, 44). While the biological actions of EGF have been studied extensively (13, 14, 17,
32), these unique characteristics of proEGF suggest that it
may subserve biological functions distinct from those of
mature EGF and the other EGF-like growth factors.
Elucidation of the sorting, processing, and steady-state
distribution of EGF-like growth factors in polarized epithelial cells, which have basolaterally restricted EGFRs,
may provide insight into modes of action of this family of
growth factors. We have previously used the Madin-Darby
canine kidney (MDCK) cell line to investigate molecular
trafficking and processing of human proTGF In the present study, we have examined the biosynthesis, sorting, and processing of human proEGF constitutively expressed in polarized MDCK cells and have identified major differences from the disposition of TGF Reagents and Antibodies
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO)
unless otherwise stated. All cell culture reagents were purchased from
Gibco Laboratories (Grand Island, NY). 35S-Translabel was purchased
from ICN Biomedicals, Inc. (Costa Mesa, CA). NHS-LC-biotin, protein
A-agarose, and streptavidin-agarose were purchased from Pierce (Rockford, IL). All electrophoresis reagents were purchased from BioRad Laboratories (Hercules, CA). N-glycosidase F, endoglycosidase H (endo H),
O-glycosidase, and neuraminidase were obtained from Boehringer Mannheim Corp. (Indianapolis, IN). Recombinant human EGF was purchased
from Collaborative Biomedical Products (Bedford, MA). Batimastat
(BB94), a matrix metalloprotease inhibitor, was kindly provided by Dr.
Peter Brown (British Biotech, Oxford, UK).
EGF antibodies used in this study included a monoclonal antibody to human EGF (mAb-EGF) (57) as well as Ab-1 and Ab-3 purchased from Oncogene Science (Uniondale, NY) and the rabbit polyclonal antibody 889, kindly provided by Dr. Stanley Cohen (Vanderbilt University, Nashville,
TN) (40). Rabbit polyclonal antibody [Ab 1417], which recognizes the amino
acid sequence 1165-1186 within the cytoplasmic domain of human proEGF
(38), was kindly provided by Dr. Barbara Mroczkowski (The Agouron Institute, La Jolla, CA). Monoclonal antibody to human EGFR (mAb-528) was
generously provided by Dr. Hideo Masui (Memorial Sloan-Kettering Cancer
Center, New York). Affinity-purified fluorescein (DTAF)-conjugated and CY3-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA).
Cells and Cell Culture
MDCK strain II cell line was obtained from Enrique Rodriguez-Boulan
(Cornell University Medical College, New York). Cells were grown in
routine cell culture and cultured on Transwell filter chambers (0.4 µM;
Costar, Cambridge, MA) as previously described (16). Transepithelial
electrical resistance was measured to assess the integrity of tight junctions
(16). All experiments were performed when electrical resistance was >200
DNA Transfection and Isolation of Expressing Clones
The wild-type proEGF cDNA isolated from human kidney was kindly
provided by Dr. Barbara Mroczkowski. The proEGF construct lacking the
cytoplasmic domain (1,057 amino acids in length) was generated by PCR
and ended in the sequence . . .LLSLWGAHYY1057.
Stable transfection of wild-type and tail minus proEGF cDNA constructs in pCB6 were performed as previously described (16). In this
study, both pools and individual clones of stably transfected cells were
used. All experiments were performed with transfected cell lines between
passage 5 and 15, and results were verified with two to three different
clonal cell lines for each construct.
Immunofluorescence
Indirect immunofluorescence and laser scanning confocal microscopy
were performed as previously described with some modifications (16). Primary antibodies used for detection of human proEGF were mAb-EGF (2 µg/ml) and polyclonal rabbit antibody Ab-3 (5 µg/ml). For double staining of proEGF and EGFR, mAb-528 (15 µg/ml) was added at the same
time as Ab-3. After washing, appropriate secondary antibodies were
added simultaneously. Laser scanning confocal microscopy was performed using a Zeiss LSM 4 confocal microscope (Thornwood, NY). Confocal images were generated by a Personal Iris graphic workstation (Silicon Graphics, Mountain View, CA) using Vital Images software.
EGF Precursor Immunoprecipitation
Immunoprecipitations from cell lysates of metabolically labeled cells were
performed at 4°C. For cells grown on Transwell filters, cells were washed,
filters were cut out, and cells were lysed as previously described (16). After insoluble material was pelleted, the supernatant was incubated for various times (1-16 h) with mAb-EGF (2 µg/ml lysate), and then immunoprecipitations were performed as previously described (16). To immunoprecipitate EGF species from conditioned media, 2 mM PMSF was added, media was
precleared, and then immunoprecipitations were performed as described
above. All immunoprecipitates were extensively washed and either stored
at Enzymatic digestion of proEGF immunoprecipitations with N-glycosidase F, endo H, O-glycosidase, and neuraminidase were performed using
the manufacturer's protocol.
Metabolic Labeling
All cell cultures were initially grown in DME/FBS. To increase proEGF
expression, all cell cultures were treated with 5 mM sodium butyrate in
DME/FBS for 16 h before labeling unless otherwise stated. In some labeling experiments, basolateral surface EGFRs were down-regulated by addition of 20 nM mAb-528 to the medium for 24 h before labeling cells, as
well as to labeling and chase medium.
For steady-state labeling, cells grown on Transwell filters were washed
with serum-free DME lacking L-methionine and L-cysteine (DME Drug Treatments and Temperature Shift
In metabolic labeling experiments, cells were exposed to either monensin
(1-10 µM) or BFA (1-30 µg/ml) during starvation, labeling, and chase.
For tunicamycin experiments, cells were pretreated with different concentrations of tunicamycin (0.1-5 µg/ml) for 4 h before as well as during starvation, labeling, and chase. For 18°C pulse-chase experiments, cells were
pulse labeled as described above, washed with 18°C medium, and then
chased at 18°C. For 18 to 37°C temperature shift experiments, cells were
pulse labeled as described above, washed with 18°C chase medium, and incubated at 18°C for 1 h. Cells were then washed with 37°C chase medium
and subsequently chased at 37°C.
Cell Surface Immunoprecipitation
Metabolically labeled cells were cooled on ice and then washed four times
with ice-cold PBS+ (PBS-0.1 mM CaCl2 and 1.0 mM MgCl2) containing
0.2% BSA (PBS-BSA). All subsequent steps were performed on ice or at
4°C. Proteins that arrived at the cell surface were detected by addition of mAb-EGF (2 µg/ml in PBS-BSA) to either the apical (500 µl) or basolateral (1.5 ml) compartments at the indicated times and incubated for 30 min with gentle rocking. The compartments not receiving antibody were
filled with PBS-BSA. Cells were then rinsed with PBS-BSA and extensively washed with four changes of PBS-BSA for 1 h. Cell surface and total lysate immunoprecipitations were performed as previously described
(16). A comparison of total and cell surface immunoprecipitates was used
to determine equal loading.
Protease Assay
Metabolically labeled cells were washed three times with ice-cold PBS+
and incubated with protease added to either the apical, basolateral, or
both compartments. For N-tosyl-L-phenylolanine chloromethylketone (TPCK)-trypsin digestion, cells were incubated for 30 min at 37°C with 10 µg/ml TPCK-trypsin or at 4°C with 100 µg/ml TPCK-trypsin. The opposite
compartment received soybean trypsin inhibitor (STI) at 200 µg/ml. After
incubation, cells were washed with PBS+ containing 200 µg/ml STI. For
papain digestion, cells were washed with PBS+, pH 6.8, incubated with papain at 1 mg/ml diluted in PBS+, pH 6.8, for 10 min at 37°C, and then extensively washed with PBS-BSA. Cell surface and total cell lysate immunoprecipitations as well as immunoprecipitations from equivalent TCA
precipitable counts were then performed as described above. For TPCK-trypsin, cells were lysed in lysis buffer containing 200 µg/ml STI.
Batimastat Treatment
Cells grown on Transwells were metabolically labeled for 2 h, washed, and
then incubated with serum-free chase medium containing different concentrations of batimastat (0.1-10 µM in DMSO) added to both compartments. Control cells received serum-free chase medium containing DMSO
alone. After chase, cells and conditioned media were collected and immunoprecipitations performed as described above.
Biosynthesis and Processing of ProEGF in MDCK Cells
To investigate the biosynthesis and processing of proEGF,
we performed pulse-chase experiments in MDCK cells
stably transfected with human proEGF. A schematic
model depicting the different cellular proEGF and soluble
EGF species identified is shown in Fig. 8. Cells grown on
Transwell filters were metabolically labeled with 35S-Translabel for 20 min and then chased for different periods of time. Total lysates were prepared, immunoprecipitated with mAb-EGF, and then subjected to SDS-PAGE
and fluorography. At 0 min of chase, a single intense band
of 175 kD was detected (Fig. 1 A). The intensity of the
175-kD band slowly diminished with a half-life of ~2 h. A
second, more diffuse band of 185 kD appeared by 1 h
chase (in other experiments, it was detected as early as 40 min), and it also decreased with time with a half-life of ~2 h.
Interestingly, the decrease in intensity of cellular 175-kD proEGF did not coincide with an equivalent increase in
the appearance of cellular 185-kD proEGF. The detection
of a soluble, high-molecular mass 170-kD EGF species in
the conditioned medium followed the appearance of 185-kD
proEGF form, suggesting that the 185-kD form may be
processed into the soluble 170-kD EGF (Fig. 2 and Fig. 6).
In contrast to the processing of proTGF
In previous studies using mouse NIH 3T3 cells transfected with human proEGF, a single N-glycosylated high-
molecular mass proEGF species was detected in total cell
extracts (39, 40). Our results are consistent with this finding, although the observation of two cellular proEGF
forms (175 and 185 kD) in MDCK cells suggests that some
additional posttranslational modifications occur. To further examine the relationship between the two cellular
proEGF species, pulse-chase experiments with subsequent
N-glycosidase F and endo H digestion were performed.
Both cellular proEGF forms were sensitive to N-glycosidase
F digestion throughout the chase period with the appearance of a single, faster-migrating 165-kD band (Fig. 1 B).
A similar, faster-migrating band of 165-kD was detected
after tunicamycin treatment (data not shown). Only the 175-kD band was sensitive to endo H digestion; it displayed an increase in mobility similar to the 165-kD form
seen with N-glycosidase F treatment (Fig. 1 C). Together,
these results suggest that the predominant cellular species
is the immature, endo H-sensitive, 175-kD proEGF form,
which does not efficiently chase into the more mature,
endo H-resistant, 185-kD proEGF form.
185-kD ProEGF Is Proteolytically Cleaved at the
Cell Surface to Release a Soluble 170-kD EGF into
the Medium
To further examine the relationship between processing of
cellular proEGF and the soluble, high-molecular mass
170-kD EGF detected in the conditioned medium, cells
grown on Transwell filters were metabolically labeled with
35S-Translabel for 2 h and then chased for 4 h to accumulate sufficient levels of soluble 170-kD EGF in the medium. Apical and basal conditioned medium were immunoprecipitated using antibodies directed to either the
proEGF extracellular domain (mAb-EGF) or cytoplasmic
domain (Ab 1417). Both proEGF domain antibodies
readily detected the two cellular proEGF forms (175 and
185 kD) in the total lysates, indicating that both cellular
forms contain the cytoplasmic domain (Fig. 2 A). In contrast, only the extracellular domain antibody was able to
immunoprecipitate the soluble 170-kD EGF species from the medium (Fig. 2 A). This result argues that the cellular
proEGF form(s) must be proteolytically cleaved within
the ectodomain to release the soluble 170-kD EGF into
the medium. This data is in agreement with previous findings of Mroczkowski and colleagues that demonstrated that
the majority of proEGF was released as a high-molecular
mass (~160-kD) species (38).
The biochemical properties of soluble, high-molecular
mass 170-kD EGF of being N-glycosylated but resistant to
endo H digestion (data not shown) are consistent with it
being cleaved specifically from the mature, endo H-resistant,
185-kD proEGF form. Using cell surface biotinylation, it
was not possible to determine if cleavage occurred at the
cell surface because of inefficient labeling of proEGF (data
not shown). In an attempt to further define whether 185-kD proEGF processing occurred at the plasma membrane,
we performed pulse-chase experiments in the presence of
different agents that block transport in the ER-Golgi-
plasma membrane pathway (monensin, BFA, and 18°C
temperature shift) (Fig. 2 B). First, we examined the role
of 18°C temperature block, which detains proteins in the
TGN, preventing their delivery to the plasma membrane
(34). In pulse-chase experiments, cells were pulse labeled
for 20 min at 37°C and chased at 18°C for 2 h or placed at
18°C for 1 h and temperature shifted to 37°C and chased
for 2 h. After the 2-h chase, cell lysate and conditioned
medium immunoprecipitations were performed. In the
18°C chase experiment, the 175-kD proEGF form was
readily detected in cell lysates throughout the chase. By
contrast, the 185-kD proEGF was never detected in cell lysates, indicating that its progression in the secretory pathway had been blocked at some point before or at the TGN
(Fig. 2 B). No soluble 170-kD EGF was detected in the
conditioned medium under these conditions. Pulse-labeled
cells that had been blocked for 1 h at 18°C and then shifted
to 37°C for the chase demonstrated kinetics of appearance
similar to the 185-kD form as seen in control pulse-chase experiments (data not shown, Fig. 4 B). At 2 h chase, both
185-kD proEGF and soluble 170-kD EGF were detected in
the cell lysate and in the conditioned medium, respectively.
Similar results were obtained in pulse-chase experiments in the presence of either monensin or BFA. The
175-kD proEGF was detected in total cell lysates, but expression of both the cellular 185-kD proEGF and the soluble 170-kD EGF in the medium were completely inhibited
by both drugs (Fig. 2 B). Together, these results suggest that the appearance of the endo H-resistant, 185-kD
proEGF form in a post-Golgi/plasma membrane compartment is required for its cleavage to release soluble 170-kD
EGF into the conditioned medium.
185-kD ProEGF Is Localized Predominantly to the
Apical Membrane Domain in Polarized MDCK Cells
To determine the cell surface distribution of proEGF in
polarized epithelial cells, MDCK cells expressing wild-type proEGF were grown on Transwell filters and examined by indirect immunofluorescence and confocal microscopy. ProEGF was localized primarily to the apical surface
of MDCK cell monolayers (Fig. 3 A). In pooled transfectants expressing very high levels of proEGF, it was also
possible to detect some proEGF staining in the lateral
membranes and to a lesser extent in the basal portion of
the basolateral membrane domain (Fig. 3 B). It must be
emphasized, however, that under all culture conditions,
proEGF was localized predominantly to the apical membrane. This is consistent with in vivo observations that
showed that proEGF is localized at the luminal surface of epithelial cells in the distal convoluted tubule of the kidney (5, 48, 51).
To biochemically characterize the apical/basolateral membrane distribution of proEGF in MDCK cells under steady-
state conditions, we used metabolic labeling combined
with cell surface immunoprecipitation. Cells grown on Transwell filters were metabolically labeled with 35S-Translabel
for either 8 or 24 h, and then cell surface immunoprecipitations were performed using an ectodomain antibody
(mAb-EGF). In agreement with the immunofluorescence
results, these studies suggested that the 185-kD cell surface
proEGF is localized primarily on the apical surface (Fig. 4
A). In both 8- and 24-h labeling experiments, the percentage
of 185-kD proEGF expressed on the apical surface was
>85%.
Results from cell surface immunoprecipitation and antibody competition experiments (data not shown) also suggested that 175-kD proEGF form was expressed at the
cell surface. This was a surprising finding since the endo
H-sensitive, 175-kD proEGF represents an immature, intracellular form of proEGF (see Fig. 8). One possible explanation for this result is that the integrity of the plasma membrane was perturbed, allowing access of EGF antibodies to intracellular proEGF forms. To address this possibility, we first demonstrated that MDCK cells expressing
proEGF were structurally and functionally polarized as
determined by polarized distribution of both apical (gp135)
and basolateral cell surface proteins (E-cadherin, Na+K+-ATPase and EGFR) (data not shown; Fig. 4 A). Next, we
used an antibody directed against the cytoplasmic domain
of proEGF (Ab 1417), which should not be able to access
and therefore bind proEGF by cell surface immunoprecipitations. Using Ab 1417, no proEGF forms were detected
in cell surface immunoprecipitations, but both cellular
proEGF forms were readily immunoprecipitated from total
cell lysates (Fig. 4 A). This result indicates that the integrity of the plasma membrane had been maintained throughout the cell surface immunoprecipitation procedure.
To confirm directly that the endo H-resistant, 185-kD
proEGF, and not the endo H-sensitive, 175-kD proEGF,
is the authentic cell surface form, we used a protease assay
that did not rely on an antibody immunoprecipitation procedure to detect cell surface proEGF forms. In this assay,
cells were metabolically labeled for 20 min and then
chased for various times. Cells were then treated with protease to cleave labeled proEGF forms that had arrived at
the cell surface. As shown in Fig. 4 B, 185-kD proEGF was
efficiently digested from both the apical and basolateral cell surfaces (~65-95%), whereas 175-kD proEGF was resistant (<10%) to proteolytic cleavage. To validate the
cell surface protease assay, we have demonstrated that the
175-kD proEGF is sensitive to protease digestion under
different experimental conditions (data not shown). To
further confirm that proEGF cleavage had occurred, total
lysates were immunoprecipitated with the antibody directed against the cytoplasmic domain of proEGF (Ab
1417). This antibody not only recognizes proEGF forms
but can detect a 30-kD cleavage product that represents
the transmembrane and cytoplasmic domains of proEGF
left within the cell after cell surface proteolytic digestion
(see Fig. 8). The 30-kD cleavage product was only detected when 185-kD proEGF was expressed at the cell surface (Fig. 4 C). Based on these experiments, we believe
that the immature, endo H-sensitive, 175-kD proEGF is
artifactually detected in the cell surface immunoprecipitation procedure because of either antibody exchange or unbound antibody binding to 175-kD proEGF after cell lysis.
In all subsequent studies, the cell surface proEGF form refers only to the 185-kD form, but results obtained with the 175-kD proEGF form are included for all experiments.
Newly Synthesized ProEGF Is Delivered Directly to
Both Apical and Basolateral Membrane Domains
To study direct delivery of newly synthesized proEGF to
the different cell surface domains in polarized MDCK
cells, we performed pulse-chase experiments combined
with cell surface immunoprecipitations. As shown in Fig. 5
A, 185-kD proEGF was delivered directly to both apical
and basolateral cell surfaces. The 185-kD proEGF appeared at the cell surface after 40-60 min of chase and was
maximally expressed by 2 h of chase (Fig. 5 A). Quantitation of 185-kD proEGF arrival to the cell surface indicates
that it is delivered in a nonpolarized fashion with an apical/basolateral ratio of 55:45.
To further verify the nonpolarized delivery of newly
synthesized proEGF to the cell surface, we performed
pulse-chase experiments combined with cell surface protease digestion. In this assay, 185-kD proEGF would be
accessible to protease digestion upon arrival at the cell
surface. If protease digestion of cell surface proEGF occurred, it would result in the appearance of the 30-kD
cleavage product in total lysates. In agreement with the kinetics for the cell surface arrival of 185-kD proEGF, the
30-kD cleavage product was not observed at early chase
times but was readily detected after 1 h of chase (Fig. 5 B).
Quantitatively, the 30-kD cleavage product appeared
equally after protease digestion of either the apical or basolateral cell surfaces (Fig. 5 C). In these protease experiments, cell integrity and polarity were maintained as measured by control cell surface proteins (data not shown).
This is further supported by the fact that protease treatment of both compartments had an additive effect on both
the loss of cell surface 185-kD proEGF and appearance of
the 30-kD cleavage product (Fig. 4). Taken together, these
results indicate that 185-kD proEGF is delivered in a nonpolarized manner in MDCK cells.
ProEGF Accumulates at the Apical Membrane Domain
The fact that newly synthesized proEGF is not sorted in
MDCK cells implies that some other mechanism is involved in establishing and maintaining the apical steady-state distribution of proEGF in polarized MDCK cells. To
determine the fate of proEGF at the apical and basolateral
membrane domains, we performed metabolic labeling
studies combined with cell surface immunoprecipitations. In these experiments, cells were labeled for 2 h and then
chased for long time periods. Cell surface immunoprecipitations, as well as apical and basal conditioned medium immunoprecipitations, were performed. The 185-kD proEGF
was expressed on the apical and basolateral membrane domains at 0 min of chase but was rapidly lost from the basolateral surface with a half-life of less than 30 min. In contrast, proEGF had relatively stable expression on the
apical cell surface during the first 2 h of chase and then
was slowly removed from the apical surface with a half-life
of 4 h (data not shown). A soluble 170-kD EGF form was
immunoprecipitated from both apical and basal conditioned medium (Fig. 6 A). Quantitatively, the rate of soluble 170-kD EGF release was more than fourfold greater in basal medium as compared to apical medium (Fig. 6 B)
and coincided with a similar rate of loss of proEGF from
the basolateral membrane domain. The steady-state, polarized distribution of proEGF at the apical membrane domain in MDCK cells observed by indirect immunofluorescence (Fig. 3) can therefore be attributed, at least in part,
to the increased rate of cleavage of proEGF from the basolateral cell surface.
Metalloprotease Inhibitor Batimastat Inhibits
Ectodomain Cleavage of ProEGF
If our hypothesis that preferential basolateral proteolytic
activity plays a role in maintaining the polarized distribution of proEGF in MDCK cells is correct, then inhibition
of ectodomain cleavage should prevent the polarized release of soluble 170-kD EGF into the basal medium and
increase levels of proEGF at the cell surface. In addition,
increased levels of cell surface proEGF at each domain
should inversely reflect rates of cleavage at the two membrane domains. In preliminary studies, we attempted to either stimulate or inhibit proEGF ectodomain cleavage under a variety of experimental conditions. We found that
endogenous proEGF proteolytic activity was not stimulated by serum or phorbol ester (PMA) and was resistant
to a wide variety of protease inhibitors. However, it was
sensitive to both EDTA and EGTA and was temperature dependent (data not shown). Because of the sensitivity of
proEGF cleavage to EDTA/EGTA and the recent findings that metalloproteases may be involved in the processing of HB-EGF (31) and proTGF
Fundamental to understanding the physiological roles of the
EGF family of growth factors is defining their unique biological functions. MDCK cells offer an excellent in vitro model
system to study the expression of EGF-like growth factors
and their interactions with basolateral EGFRs in polarized
epithelial cells. In the present study, we demonstrate that
the biosynthesis, sorting, and processing of proEGF in polarized MDCK cells differ significantly from proTGF Ectodomain Cleavage of ProEGF Occurs at the
Plasma Membrane
The membrane-anchored forms of two EGF-like ligands,
proTGF ProEGF Is Not Sorted in MDCK Cells but Accumulates
at the Apical Cell Surface
Establishment and maintenance of the polarized distribution of membrane proteins in epithelial cells is regulated
by several mechanisms. In MDCK cells, proteins can be
sorted directly from the TGN to the apical and basolateral
membrane domains (18, 37, 49, 55). For several basolateral membrane proteins, sorting signals have been identified in the cytoplasmic domain. Some but not all of these
sequences overlap with endocytic motifs. Apical sorting
signals are less well defined. Sorting information is conferred by a glycosyl-phosphatidyl-inositol (GPI) anchor for one specific group of apical membrane proteins; for
non-GPI-anchored proteins, apical sorting information
may be located in the extracellular domain and involve
posttranslational modifications, such as N-glycosylation (18, 20, 37). Other membrane proteins may not be sorted preferentially but may exhibit cell surface selectivity because of differential residency times. For example, both
Na+K+-ATPase and E-cadherin can be retained selectively at the basolateral membrane by interactions with
the basolateral membrane cytoskeleton (23, 35, 56). Similarly, a subunit of the rat epithelial (amiloride-sensitive)
Na+ channel may localize to the apical membrane because
of interactions of the SH3 domain within its cytoplasmic
domain and Our results provide another mechanism by which a polarized distribution of membrane proteins in epithelial
cells can be achieved. Newly synthesized proEGF is delivered equally to both apical and basolateral membrane domains (Fig. 5), indicating that proEGF is not sorted at the
level of the TGN. If proEGF is not sorted, how is its apical
localization established and maintained in MDCK cells?
The more rapid disappearance of proEGF from the basolateral cell surface combined with a concomitant accumulation of high-molecular mass, soluble 170-kD EGF in the
basal medium (Fig. 6) indicates an increased level of proteolytic activity at the basolateral membrane domain. We
can exclude an interaction between the proEGF cytoplasmic domain and the apical membrane cytoskeleton contributing to this effect since removal of the cytoplasmic domain of proEGF did not affect these results (data not
shown). Based on these results, we submit that differences
in proteolytic activity alter the residency time of proEGF
at the two membrane domains. Under steady-state conditions, this would be reflected as an enrichment of proEGF
at the apical cell surface. Thus, differential processing at
apical and basolateral cell surfaces offers a novel mechanism to maintain a polarized distribution of proteins at the
plasma membrane. A schematic model of preferential basolateral cleavage of proEGF in polarized MDCK cells is
shown in Fig. 8.
Ectodomain Cleavage of ProEGF Is Sensitive to a
Metalloprotease Inhibitor, Batimastat
Many membrane-anchored proteins undergo limited proteolysis to release ectodomains into the extracellular medium (1, 19). Ectodomain cleavage can also be stimulated
by the protein kinase C activator, PMA, for some cell surface proteins, including membrane-anchored forms of proTGF Our initial studies to characterize the proEGF proteolytic activity demonstrated that proEGF proteolytic
cleavage was not activated by serum or phorbol esters but
was sensitive to EDTA and EGTA and was temperature
dependent (data not shown). The inability to stimulate
ectodomain cleavage of proEGF by various other activators meant that our analysis of potential inhibitors was dependent on the slow basal rate of proEGF cleavage with a
half life of ~2 h. This contrasts to PMA-activated release
of proTGF Using batimastat to inhibit proEGF processing, we demonstrate that preferential basolateral ectodomain cleavage
of proEGF plays an important role in establishing and
maintaining a polarized apical distribution of proEGF in
MDCK cells. Batimastat dramatically inhibited the polarized basolateral release of soluble 170-kD EGF and
caused a concordant increase of proEGF cell surface expression at the basolateral membrane domain. The ability
of batimastat to inhibit apical proEGF cleavage also suggests that some metalloprotease activity is expressed at the
apical membrane domain in MDCK cells. It is also important to note that the increase in proEGF cell surface expression observed at the basolateral membrane domain after batimastat treatment was quantitatively less than
would be predicted from the inhibition of release of high-
molecular mass 170-kD EGF into the basolateral medium.
This difference may be due, in part, to the experimental
procedures used which relied on the spontaneous, basal
rate of proEGF cleavage and a 4-h chase to accumulate
high-molecular mass 170-kD EGF in the medium. We therefore cannot rule out the possibility that upon inhibiting the cleavage of cell surface proEGF, the cell surface
fate and steady-state distribution of proEGF may be influenced by other factors such as membrane turnover, recycling, transcytosis, or membrane retention. Interestingly, a
mutant proEGF lacking the cytoplasmic domain was
sorted and cleaved in an identical manner to wild-type
proEGF, suggesting that at least the proEGF cytoplasmic domain is not involved in any of these processes. Despite
these differences, the dramatic inhibition of proEGF release by batimastat strongly suggests a role for a metalloprotease-sensitive pathway in the preferential basolateral
cleavage and apical enrichment of proEGF in polarized
MDCK cells.
How is differential processing of proEGF maintained at
the basolateral membrane domain in MDCK cells? Matrix
metalloproteases are a family of homologous enzymes that
can be distinguished by their substrate specificity and
can be divided structurally into secreted and membrane-
anchored forms (11, 25). Disintegrin-metalloproteases or
adamalysins (54) represent another family of membrane-anchored metalloproteases. A new member of the adamolysin class of metalloproteases is the tumor necrosis factor- Biological Consequences of Differential Sorting
and Processing of ProEGF Compared to ProTGF Dynamic recycling of EGFRs has been demonstrated in
many polarized epithelial cell types. For example, in MDCK
cells overexpressing proTGF What are the biological consequences of proEGF expression at and release from the apical membrane domain? In polarized epithelial cells, apical and basolateral
membrane domains are separated by tight junctions that
create a barrier that prevents diffusion of ions and macromolecules across the monolayer. Consequently, high-
molecular mass EGF released into the apical medium cannot access basolateral EGFRs but rather bathes the apical
cell surface. Similarly, membrane-anchored proEGF enriched at the apical cell surface cannot interact with
EGFRs at the basolateral cell surface. In normal polarized
epithelial cells, this segregation of apical ligand from basolateral EGFRs may be important to limit the capacity of
EGFR signaling. However, in situations where epithelial
cells depolarize, such as during cell transformation,
EGFRs would have direct access to apical ligand, which
may cause enhanced EGFR activation and promote cell
growth.
The exclusive expression of proEGF on the luminal surface of epithelial cells in the distal convoluted tubule of
the kidney (5, 48, 51) and on the luminal surface of alveolar cells in the mammary gland (10, 26), as well as the
release of high-molecular mass proEGF into the luminal
environment as seen in milk and urine (38, 44), suggests
that apical proEGF does perform some relevant biological
function(s). In the gastrointestinal tract, it has been proposed that mature EGF found in the lumen may act as a surveillance factor that monitors the integrity of the epithelial monolayer, and at sites of damage or injury to the
monolayer, luminal EGF would be able to access and activate basolateral EGFRs (46). An analogous scenario could
be envisioned for high-molecular mass 170-kD EGF. Alternatively, high-molecular mass EGF may act as a reservoir
of latent growth factor in the luminal environment, which
upon processing would release mature high-affinity 6-kD EGF. In urine, endoproteases and several kallikreins have
been identified that could subserve this function (33). In
either situation, EGF released into the luminal environment could be presented to distinct populations of EGFRs
possibly far removed from their site of synthesis. Significantly, these EGFR populations would not be accessible
to other basolaterally targeted EGF-like growth factors,
like TGF (TGF
), amphiregulin, heparin binding EGF-like
growth factor, betacellulin and epiregulin (3, 17, 32).
These growth factors are all synthesized as glycosylated,
membrane-anchored precursors that contain at least one
EGF-like repeat in their extracellular domains. A distinctive feature of these membrane-anchored growth factor
precursors is that they are biologically active at the cell
surface, although they can be proteolytically cleaved from
the cell surface to release soluble, diffusible factors (3, 17, 32).
(16). In polarized MDCK cells, newly synthesized human proTGF
is directly delivered to the basolateral cell surface, where it is sequentially cleaved to release mature TGF
into the
basal medium. Overexpression of TGF
did not cause
down-regulation of EGFRs due to the efficient recycling
of EGFRs in polarized MDCK cells. The colocalization of
proTGF
with EGFRs to lateral membranes of polarized
MDCK cells, together with its efficient consumption by
basolateral EGFRs, suggests that TGF
acts only as a basally restricted, locally acting autocrine factor.
.
Under steady-state conditions, 185-kD proEGF is found
predominantly on the apical cell surface. It is not sorted,
but is delivered equally to the apical and basolateral membrane domains. At the cell surface, proEGF is cleaved
proteolytically to release a high-molecular mass soluble
170-kD EGF that accumulates in the extracellular medium
and does not appear to interact with the EGFR. We show
that preferential ectodomain cleavage of proEGF at the basolateral cell surface accounts, in part, for the increased residency time of proEGF at the apical membrane and its
accumulation at that surface. We suggest that this preferential proteolytic activity, which is sensitive to the metalloprotease inhibitor, batimastat, provides a novel mechanism to regulate steady-state distribution of membrane
proteins in polarized MDCK cells.
Materials and Methods
·cm2. To enhance proEGF expression, cells were treated with 5 mM sodium butyrate for 16 h before any experimental procedure.
70°C or directly analyzed under reducing conditions on 7.5% SDS-PAGE. Gels were treated with Amplify (Amersham Corp., Arlington
Heights, IL), fixed, dried, and fluorography was performed. Scanning densitometry was analyzed by the PhosphorImager and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). The specificity of mAb-EGF for
human proEGF precursor and other proEGF immunoreactive species was
confirmed by preincubation of antibody with excess recombinant human
EGF (10 or 100 ng per immunoprecipitate) before the immunoprecipitation procedure.
) and
then incubated for 8-24 h in DME
containing 10% FBS and 100 µCi/ml
35S-Translabel added to the basal compartment. The apical compartment received 1.5 ml of the same medium lacking 35S-Translabel. For pulse and
2-h labeling, cells grown on Transwell filters were rinsed twice with
DME
and then starved for 30 min in the same medium. Cells were then labeled by placing filters on 100 µl of serum-free DME
containing 1-2
mCi/ml 35S-Translabel. 1 ml of the same medium lacking 35S-Translabel
was added to the apical compartment. In pulse-chase experiments, cells
were pulse labeled for the specified time at 37°C and washed once with
18°C chase medium (DME/FBS containing 10 times excess L-methionine
and L-cysteine). Media was then replaced with 37°C chase medium and
cells were chased for various times.
Results
in MDCK cells
(16), no mature 6-kD EGF was detected in cell lysates or
conditioned medium (data not shown).
Fig. 8.
A proposed model
for the establishment and
maintenance of an apical distribution of proEGF in polarized MDCK cells. (A) Biosynthesis and sorting of
proEGF in MDCK cells. 1.
175-kD proEGF represents the endo H-sensitive, intracellular form of proEGF.
175-kD proEGF is modified
during transit through the
secretory pathway to form 185-kD proEGF. 2. Newly
synthesized 185-kD proEGF
is not sorted at the level of
the TGN but is delivered
equally to both apical and
basolateral membrane domains. 3. 185-kD proEGF
represents the endo H-resistant, cell surface form of
proEGF. 4. Ectodomain cleavage of cell surface 185-kD proEGF. 5. 170-kD EGF represents a soluble high-molecular mass EGF obtained by ectodomain cleavage of cell surface 185-kD proEGF. 6. 30-kD cleavage product represents the cytoplasmic and transmembrane domains found after cell surface protease digestion. (B) In polarized MDCK cells, preferential ectodomain cleavage of cell surface proEGF at the basolateral membrane domain leads to an apical enrichment of proEGF. Enhanced basolateral processing also
leads to an accumulation of soluble 170-kD EGF in the basolateral medium. A matrix metalloprotease inhibitor, batimastat, dramatically inhibits processing of 185-kD proEGF at the cell surface.
[View Larger Version of this Image (20K GIF file)]
Fig. 1.
Biosynthesis and processing of proEGF forms in polarized MDCK cells. (A) MDCK cells expressing proEGF were
grown on Transwell filters and metabolically labeled with 35S-Translabel for 20 min. The label was then chased for different periods of time. Total cell lysates were prepared and immunoprecipitated with mAb-EGF. (B and C) Pulse-chase experiments
and immunoprecipitations were performed as described above.
Immunoprecipitates were washed with the appropriate buffers,
divided into equal parts, and digested with either N-glycosidase F
(B) or endo H (C) as described in Materials and Methods.
[View Larger Version of this Image (36K GIF file)]
Fig. 2.
ProEGF is cleaved in its ectodomain to release a high-
molecular mass soluble 170-kD EGF into the conditioned medium. (A) MDCK cells transfected with proEGF were grown on
Transwell filters and metabolically labeled with 35S-Translabel
for 16 h. After labeling, conditioned medium from apical and basolateral compartments was immunoprecipitated with either the
ectodomain (Ecto; mAb-EGF) or cytoplasmic domain (Cyto; Ab
1417) EGF antibodies. Total cell lysates were also immunoprecipitated with these EGF antibodies. (B) Filter-grown MDCK
cells expressing proEGF were metabolically labeled with 35S-Translabel for 20 min. The label was then chased for 2 h. Cells were either untreated (control) or treated with 18°C temperature, 18°C 37°C temperature shift, monensin, or BFA. Monensin (3 µM) and BFA (15 µg/ml) were added during starvation and
throughout the pulse-label and chase. For 18°C temperature experiments, cells were either chased at 18°C or placed at 18°C for 1 h
and then shifted to 37°C and chased for 2 h. Total cell lysate immunoprecipitations were performed using mAb-EGF. Apical
and basolateral conditioned media were collected and immunoprecipitated. Only results of conditioned medium from basolateral compartment are shown.
[View Larger Version of this Image (26K GIF file)]
Fig. 6.
185-kD proEGF is preferentially cleaved from the basolateral cell surface in MDCK cells. (A) MDCK cells transfected
with proEGF were grown on Transwell filters and labeled for 2 h
with 35S-Translabel. Cells were then washed and chased for time
periods indicated. After the chase, cell surface (data not shown)
and media (A) immunoprecipitations were performed using
mAb-EGF. (B) Quantitation of soluble 170-kD EGF released
into apical (open circles) and basolateral (closed circles) medium.
Average of two separate experiments ± SD are shown and expressed in arbitrary units calculated from total soluble 170-kD
EGF released at each time point.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Cell surface 185-kD proEGF is expressed predominantly at the apical membrane domain in polarized MDCK cells.
(A) For cell surface immunoprecipitations using an EGF
ectodomain antibody, MDCK cells expressing proEGF were
grown on Transwell filters and labeled for 8 h with 35S-Translabel
in the presence of 10% FBS. After labeling, cell surface and total
cell lysate immunoprecipitations with the EGF ectodomain antibody (Ecto Ab; mAb-EGF) were performed and subjected to
SDS-PAGE and fluorography. For cell surface immunoprecipitations using EGF cytodomain and EGFR antibodies, cells were
metabolically labeled with 35S-Translabel for 2 h, and then cell
surface and total cell lysate immunoprecipitations were performed using EGF cytodomain (Cyto Ab; Ab 1417) and EGFR
(mAb-528) antibodies, respectively. Protease assay was performed using either TPCK-trypsin (B) or papain (C). MDCK
cells expressing proEGF were grown on Transwell filters, metabolically labeled with 35S-Translabel for 20 min, and then chased
for 2 h to allow labeled proEGF forms to accumulate on the cell
surface. Cells were treated with protease added to either the apical (Ap), basolateral (Bl), or both (AB) compartments. (B) After
protease treatment, cell surface and total cell lysate (not shown)
immunoprecipitations were performed using the EGF ectodomain
(mAb-EGF) antibody. (C) After protease treatment, total lysates
were divided equally and immunoprecipitated with the EGF
ectodomain (mAb-EGF) and cytodomain (1417) antibodies. Apical, basolateral or both membrane domains are represented by
Ap, Bl, and AB, respectively, in this and all subsequent figures.
[View Larger Version of this Image (30K GIF file)]
Fig. 3.
ProEGF is localized predominantly
at the apical cell surface in polarized MDCK
cells. (A) MDCK cells transfected with
proEGF were cultured for 4 d on Transwell filters and stained with polyclonal rabbit EGF antibody (Ab-3). Note intense staining at the
apical membrane domain. (B) A pool of transfected MDCK cells expressing proEGF was
grown on Transwell filters and stained with
polyclonal rabbit antibody to EGF (Ab-3) and
mouse monoclonal antibody to EGFR (mAb-528). ProEGF and EGFR are visualized in red
and green, respectively. Yellow represents
colocalization of both proteins. Arrowheads indicate the basal surface of the monolayer.
[View Larger Version of this Image (50K GIF file)]
Fig. 5.
Newly synthesized 185-kD proEGF is delivered equally
to both apical and basolateral membrane domains in polarized
MDCK cells. MDCK cells expressing proEGF were grown on
Transwell filters and pulse-labeled with 35S-Translabel for 20 min
and then chased for 0, 30, 60, or 120 min as indicated. (A) After
the chase, cell surface and total cell lysate immunoprecipitations
were performed using mAb-EGF. (B) After the chase, cells were
treated with TPCK-trypsin added to either the apical or basolateral compartments. After protease treatment, cell surface immunoprecipitations were performed using the EGF ectodomain
(mAb-EGF) antibody. Total cell lysates were divided equally and
immunoprecipitated with the EGF ectodomain (mAb-EGF) (not
shown) and cytodomain (Ab 1417) antibodies, respectively. (C)
Quantitation of the appearance of 30-kD protease cleavage product after protease digestion of either apical (open circles) or basolateral (closed circles) membrane domains during pulse-chase experiment. The results are expressed in arbitrary units calculated from total amounts of 30-kD at each time point determined after subtracting control values. Average of three separate experiments ± SD are shown.
[View Larger Version of this Image (19K GIF file)]
(2), we next examined
the effects of the metalloprotease inhibitor, batimastat, on
proEGF cell surface cleavage. As shown in Fig. 7 A, batimastat dramatically inhibited the release of soluble 170-kD EGF from both membrane domains. Quantitatively,
batimastat inhibited release of soluble 170-kD EGF into
the apical and basolateral medium by 7 and 60%, respectively (Fig. 7 B). In addition, batimastat caused an increase
in the cell surface expression of 185-kD proEGF at both
membrane domains even though the inhibitor did not
completely block release of soluble 170-kD EGF. Cell surface expression of proEGF increased at the apical and basolateral membrane domains by 150 and 280%, respectively (Fig. 7 C). Taken together, these results support our
contention that preferential basolateral cleavage of proEGF
directly contributes to the polarized steady-state distribution of proEGF in MDCK cells. A model for the maintenance of an apical steady-state distribution of proEGF in
polarized MDCK cells is proposed in Fig. 8.
Fig. 7.
Batimastat inhibits cleavage of proEGF. MDCK cells
expressing proEGF were grown on Transwell filters and labeled
with 35S-Translabel for 2 h and then chased for 4 h in the absence
or presence of 5 µM batimastat. (A) After the chase, media, cell surface, and total cell lysate immunoprecipitations were performed using mAb-EGF. Results for soluble 170-kD EGF released into medium are shown. (B) Quantitation of soluble 170-kD EGF released into apical and basolateral medium. Average
of two separate experiments ± SD are shown and expressed in
arbitrary units calculated from controls that received no batimastat. The values for apical and basolateral compartments are calculated from total soluble 170-kD released. (C) Quantitation of
cell surface 185-kD proEGF expressed on the apical and basolateral membrane domains. Average of two separate experiments ± SD are shown and expressed in arbitrary units calculated from
controls that received no batimastat. The values for apical and
basolateral compartments are calculated separately. Cell surface
values were determined from total lysate immunoprecipitates.
[View Larger Version of this Image (15K GIF file)]
Discussion
and
that these differences provide a conceptual framework to
understand the actions of these polypeptide growth factors.
In addition, we propose that apical enrichment of human
proEGF in MDCK cells involves preferential basolateral processing sensitive to a metalloprotease inhibitor, batimastat.
and proHB-EGF, can be cleaved from the cell
surface in response to the protein kinase C activator, PMA
(8, 22, 31, 41, 42, 47). In most cell types, including MDCK
cells, proTGF
is sequentially cleaved at proximal and distal sites within the extracellular domain to release mature
TGF
. The distal cleavage site is activated by phorbol esters to release soluble growth factor (41, 42). Within the
plasma membrane compartment, proTGF
processing can
be activated independent of cytosol and does not require
membrane traffic (7, 8). In vitro and in vivo studies have
suggested that proEGF is proteolytically cleaved to release a high-molecular mass proEGF form (27, 30, 38,
44). Using domain-specific antibodies in the present study,
we demonstrate that mature, 185-kD proEGF is cleaved
within its ectodomain to release a high-molecular mass,
soluble 170-kD EGF form. Unlike TGF
, proEGF is only
cleaved distally to release high-molecular mass, soluble
170-kD EGF and therefore no mature 6-kD EGF is detected in the conditioned medium. The release of high-
molecular mass proEGF species from purified membranes
of NIH 3T3 cells expressing proEGF and from rat kidney
membranes by autolysis suggested that the proteolytic activity may be membrane associated (40, 44). To determine
the cellular location of proEGF ectodomain cleavage in
MDCK cells, we examined the transport and processing of
proEGF in the presence of 18°C temperature block, monensin, or BFA (12, 29, 34, 45). Using these experimental
procedures to block transport in the ER-Golgi-plasma
membrane pathway, we show that proteolytic cleavage of
185-kD proEGF only occurs in a post-Golgi/plasma membrane compartment.
-spectrin in the apical membrane cytoskeleton (50).
and proHB-EGF (8, 22, 31, 41, 42, 47). Activated
proteolytic cleavage of proTGF
can also be induced by a
variety of other factors, including serum and Ca2+ (8, 42),
and is dependent on the COOH-terminal valine in the cytoplasmic tail (7, 8). Earlier studies on proTGF
processing indicated that certain specific serine protease inhibitors could block PMA-induced cleavage of the proTGF
ectodomain (43). However, more recent data suggests that
these inhibitors block release by interfering with maturation and transport of the protein to the cell surface rather
than by inhibiting proteolytic activity at the plasma membrane (2). The specificity of this proteolytic activity has
only recently been established with the demonstration that
a specific matrix metalloprotease inhibitor, TAPI, can inhibit PMA-induced release of proTGF
(2) and proHB-EGF (31).
, which has a short half-life of 15 min (41, 42).
ProEGF cleavage was not blocked by a variety of specific
protease inhibitors, including pepstatin (5 µg/ml), leupeptin (5 µg/ml), PMSF (2 mM), elastatinal (100 µM), soybean trypsin inhibitor (200 U/ml), TPCK (100 µM), 3,4-DCI (100 µM), and aprotinin (200 KIU/ml) (data not shown). Interestingly, the serine protease inhibitor, aprotinin, which at very high concentrations is reported to inhibit release of EGF in an isolated perfused kidney model
(27), did not block cleavage of proEGF in MDCK cells. In
light of these negative results, together with the sensitivity
of proEGF processing to EDTA/EGTA and the inhibition
of PMA-stimulated processing of proHB-EGF and proTGF
by a matrix metalloprotease inhibitor (2, 31), we examined the effects of a specific metalloprotease inhibitor
on proEGF cleavage. We found that the synthetic matrix
metalloprotease inhibitor, batimastat (15, 21), effectively
inhibited spontaneous cleavage of proEGF at a dose range
of 2-10 µM (data not shown, Fig. 7).
(TNF-
)-converting enzyme, which specifically cleaves the
TNF-
precursor (6, 36). Interestingly, TNF-
-converting
enzyme is inhibited by the metalloprotease inhibitor, TAPI (6), which can also inhibit both PMA-induced and
spontaneous cleavage of proTGF
and proHB-EGF (2,
31). One possibility for the preferential basolateral cleavage of proEGF in polarized MDCK cells therefore is that
the metalloprotease(s) involved also displays a polarized
basolateral distribution. At present, the sorting, membrane distribution, or secretion of matrix metalloproteases in polarized epithelial cells has not been defined. Alternatively, most matrix metalloproteases are synthesized in a
latent form and require cleavage of the amino-terminal
domain to activate the enzyme (11). It is therefore possible
that cofactors involved in regulating the catalytic activity
of the protease may have a polarized distribution. In this
context, it is interesting to note that EGFR activation can
induce ectodomain cleavage of proTGF
(4). In polarized
MDCK cells, EGFRs are expressed on the basolateral membrane domain (16). In this scenario, preferential basolateral cleavage of proEGF would not require the metalloprotease to have a polarized distribution because direct
EGFR activation might lead to localized stimulation of
proteolytic activity at the basolateral cell surface of polarized epithelial cells. Although the exact mechanism(s) involved in preferential basolateral cleavage of proEGF in
polarized MDCK cells awaits identification of the specific metalloprotease(s), the present results do raise the possibility that ectodomain shedding of other membrane proteins may be regulated differently at apical and basolateral
membrane domains in polarized epithelial cells.
in
Polarized Epithelial Cells
, the inability to down-regulate basolateral EGFRs concomitant with the high rate of
TGF
consumption is due to efficient recycling of EGFRs
to the cell surface (16). In fact, immunoreactive TGF
could only be detected in the basolateral medium upon
EGFR antibody blockade (16). Hence, one distinguishing
feature of MDCK cells expressing proEGF is the accumulation of soluble 170-kD EGF in the medium after its release from the cell surface (Figs. 6 and 7). Moreover, antibody blockade of EGFRs did not alter the levels of soluble
170-kD EGF found in the medium (Dempsey, P.J., unpublished observation). Species differences between EGF and
its receptor cannot account for the lack of growth factor
consumption because human and rodent EGF bind with
high affinity to endogenous canine EGFRs on MDCK
cells (9, 16, 24). Although studies using NIH 3T3 cells stably expressing proEGF demonstrated that high-molecular mass EGF released from the cell surface is biologically active (40), more recent studies using both in vivo and in
vitro EGFR kinase activation and mitogenic assays have
shown that soluble, high-molecular mass EGF isolated
from human milk or urine is less biologically active than
mature 6-kD EGF (38, 44). Therefore, it is reasonable to
assume that accumulation of soluble 170-kD EGF in the
basal medium of MDCK cells is a direct result of it being a
low-affinity ligand for the EGFR (38) and once again underscores differences between disposition of EGF and TGF
.
. It is also possible that proEGF may subserve
functions within the apical compartment independent of the EGFR. For example, it has been suggested that
proEGF may act as a receptor (17). Similarly, recent studies with high-molecular mass urinary proEGF indicate
that it may promote cell adhesion (44). To effectively perform these functions, it may be necessary for proEGF to
avoid interactions with the EGFR. Taken together, these
results demonstrate marked differences in the disposition of EGF and TGF
in polarized epithelial cells and provide
new insights into possible distinct actions of these growth
factors.
Received for publication 6 March 1996 and in revised form 14 May 1997.
Address all correspondence to Dr. Peter J. Dempsey, Departments of Medicine and Cell Biology, GI Cancer Program, CC-2218 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2583. Tel.: (615) 343-0171. E-mail: peter.dempsey{at}mcmail.vanderbilt.eduWe thank Drs. Tom Daniels and Christine Saunders, as well as members of the Coffey laboratory, for helpful discussions and critical reading of the manuscript. We would also like to thank Dr. Barbara Mroczkowski, Dr. Hideo Masui, and Dr. P. Brown for supplying reagents used in this study.
This work was supported by National Institutes of Health Grant CA46413 (to R.J. Coffey) and by the Joseph and Mary Keller Foundation. R.J. Coffey is a Veterans Administration Clinical Investigator. Analyses were performed in part through use of the Vanderbilt University Medical Center Cell Imaging Resource (supported by CA68485 and DK20593).
BFA, brefeldin A;
EGFR, epidermal
growth factor receptor;
endo H, endoglycosidase H;
MDCK, Madin-Darby canine kidney cells;
TGF, transforming growth factor-
;
TPCK, N-tosyl-L-phenylalanine chloromethylketone.
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