(Received for publication, October 11, 1996, and in revised form, November 12, 1996)
From the Departments of Anatomy and Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0452
Hepatocyte growth factor (HGF) and E-cadherin are
important for epithelial morphogenetic events. We examined the effects
of HGF on E-cadherin localization and interaction with -catenin in
polarized Madin-Darby canine kidney (MDCK) cell monolayers grown on
filters. Surface biotinylation experiments showed that HGF increases
apically accessible E-cadherin. Confocal immunofluorescence microscopy
of HGF-treated cells showed localization of E-cadherin at membrane
domains contacting the apical compartment and an increase in
accessibility of apically applied antibodies to lateral E-cadherin below the tight junction. Coimmunoprecipitation of
-catenin/E-cadherin complexes showed that the amount of E-cadherin
associated with
-catenin increased during the first 24 h of HGF
treatment with a return to baseline values after 48 and 72 h.
Metabolic labeling showed that HGF increased the synthetic rate of
-catenin and the amount of newly synthesized E-cadherin associated
with immunoprecipitated
-catenin, with the peak effect occurring
after 12 h of treatment and returning to baseline after 24 h.
HGF treatment inhibited transcytosis of immunoglobulin A by the
polymeric immunoglobulin receptor. We conclude that HGF treatment of
polarized MDCK cells grown on filters decreases cell polarity and
alters E-cadherin/
-catenin interaction and synthesis.
Hepatocyte growth factor (HGF)1 is a polypeptide growth factor with pleiotrophic functions which, depending on target cells and tissues and stage of development, can include mitogenesis, cell motility, and the development and regeneration of organs (1). These HGF-induced events are mediated by activation of c-met, the tyrosine kinase receptor for HGF. Two in vitro models used previously for the characterization of motogenic and morphogenic events induced by HGF use the Madin-Darby canine kidney (MDCK) epithelial cell line. In the first model, MDCK cells were grown on impermeant supports as small colonies at low density. When exposed to HGF, these cells assumed a fibroblastoid morphology and scatter away from the colonies (hence the synonym of scatter factor for HGF) (2, 3). In the second model, MDCK cells were grown as hollow cysts in type I collagen gels. When exposed to HGF, they formed complex branching tubules extending out from the cysts, mimicking the normal branching tubule morphogenesis that occurs during the development of many epithelial organs (4, 5).
To analyze directly the effects of HGF on polarized epithelial cell functions that may be important for epithelial cell rearrangements during HGF-induced morphogenesis and motogenesis, we tested the effects of HGF on MDCK cells cultured on permeable supports. MDCK cells cultured on permeable filter supports form well polarized monolayers with apical and basolateral membrane domains separated by a functional tight junction belt (4). This widely used model allows for apical and basolateral surface domain-specific techniques such as surface biotinylation, surface immunolabeling, and surface immunoprecipitation to study cell surface polarity. In epithelial cells, the response to HGF and cell polarity are interdependent. For example, in polarized MDCK cells, the HGF receptor, c-met, is localized to the basolateral cell surface (5), and cells respond to basolateral but not apical HGF.2 However, epithelial cells that are less well polarized, such as MDCK cells cultured on plastic, scatter in response to HGF in the apical medium and acquire a fibroblastic morphology (2, 3). We hypothesized that the permeable filter support model system would be useful for characterizing HGF-induced effects on epithelial cell polarity that might provide insight into more complex HGF-induced epithelial morphogenetic events such as tubulogenesis, organ development, and tissue repair.
These morphogenetic events are likely to involve complex changes in
cell-cell interactions. E-cadherin is a 120-kDa transmembrane protein
that is primarily responsible for homotypic adhesion between adjacent
epithelial cells (6). In well polarized epithelia, E-cadherin is
localized to the basolateral membrane below the tight junction, and the
extracellular domain of E-cadherin is, therefore, inaccessible from the
apical environment. E-cadherin activity is necessary to maintain the
adherens junctions, which are characteristic of polarized epithelial
cells, as well as for the activity of other intracellular junctions
including zonula occludins (tight junctions) and desmosomes (7).
E-cadherin is also believed to play a critical role in morphogenetic
events through the regulation of cell-cell adhesion (8, 9). To exhibit
functional activity, E-cadherin forms complexes with cytosolic proteins
called catenins (,
, and
), which link E-cadherin to the actin
cytoskeleton (10, 11).
- and
-catenin are homologous to
armadillo, a Drosophila segment polarity gene
(12). Both of these catenins have been shown to modulate cell-cell
adhesion (13, 14).
-Catenin is involved in linking membrane proteins to the cortical cytoskeleton at sites of cell-cell contact and is also
required for cell adhesion (15, 16). Because of the importance of
E-cadherin in epithelial structure and its suspected role in
morphogenesis, we have now studied the effect of the HGF on E-cadherin
localization and interaction with
-catenin in polarized MDCK cells
grown on permeable filter supports.
MDCK type II cells as well as MDCK type
II cells expressing the transfected rabbit polymeric immunoglobulin
receptor (pIgR) were grown in MEM containing Earle's balanced salt
solution (Cellgro, Mediatech, Inc., Washington, DC) supplemented with
5% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin,
100 mg/ml streptomycin, and 0.25 µg/ml amphotericin B in 5%
CO2/95% air at 37 °C. Cells grown on Transwells
(Costar, Cambridge, MA) were seeded at confluency. Cell monolayers were
used for experiments after 3 days of culture with daily media change.
Hybridoma cells secreting mouse anti-E-cadherin mAb (rr1), which
recognizes an extracellular epitope, were a kind gift from B. Gumbiner
(Sloan Kettering, New York, NY)(17). Rat mAb R40.76 against ZO-1, a
peripheral membrane protein associated with the cytoplasmic aspect of
tight junctions (18, 19), was obtained from B. Stevenson (University of
Alberta). Mouse mAb against -catenin was obtained from Transduction Laboratories (Lexington, KY). Secondary antibodies for
immunofluorescence were goat anti-mouse-fluorescein-5-isothiocyanate
and goat anti-rat-Texas Red from Jackson ImmunoResearch Laboratories
(West Grove, PA). Recombinant human HGF was generously provided by R. Schwall (Genentech, South San Francisco, CA).
Biotinylation of MDCK cells grown on Transwells was performed essentially as described previously (5). Briefly, cells were washed thoroughly with ice-cold Hank's balanced salt solution, pH 7.4, and the apical or basolateral cell surface was then derivatized twice for 15 min at 4 °C using a sulfonated N-hydroxysuccinimide ester of biotin (Sulfo-NHS-Biotin; Pierce) (0.5 mg/ml in Hank's balanced salt solution). The reaction was quenched by washing three times with ice-cold MEM, containing Hank's balanced salts, 0.6% BSA, and 20 mM HEPES (MEM/BSA), pH 7.3. Cells were then lysed, and the appropriate protein was immunoprecipitated as described below.
ImmunoprecipitationMDCK cells grown on 24-mm Transwells
with or without HGF treatment were rinsed twice with Dulbecco's
phosphate-buffered saline containing Mg2+ and
Ca2+ (PBS+) at 4 °C. Filters were cut from
their support and placed in 0.8 ml of 20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 0.1% SDS, 1% TX-100, 1% droxycholic
acid, and 5 mM EDTA (RIPA buffer) containing inhibitors of
proteases (2 mM phenylmethylsulfonyl fluoride, 50 µg/ml
pepstatin, 50 µg/ml chymostatin, and 10 µg/ml antipain) for 15 min
on ice. Protein concentration of cell lysates was determined by using a
Pierce BCA kit. Cell lysates were rotated 1.0 h at 4 °C with E-cadherin mAb (rr1 hybridoma conditioned media) or -catenin mAb.
Immunocomplexes were collected with affinity-purified rabbit anti-mouse
IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA) coupled to
protein A-Sepharose beads. Immunoprecipitate beads were washed three
times with RIPA buffer, one time with 140 mM NaCl, 20 mM tetraethylammonia chloride, pH 8.6, 5 mM
EDTA, pH 8.0, 0.1% Trasylol, and 0.02% NaN3 (final wash
buffer), and samples were eluted by boiling in Laemmli buffer
containing 100 mM dithiothreitol.
Cells were cultured on Transwell filters and cooled to 4 °C in MEM/BSA and exposed to apical or basolateral rr1 conditioned media for 1 h. After this incubation, cells were quickly washed three times at 4 °C with PBS+ and lysed in RIPA buffer as described in the immunoprecipitation protocol.
Electrophoresis and BlottingImmunoprecipitates were
electrophoresed on 8% SDS-PAGE minigels and transferred to Immobilon P
filters (Millipore Corp., Bedford, MA). Filters containing biotinylated
samples were blocked at room temperature for 60 min with 1 M glucose, 10% glycerol, and 0.5% Tween 20 solution (TGG)
containing 3% BSA and 1% milk. The filter was then washed two times
with PBS containing 0.5% Tween 20 and incubated for
1 h at room temperature with streptavidin-horseradish peroxidase
diluted 1:1000 in TGG and 0.3% BSA. Filters were washed five times for
15 min each with PBS
containing 0.5% Tween 20. Nonbiotinylated filter samples were blocked for 1 h at room
temperature with PBS
, 5% milk, and 0.1% Tween 20 (block
solution) and probed with mAb rr1 conditioned media diluted 1:2 in
block solution or mAb
-catenin diluted 1:5000 in block solution. The
filters were washed five times for 5 min each with PBS
,
0.1% Tween 20 (wash solution), and probed with goat-anti-mouse horseradish peroxidase diluted 1:30,000 in block solution for 1 h.
Filters were washed five times for 5 min each with wash solution. All
filters were visualized on Kodak X-OMAT AR film with an enhanced chemiluminescence kit (ECL, Amersham Corp.). Autoradiographs were scanned and saved as Adobe Photoshop files with a UMAX PowerLook II
scanner. Densitometry was performed using NIH Image Version 1.60 (NIH
Image is in the public domain).
MDCK cells grown on 6-mm Transwell filters were fixed with paraformaldehye. Cells were fixed with ice-cold 4% paraformaldehyde in PBS+ for 20 min. After washing the filters three times with PBS+, the cells were quenched with 75 mM NH4Cl and 20 mM glycine, pH 8.0, with KOH (quench solution) for 10 min at room temperature. Filters were washed one time with PBS+ and permeabilized with PBS+, 0.7% fish skin gelatin, and 0.025% saponin (PFS) for 15 min at 37 °C. Filters were labeled with rr1 or ZO-1 mAb diluted in PFS 1:2 and 1:100, respectively, for 1 h at 37 °C. Filters were then washed four times for 5 min each with PFS at room temperature and then labeled with the appropriate secondary Ab diluted 1:100 in PFS for 1 h at 37 °C. Filters were rinsed four times for 5 min each with PFS, one time with PBS+, 2 times with PBS+ containing 0.1% TX-100, and one time with PBS+. Cells were postfixed in 4% paraformaldehyde for 15 min at room temperature. Filters were cut from the support with a scalpel and mounted in Vectashield mounting medium (Burlingame, CA).
For surface fluorescence labeling, the living cells were cooled to 4 °C on ice and exposed to rr1-conditioned media from the apical or basolateral surface for 1 h at 4 °C. The filters were then washed three times with ice-cold PBS+ and subjected to fixation and fluorescent labeling as described above.
Scanning Laser Confocal Analysis of Fluorescently Labeled CellsThe samples were analyzed using a krypton-argon laser coupled with a Bio-Rad MRC1000 confocal head, attached to an Optiphot II Nikon microscope with a Plan Apo 60 × 1.4 NA objective lens. The samples were scanned individually or simultaneously for fluorescein-5-isothiocyanate or Texas Red with excitation/emission wavelengths of 488/520 and 568/615, respectively. The data were analyzed using Comos software. Images were converted to tagged information file format (TIFF), and contrast levels of the images were adjusted by using the Photoshop program (Adobe Co., Mountain View, CA) on a Power Macintosh 9500 (Apple, Cupertino, CA). Conventional immunofluorescent images were obtained with a Leitz microscope equipped with a 35-mm camera. The Leitz microscope generated photomicrographs were converted to TIFF images with a Polaroid SprintScan 35/ES.
Metabolic LabelingFilter-grown cells were starved in MEM lacking cysteine and methionine (obtained from UCSF Cell Culture Facility and supplemented with 5% fetal bovine serum for 20 min, then pulse-labeled for 15 min in the same medium supplemented with [35S]cysteine and [35S]methionine (250 µCi/ml Tran35Slabel, ICN Biochemicals, Irvine, CA) as described previously (20). 35S incorporation into immunoprecipitated proteins was analyzed by SDS-PAGE and quantitated with a PhosphorImager (Molecular Dynamics).
Transcytosis Experiments with 125I-labeled dIgATranscytosis of dimeric immunoglobulin A (dIgA) assays were performed as described previously (21).
StatisticsData are presented as means ± S.E. Each
experiment was performed at least three times. The paired two-sample
for means t test was used to determine the probability
(p) that the sample means are equal. A p 0.025 was considered to be significant. Statistical analyses were
carried out with Microsoft EXCEL version 5.0a (Microsoft Corp.).
In polarized epithelia, E-cadherin is
basolaterally localized and, therefore, is not accessible to reagents
in the apical environment that cannot cross the tight junction. Because
E-cadherin function is important in cell-cell adhesion during
development, we hypothesized that E-cadherin localization is altered
during HGF-induced morphogenetic events. We, therefore, examined the effect of HGF treatment on the localization of E-cadherin in polarized MDCK cell monolayers grown on filters by domain-selective surface biotinylation. Cells were plated at confluency on 24-mm Transwell filters and cultured for 3 days with daily media changes before introducing recombinant human HGF at a concentration of 100 ng/ml into
the basolateral compartment. After treatment for 24 h, apical or
basolateral membrane domains were surface biotinylated, and cell
membrane proteins were solubilized in RIPA buffer. E-cadherin in the
total cell lysates from individual filters was immunoprecipitated with
anti E-cadherin mAb rr1. Biotinylated E-cadherin was detected on
immunoprecipitate blots by probing with streptavidin-horseradish peroxidase and developed using ECL. The results are shown in Fig. 1 and demonstrate that HGF treatment increased apically
exposed, biotin-derivatized E-cadherin. Total E-cadherin exposed at the basolateral surface was also increased, but to a lesser degree, and is
likely due to a total increase in lateral membrane domain E-cadherin
induced by HGF (see below). Because HGF elicits its biological effect
by stimulation of c-met, a receptor tyrosine kinase, we also
examined the effect of another tyrosine kinase agonist, epidermal
growth factor. Whereas epidermal growth factor also increased total
basolateral biotinylated E-cadherin, there was no effect on the amount
of apically exposed, biotinylated E-cadherin (Fig. 1). Thus, the
increase in apically biotinylated E-cadherin is specific to stimulation
by HGF and is not due to nonspecific receptor tyrosine kinase
activation.
Surface Labeling and Immunofluorescence Microscopy of Control and HGF-treated MDCK Cells Grown on Filters
To visually characterize
the HGF-induced, apically accessible E-cadherin, we added the
anti-E-cadherin mAb rr1 to either the apical or basolateral surface of
live cells at 4 °C and detected binding of this antibody by
immunofluorescence microscopy. The results of this experiment are shown
in Fig. 2. In non-HGF-treated control cells (Fig.
2a), basolaterally applied rr1 clearly labeled E-cadherin,
whereas apically applied rr1 failed to label E-cadherin. These findings
confirm that under control conditions, the MDCK cell monolayer has
competent tight junctions that exclude diffusion of rr1 from the apical
compartment to the basolateral compartment. In contrast, cells treated
for 24 h with HGF (Fig. 2b) showed an increase (2-3%
of the total number of cells) in labeling by apically applied rr1 of
discrete patches of membrane at the lateral borders of individual cells
of the monolayer. When viewed in the x-y axis
(i.e. a section parallel to the plane of the filter), the
apically accessible E-cadherin appeared to be in the lateral domain.
Confocal microscopy allowed us to reconstruct x-z section views (i.e. perpendicular to the plane of the filter) of the
apically accessible E-cadherin (Fig. 2c) and the tight
junction marker ZO-1 (Fig. 2c). After 24 h of HGF
treatment, apically accessible E-cadherin was detected below the tight
junction.
Fig. 3 shows that with extended exposures to HGF, an
increasing percentage of cells expressed E-cadherin, which was
accessible from the apical compartment for surface immunolabeling. Fig.
3 was taken with a conventional fluorescence microscope, because it
allowed a slightly different and informative overview of the distribution of apically accessible E-cadherin in this case. After 48 h of HGF exposure, approximately 15-20% of the cells
expressed apically accessible E-cadherin, whereas after 72 h of
HGF exposure, 30-40% of the cells expressed apically accessible
E-cadherin.
Confocal microscopy x-z section views shown in Fig.
4 of control and 48- or 72-h HGF-treated cells were
co-stained for ZO-1 (Fig. 4) and either apically accessible E-cadherin
(Fig. 4, b, c, and e) or basolaterally accessible
E-cadherin (Fig. 4, a and d). These pictures
showed that in addition to increasing access through the tight junction
to E-cadherin in lateral membranes below the tight junction, the longer
periods of HGF exposure (Fig. 4, c, 48 h and
e, 72 h) also induced mislocalization of E-cadherin to
membrane domains above the tight junction that are in direct contact
with the apical compartment. The images of basolaterally accessible
E-cadherin for control (Fig. 4a) and 72 h of HGF
treatment (Fig. 4d) demonstrate that HGF treatment induced
other morphological changes, i.e. increased thickness of the
monolayer and more tortuous interrelationships between adjacent cells
(increased lateral membrane surface).
Effect of HGF Treatment on E-cadherin Associated with Immunoprecipitated
E-cadherin functional activity is
dependent upon association with catenins. -Catenin represents an
important link between epidermal growth factor-induced signal
transduction and cadherin function (22). We, therefore, examined the
effect of HGF on the association of E-cadherin with immunoprecipitated
-catenin. Fig. 5A shows that during HGF
treatment, the amount of E-cadherin associated with immunoprecipitated
-catenin increased over a 24-h period (at 24 h, the amount was
313 ± 5.7% (p = 0.0008)). Fig. 5B
shows the amount of E-cadherin associated with immunoprecipitated
-catenin after 24, 48, and 72 h of HGF treatment. Again,
E-cadherin associated with
-catenin is increased after 24 h of
HGF treatment, but after 48 or 72 h of HGF treatment, this amount
returned to approximately baseline. A slight increase in the amount of
total immunoprecipitated
-catenin is detectable after HGF treatment (at 48 h, the amount was 115 ± 12% of control) but is not
statistically significant (data not shown). These results demonstrate
that HGF modulates E-cadherin and
-catenin interaction during
morphogenetic events.
HGF Treatment of Polarized MDCK Cell Monolayers Increases the Synthetic Rate of
Because the previous experiments provided evidence
that HGF increased the amount of E-cadherin associated with -catenin
in MDCK cells and, to a lesser extent, the amount of
-catenin, we examined the effect of HGF treatment on the biosynthetic rates of
-catenin and E-cadherin associated with
-catenin. (We did not
examine the biosynthesis of total E-cadherin for technical reasons.)
Polarized monolayers of MDCK cells were treated with HGF for 1, 3, 6, 12, or 24 h and pulse-labeled with [35S]cysteine and
[35S]methionine for 15 min, and cell lysates were
immediately collected. Equal amounts of RIPA buffer solubilized protein
were subjected to immunoprecipitation by
-catenin mAb. Amounts of
newly synthesized E-cadherin and
-catenin in the immunoprecipitates
were quantified by PhosphorImager analysis. The results (Fig.
6) show that HGF treatment of polarized MDCK cells
increased the synthetic rates of
-catenin and E-cadherin found in
the
-catenin immunoprecipitates. The peak effect on synthesis was
observed at approximately 12 h (p < 0.003 for
both E-cadherin and
-catenin) and returned to near baseline by
24 h. These results demonstrate that HGF increases the relative
rate of
-catenin synthesis. Moreover, HGF also increases the amount
of newly synthesized E-cadherin molecules found in
-catenin
complexes. This observation suggests that HGF is stimulating the rate
of E-cadherin synthesis and/or the rate at which E-cadherin interacts
with new and existing pools of
-catenin.
HGF-induced Apically Accessible E-cadherin Is Associated with
The -catenin immunoprecipitation results
demonstrate that HGF modulates the amount of E-cadherin associated with
-catenin. To determine if
-catenin is associated with HGF-induced
apically accessible E-cadherin, control cultures and cells treated with HGF for 24 or 48 h were subjected to apical and basolateral
surface immunoprecipitation with rr1. Amounts of
-catenin associated in the immunoprecipitate complexes were determined by Western blot.
Fig. 7 shows the results of such an experiment, from
which we can draw the following conclusions: (a) these
results confirmed that HGF increased the amount of apically accessible
E-cadherin. However, this population of E-cadherin was not well
visualized until 48 h of treatment, suggesting that this method is
not as sensitive as surface biotinylation and surface
immunofluorescence labeling; (b) these results demonstrated
that both the apically and basolaterally accessible E-cadherin are
associated with
-catenin. Because this protocol did not effectively
immunoprecipitate apically accessible E-cadherin at 24 h, the time
period in which increasing amounts of E-cadherin are associated with
-catenin, we cannot absolutely rule out changes in
-catenin
associated with apically accessible E-cadherin at this time point;
(c) the results show that HGF induced an increase in
E-cadherin/
-catenin complexes at the basolaterally accessible cell
surface, consistent with the observation of increased lateral membrane
surface area (Fig. 4).
Effect of HGF Treatment on pIgR Transcytosis
The HGF-induced
increase in apically accessible E-cadherin suggests a significant
change in cell polarity. In a preliminary attempt to gain insight into
the effects of HGF on polarized membrane traffic, we examined the
effect of 48-h HGF treatment on the transcytosis of iodinated dIgA by
MDCK cells expressing the rabbit pIgR. Under normal conditions, these
cells exhibit functional polarity by trancytosing dIgA from the
basolateral to the apical compartment, although a small fraction of the
dIgA recycles to the basolateral compartment. Fig. 8
illustrates the results of such an experiment. HGF treatment of the
pIgR-expressing MDCK monolayers significantly inhibited the basolateral
to apical transcytosis of 125I-labeled dIgA but did not
influence basolateral recycling. To a lesser degree, a similar pattern
of transcytosis inhibition was observed after 24 h of HGF
treatment (data not shown). These results suggest that HGF alters the
polarity of filter-grown MDCK cells, at least in part, by changing the
polarized membrane traffic.
Our results provide evidence that HGF treatment of polarized MDCK
cell monolayers grown on filters induced morphological changes, including an increase in apically accessible E-cadherin. The mechanism by which this occurs appears to be through localization of E-cadherin to the membrane domain in contact with the apical compartment and
modulation of tight junction integrity. HGF treatment also modulated
the association of E-cadherin with immunoprecipitated -catenin and
stimulated the de novo synthesis rates of
-catenin. In
addition to altering the polarity of MDCK cell monolayers, HGF also
altered polarized membrane traffic, as determined by the transcytosis
of dIgA by the pIgR. Collectively, these findings demonstrate that this
model of HGF treatment of polarized MDCK cells grown on filters
provides a system by which complicated morphogenetic events in
polarized cells can be dissected with regard to structural,
biochemical, and functional polarity. This model will serve in the
understanding of more complex processes, including the loss of polarity
in disease processes such as carcinomas (23), regaining epithelial
polarity in tissue regeneration following injuries such as acute
tubular necrosis (24), and epithelial morphogenesis in normal and
abnormal (e.g. polycystic kidney disease) development of the
kidney (25).
The importance of E-cadherin in epithelial morphogenetic events is not
restricted to mammalian systems. Recently, two groups simultaneously
reported that the Drosophila gene shotgun that encodes the first classic cadherin isolated from invertebrates plays a
crucial role in the developing Drosophila embryo (26, 27).
Their results seem congruent with ours, in that they found that the
continued zygotic expression of the Drosophila cadherin is
required for epithelial cell rearrangement during morphogenesis. This
suggests that our observed increase in the expression of E-cadherin in
MDCK cells exposed to HGF may similarly be important for morphogenesis
in mammalian epithelia. The Drosophila cadherin also
interacts with catenins, and it is likely that these interactions are
also modulated during invertebrate morphogenetic events such as
embryonic development. Evidence supporting this hypothesis has been
reported by two groups, demonstrating the requirement of the
Drosophila -catenin homolog Armadillo for the formation of Drosophila adherens junction and epithelial polarity (28, 29).
By inducing localization of E-cadherin to the membrane domain facing the apical compartment and increasing apical accessibility via tight junctions, HGF decreases the degree of polarization in previously well polarized MDCK cells. Because alterations in epithelial polarity are associated with pathologic states such as carcinoma (23), this model has potential in the characterization of cellular events during carcinoma induction. Recently, it was shown that estrogen activated c-JunER protein-induced loss of epithelial polarity, a disruption of intercellular junctions, and the formation of irregular multilayers in mammary epithelial cells (30). The authors of this paper suggested that these processes may be important for both normal breast development and as initiating steps in the induction of breast carcinogenesis. Epithelial invasiveness is associated with down-regulation of E-cadherin amounts (31, 32); however, the present findings showed an increase in apparent amounts of apical and basolateral E-cadherin by HGF treatment. This finding, coupled to the loss of epithelial polarity, raises the possibility of E-cadherin dysfunction in the present model and/or suggests that under certain conditions, epithelia can lose functional and structural polarity (a phenotype seen in carcinomas) with a paradoxical increase in E-cadherin amounts.
HGF also reduces transcytosis of the pIgR (33). Basolateral recycling of dIgA by the pIgR was not affected by HGF treatment, suggesting that HGF treatment specifically alters the machinery necessary for efficient basolateral to apical translocation of dIgA. We do not know if this is due simply to the increased distance that the pIgR and dIgA must travel across the thicker monolayer. This result is intriguing because transcytosis to the apical surface is an essential mechanism by which epithelial polarity is established and maintained, particularly during the early steps in development of the polarized phenotype (34). Clearly, HGF affects the basic mechanisms of membrane traffic involved in the development of polarity.
Treatment of polarized MDCK cells with HGF increased the amount of
E-cadherin associated with immunoprecipitated -catenin during the
first 24 h of exposure with a return to baseline levels at 48 and
72 h of HGF treatment. Thus, HGF altered
-catenin protein interactions with E-cadherin in MDCK cells grown on filters. It should
be noted that at 24 h, there is only a very small amount of
apically accessible E-cadherin, which does not become prominent until
48-72 h. Therefore, the response to HGF can be viewed as having two
phases: 1) an early phase that peaks at 24 h and is characterized
primarily by an increase in the amount of E-cadherin associated with
-catenin; and 2) a late phase that peaks at 48-72 h and is
characterized by the return of both
-catenin synthetic rate and
association between
-catenin and E-cadherin to baseline levels, and
a large amount of apically accessible E-cadherin. Assembly of
E-cadherin/
-catenin complexes in polarized MDCK cells is a dynamic
process (20) involving existing and newly synthesized components.
Therefore, it is possible that HGF modifies this assembly process
either through alteration in cadherin and catenin synthetic rates
and/or modification of existing cadherins and catenins. We observed
that HGF increases the relative rate of
-catenin synthesis.
Moreover, HGF also increases the amount of newly synthesized E-cadherin
molecules found in
-catenin complexes. This observation suggests
that HGF is stimulating the rate of E-cadherin synthesis and/or the
rate at which E-cadherin interacts with new and existing pools of
-catenin. Weidner et al. (3) reported that HGF treatment does not increase E-cadherin synthesis. However, in that study, the
MDCK cells were grown nonconfluently on impermeant supports, and
E-cadherin synthesis was measured between 17 and 20 h of HGF treatment, a time when effects of HGF on synthesis rates of E-cadherin may have already declined to near baseline. In human carcinoma cells,
HGF has been shown to modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins, including
-catenin (35). Thus, tyrosine phosphorylation of
-catenin may play an important role in modulation of the
E-cadherin/
-catenin complex assembly and composition in the
HGF-treated MDCK monolayers and will be a subject for future
investigation.
We thank Lindsay Hinck and Inke Näthke for thoughtful comments and discussion. We also thank Barry Gumbiner and Bruce Stevenson for providing hybridoma lines and Ralph Schwall at Genentech for generous gifts of recombinant human hepatocyte growth factor. We also thank the members of the Mostov laboratory for stimulating discussions.