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INTRODUCTION |
The plasma membrane of epithelial cells is polarized into two
domains; apical and basolateral, each of which has characteristic protein and lipid composition (1-3). Although targeting sequences were
found in the structure of several polarized proteins, recent studies
have shown that some proteins are polarized in a cell type-specific
manner; for instance the Na,K-ATPase is targeted to the apical membrane
of retinal pigment epithelium but to the basolateral membrane of most
other epithelia (4). Several other proteins exhibit this flexibility in
targeting; the most dramatic example is the targeting of the
proton-translocating ATPase and the Cl:HCO3 exchanger of
the intercalated cell of the renal tubule (reviewed in Ref. 5). These
cells exist in a spectrum of forms. One extreme, the
type, has an
apical H+-ATPase and a basolateral anion exchanger that is
an alternately spliced form of the erythroid band 3 (kAE1)1 and hence is capable
of trans-epithelial secretion of H+. In contrast, the
form secretes HCO3 by a basolateral H+-ATPase
and an apical kAE1 (6). (However, one study did not find kAE1 by
immunoblot analysis but confirmed the presence of its mRNA (7).) In
an immortalized clonal cell line (8), we demonstrated that
cells
can be converted to an
form by changes in the seeding density (9)
reproducing a previous demonstration in vivo induced by
changes in the acid content of the diet (10). The development of the
clonal cell line allowed us to begin to uncover the biochemical basis
of this plasticity.
When the immortalized cells were seeded on filters at subconfluent
density, they eventually formed epithelial monolayers capable of
secreting HCO3 into the apical medium. These cells did not exhibit any apical endocytic activity; they had apical kAE1 and a
basolateral H+-ATPase (9). Remarkably, when the same cells
were seeded at confluent density, their phenotype was that of the
cells; i.e. they had vigorous apical endocytosis, apical
H+-ATPase, and basolateral kAE1, changes that occurred
within 24 h of seeding. Both phenotypes were stable in culture for
as long as the cells were observed. Hence, it was not the assumption of cell to cell contact but rather the initial seeding density that rapidly induced a binary switch in phenotype. More recently, we discovered that low density cells were flat, and high density cells
were columnar. Furthermore, the apical cytoskeleton was dramatically
different in the two phenotypes; low density cells had sparse
microvilli, no apical actin, villin, or cytokeratin 19, whereas high
density cells had exuberant microvilli, abundant sub-apical actin,
villin, and cytokeratin 19 (11). These studies show that the transition
from
to
phenotype was remarkably similar to terminal
differentiation of epithelia, especially as seen in the intestine
during the transition from crypt to villus (12).
When low density cells were seeded on the extracellular matrix (ECM) of
high density cells, they assumed all the characteristics of high
density cells. By using the induction of apical endocytosis as an
assay, we purified a protein from high density ECM that was capable of
converting the polarity of the low density cells (9). This protein, now
termed hensin (for change in body in Japanese, see Ref. 13), is
composed of three types of domains, SRCR (14), CUB (15), and Zp domains
(16). Hensin is a protein widely expressed in epithelial organs and
brain. During the purification of hensin, it became clear that the
protein traveled on gel filtration at a mobility suggestive of a size
much higher than that predicted by its molecular mass of 230 kDa. To
purify hensin to homogeneity required treatment with SDS and 4 M urea (13), but this denatured, if pure, hensin did not
induce apical endocytosis in low density cells. However, a polyclonal
antibody generated against a fusion protein composed of two SRCR
domains prevented the induction of endocytosis in high density cells
(13). These results demonstrated that hensin is necessary for activity,
but whether it is also sufficient by itself is at present unknown.
In the present paper, we demonstrate that hensin is secreted as a
soluble protein into the basolateral medium by the two phenotypes. Low
density cells secrete only monomeric hensin, whereas high density cells
secrete a soluble form of hensin that is multimeric. In addition, high
density cells (but not low density cells) secrete a form of hensin that
is retained in the ECM as a higher order multimer. We refer to this
insoluble ECM form of hensin as precipitated hensin pending further
analysis of its structure. Many ECM proteins such as collagen and
fibronectin are retained in the ECM because they form fibrils composed
of a large number of monomers associated in a specific architectural
pattern as fibers or networks. Procedures that disaggregate the ECM
type of multimer prevent hensin from inducing apical endocytosis in low
density cells. These results demonstrate that only multimeric insoluble
hensin is capable of inducing endocytosis, reversing the polarity, and
inducing terminal differentiation of intercalated cells.
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MATERIALS AND METHODS |
35S Labeling, Pulse-Chase, and
Immunoprecipitation--
Clone C of
-intercalated cells were
maintained at 32 °C as described (8, 9). The cells were seeded on
0.45-µm polycarbonate filter (Transwell, Corning Costar Corp.,
Cambridge, MA) at a density of 2 × 104/cm2 (low density) or 4 × 105 cells/cm2 (high density) and cultured at
40 °C to inactivate T antigen. Cells were seeded at high density on
polycarbonate filters and grown for 24 h at 40 °C. Media were
changed to minimum Eagle's medium without methionine and cysteine
(Sigma) for 2 h and pulse-labeled with 100 µCi/ml
35S (NEN Life Science Products Express
35S-protein labeling mix) for 10 min at 40 °C followed
by chase with regular medium supplemented with 10 mM
L-methionine. Cells were lysed in 1% SDS, 1 mM
EDTA, 1% Triton X-100, 10 mM Tris-HCl (pH 8.0) and boiled
for 3 min. Insoluble materials were removed by a brief centrifugation
(14,000 × g for 5 min at room temperature), and the
protein concentration of the supernatants was determined by the
Bradford reagent (Bio-Rad). An equal amount of protein was taken from
each sample, diluted 10-fold with 10 mM Tris-HCl (pH 8.0),
and used for immunoprecipitation. Alternatively, cells and ECM were
extracted separately and used for immunoprecipitation as described
below. Tunicamycin (Sigma) (0.5 mg/ml in 10 mM NaOH) was
freshly prepared and added to media of high density cells to a
concentration of 2.5 µg/ml; an equal volume of 10 mM NaOH was added to the control media. Cells were preincubated for 5 h
and then pulse-labeled with 35S for 10 min followed by
chase up to 2 h in the continuous presence of tunicamycin.
Filter-grown high density cells were labeled with 35S for
12 h and extracted with buffer A (1% Triton X-100, 1 mM calcium chloride) for 1 h at 4 °C on a rotary
shaker. Cell extracts were removed, and filters were scraped in this
solution with a cell scraper to remove loosely attached materials and
washed thoroughly with the same solution for another hour at 4 °C.
Insoluble materials remaining on these filters were extracted with 4 M guanidine hydrochloride, 50 mM sodium acetate
(pH 6.5), 5 mM EDTA, 0.5% CHAPS at 4 °C overnight, and
this was referred as the ECM fraction (9). Both cell and ECM extracts
were dialyzed against 50 mM Tris-HCl (pH 8.0) at 4 °C
overnight. To these samples, one-tenth volume of buffer B (1% SDS, 1%
Triton X-100, 10 mM Tris-HCl (pH 8.0), 1 mM
calcium chloride) was added, and they were subjected to
immunoprecipitation. Conditioned media were collected and spun at
5,000 × g for 5 min at 4 °C, and the top two-thirds
of the supernatants were saved and mixed with one-tenth volume of
buffer A before immunoprecipitation. To materials ready for
immunoprecipitation, guinea pig anti-hensin serum (13) was added to a
dilution of 1:500 and incubated at 4 °C for 1 h followed by
addition of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) at
4 °C for another 1 h. Beads were washed three times with a
buffer containing 0.1% SDS, 0.1 mM EDTA, 0.1% Triton
X-100, and 10 mM Tris-HCl (pH 8.0) and boiled in sample buffer for 3 min and then subjected to SDS-PAGE. Gels were fixed with
10% acetic acid, 10% methanol, soaked in Amplify solution (Amersham
Pharmacia Biotech), dried, and exposed to x-ray film (Kodak X-Omat,
Eastman Kodak Co.) at
80 °C. Densitometric scanning was performed
by a Computing Densitometer model 300A (Molecular Dynamics).
Apical Endocytosis--
Monolayers plated at various densities
were exposed to
HCO3
/CO2-free medium
containing 2-5 mg/ml horseradish peroxidase and 10% fetal bovine
serum on the apical side of the monolayer only. The cells were
incubated on a rotary shaker for 10 min at 40 or 4 °C. At the end of
the incubation, the cells were transferred to ice-cold medium, washed
10 times at 4 °C on a shaker, and solubilized in 1% Triton. The
activity of horseradish peroxidase was measured as the initial rate of
hydrolysis of o-dianisidine (Sigma) in a Beckman DU-2
spectrophotometer. The volume of endocytosis was calculated by
constructing a standard curve of horseradish peroxidase activity in
dilutions of the starting material. Apical endocytosis was taken to be
the difference between uptake at 40 °C minus that at 4 °C.
Preparation of DMMA-treated ECM-coated Filters--
High density
cells seeded on transwells were extracted with buffer A, and filters
were pre-equilibrated with a solution containing 50 mM
Hepes-KOH (pH 8.5). 5 mM dimethylmaleic anhydride (DMMA, Sigma), dissolved in the same buffer was added to the filters and
incubated at 4 °C for 1 h on a rotary shaker. The filters were
washed thoroughly with 50 mM Hepes-KOH (pH 8.5) to remove excess DMMA and then incubated with 50 mM Hepes-KOH (pH
6.7) at 4 °C overnight. These filters were first equilibrated with
Dulbecco's modified Eagle's medium at 4 °C overnight before cells
were seeded on them at low density.
Sucrose Density Gradient Analysis of Hensin--
Conditioned
media were collected from low density or high density cells and
concentrated 10-fold using Centriprep-10 (Amicon, Beverly, MA) and
divided into Eppendorf tubes. Each tube received 5 mM DTT
or 5 mM EDTA. For the DMMA (5 mM) studies, the
samples were incubated at 4 °C for an hour and then dialyzed against
50 mM Hepes-KOH (pH 8.5) at 4 °C overnight, and one-half
of the sample was further dialyzed against 50 mM Hepes-KOH
(pH 6.7). All samples were loaded on 12 ml of 5-30% sucrose gradient
in 50 mM Hepes-KOH (pH 7.5, 8.5, or 6.7) and
ultracentrifuged at 100,000 × g for 16 h at
4 °C. Proteins in each fraction (1 ml) were precipitated by 6%
trichloroacetic acid, dissolved in a sample buffer, subjected to 7.5%
SDS-PAGE followed by Western blotting with anti-hensin serum.
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RESULTS |
Hensin Is a Secretory Glycoprotein That Is Retained in the ECM of
High Density Cells--
Pulse-chase experiments showed that the
molecular weight of hensin increased after its synthesis, and the
intensity of the band eventually decreased with prolonged periods of
chase (Fig. 1A). Hensin was
secreted into the basolateral medium, partially explaining the reduced
intensity during the chase period (Fig. 1C). The molecular
weight shift was abolished when the cells were pretreated with
tunicamycin indicating that hensin is N-glycosylated (Fig.
1B).

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Fig. 1.
Hensin is a secretory glycoprotein.
A, cells were seeded at high density and cultured for 2 days. They were pulse-labeled with [35S]methionine for 10 min and then chased for the indicated times. The same fraction of
lysate protein was taken from each sample for immunoprecipitation with
anti-hensin serum followed by SDS-PAGE and fluorography. B,
high density cells were preincubated in the presence or absence of 2.5 mg/ml tunicamycin for 5 h before pulse-chase. Whole cell lysate
was made from each sample and treated as in A. C,
high density cells were labeled with 35S for 3 h
followed by chase for 3 h. Apical and basolateral media were
collected separately and immunoprecipitated.
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Surprisingly, hensin was synthesized not only in high density cells but
also in low density cells; indeed low density cells consistently
synthesized it at a higher rate (Fig. 2)
(11). Cells were labeled with [35S]methionine, and hensin
was chased into the cell lysate, ECM, and medium in both low density
and high density cells. (ECM is here defined as the
guanidine-extractable material remaining on the filter after Triton
solubilization.) While hensin accumulated in the ECM of high density
cells, very little was seen in low density cells (Fig. 2). These
results confirmed our original finding that high density ECM, but not
that of low density, contained hensin (9, 13). Basolateral secretion
was seen in both phenotypes, but the degree of polarization was not
high since these cells were examined only 2 days after seeding, a time
before the development of impermeable monolayers as assayed by the
trans-epithelial flux of [14C]inulin (data not shown).
Recent immunocytochemical studies documented that hensin is indeed an
ECM protein in high density; it was accessible to externally added
antibodies without the necessity for permeabilizing the cells, and it
co-localized in confocal images with an authentic ECM protein, collagen
IV (11). Fig. 3 shows that hensin began to accumulate in the ECM within 3 h of seeding the cells at high density. In low density cells, there was a large amount of hensin, but
it was restricted to intracellular vesicles (Fig. 3).

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Fig. 2.
Hensin localizes in ECM only in high density
cells. Low density (LD) and high density
(HD) cells were labeled with 35S for 12 h,
and apical (Ap) and basolateral (BL) media were
collected separately. Cells were extracted with 1% Triton X-100
(Tx), 1 mM CaCl2, and the ECM
deposits were extracted by 4 M guanidine hydrochloride and
subjected to immunoprecipitation. The intensity of the hensin band in
three independent experiments was quantitated by densitometry and
presented as percentage of the total hensin produced.
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Fig. 3.
Distribution of hensin in intercalated cells
seeded at high and low densities for different times.
Magnification, × 400.
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Soluble Hensin Exists in Different Forms in the Two
Phenotypes--
Hensin was present in high density cells in both
soluble (i.e. secreted into the media) and insoluble forms
(i.e. present in the ECM). However, low density cells seemed
to produce only the soluble form raising the possibility that hensin
was degraded in ECM of low density but not that of high density cells.
However, detailed experiments on the rate of proteolysis of hensin, the secretion of matrix metalloproteinases, and the effect of protease inhibitors on the half-life of hensin failed to reveal any difference between the two phenotypes (data not shown).
To examine whether even the soluble hensin might exist in different
forms in the two phenotypes, we separated secreted hensin by sucrose
density gradient centrifugation. We found that hensin secreted by low
density cells was recovered in fractions 3 and 4, as would be predicted
from monomeric hensin (Fig. 4). However, as much as 30% of hensin secreted into the media of high density cells
was recovered in fractions 7 and 8 and in the pellet (Fig. 4). Fraction
7 corresponds to an apparent molecular mass of 700,000, double (or
perhaps even quadruple) the apparent molecular weight of hensin.
Because hensin is a cysteine-rich protein, we treated the high density
media with dithiothreitol (DTT), a reagent that reduces di-sulfide
bonds; all multimeric hensin was reduced to the monomeric form (Fig. 4,
lane DTT). These results, however, cannot distinguish
whether the multimerization was produced by inter-molecular or by
intra-molecular disulfide bonds. Recent studies on the structure of
SRCR domains demonstrated that intra-molecular disulfide bonds are
critical for formation of the native structure of the domain (17).

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Fig. 4.
Separation by sucrose density gradients shows
that soluble hensin exists in different forms. Conditioned media
collected from low density (LD) or high density
(HD) cells were treated with various reagents as shown at
4 °C for 1 h. The sample treated with DMMA was dialyzed against
50 mM Hepes-KOH (pH 8.0) at 4 °C, and half of it was
further dialyzed against 50 mM Hepes-KOH (pH 6.7) at
4 °C overnight. These samples were loaded on 5-30% sucrose
gradient and ultracentrifuged at 100,000 × g for
16 h at 4 °C. Proteins from each fraction were precipitated by
5% trichloroacetic acid, electrophoresed, and blotted with anti-hensin
serum. Fraction 12 corresponds to the bottom of the gradient. Aldolase
(158 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) were used as
marker proteins.
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DMMA is a useful reagent that reacts with the
-amino group of lysine
to produce a maleyl lysine, thereby converting it from a cation to an
anion. This large change in electrostatic potential frequently results
in disaggregation of subunits or large conformational changes (18).
Maleyl lysine is stable at neutral or alkaline pH but is rapidly
hydrolyzed in acidic media resulting in the regeneration of the
cationic group of lysine. We used DMMA treatment to probe the
association and dissociation of hensin. When the conditioned medium of
high density cells was pretreated with DMMA at pH 8.0, hensin was
substantially reduced in fractions 7 and 12 indicating that hensin
unfolded to assume the monomeric form (Fig. 4, DMMA pH 8).
These results demonstrate that hensin does not form multimers by
inter-molecular disulfide bonds. When this sample was further dialyzed
against a buffer of pH 6.7, 25% of the total amount of hensin appeared
in fraction 7 demonstrating that when high density hensin is
disassembled, it could spontaneously form back into multimers given the
right conditions, i.e. conversion of maleyl lysine back to
lysine. Furthermore, that the association of multimeric hensin is
reversible suggested that the conformation of hensin has been altered
in some permanent way by the high density state.
ECM Hensin Is a Higher Order Multimer--
Soluble multimeric
hensin of high density cells must undergo additional multimerization,
fibril formation, or association with other ECM proteins before being
able to localize in the ECM. Because divalent ions are often critical
for stabilization of tertiary structure, we tested their effect on
hensin extraction. High density ECM when treated with calcium (or other
divalent ions) contained more hensin than those exposed to the
chelators EDTA or EGTA (Fig. 5),
suggesting that the localization of hensin in the ECM likely involves a
calcium-mediated aggregation or a change in conformation. However, EDTA
and EGTA did not change the multimeric state of soluble hensin secreted
into the media (Fig. 4), suggesting that the effect of calcium in
retaining hensin in the ECM might involve mechanisms other than
multimerization; perhaps it had an effect on association with other ECM
proteins.

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Fig. 5.
Extraction condition affects binding of
hensin in ECM. High density cells were labeled with
35S for 12 h. Cells were extracted first with Triton
(Tx) with 1 mM calcium chloride or chelating
agents, and the ECM deposits were subsequently extracted and
immunoprecipitated.
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When the ECM of high density cells was isolated (using deoxycholate
extraction rather than guanidine to avoid excessive denaturation) and
separated on sucrose density gradients, hensin remained in the pellet.
Treatment of ECM by DTT resulted in a partial solubilization, where
some hensin was now present in fractions as light as a tetramer, although the majority of hensin remained at a higher order (Fig. 6). These results demonstrate that ECM
hensin precipitates in a very high density fraction suggesting that it
is a multimer of much higher order than a tetramer.

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Fig. 6.
Separation by sucrose density gradient of ECM
hensin. High density (HD) cells were extracted with 1%
Triton X-100 in the presence of 1 mM calcium chloride, and
the ECM deposits were extracted with 1% deoxycholate, 50 mM Tris-HCl (pH 7.5) at room temperature followed by
loading on 5-30% sucrose gradients and Western blotting. Half of the
sample was treated with 5 mM DTT for 1 h at 4 °C
before loading onto the gradient.
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Only Insoluble Polymeric ECM Hensin Is Capable of Inducing the
Change in Phenotype--
Induction of apical endocytosis is an early
and consistent marker for the change in polarity; high density cells
have a vigorous rate, whereas low density cells have no apical
endocytosis (Fig. 7A). Low
density cells seeded on high density matrix develop apical endocytosis
(Fig. 7A). Incubation of high density cells in media containing a polyclonal antibody to hensin inhibited the development of
apical endocytosis (Fig. 7A) (13). These results demonstrate that hensin in the ECM of high density cells is necessary for induction
of apical endocytosis and, by extension, reversal of the polarity of
several proteins in the low density cells.

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Fig. 7.
Apical endocytosis. A, apical
endocytosis of horseradish peroxidase; LD, low density
(n = 30); HD, high density
(n = 67); Ab, anti-hensin antibody
(n = 6); LD/HD ECM (n = 42),
endocytosis in cells plated at low density on high density ECM:
+F3-4 (n = 9), +F7-8
(n = 9), and +F12 (n = 9),
fractions 3, 4, 7, 8, and 12, respectively, isolated from sucrose
density gradients of media conditioned by high density cells and added
to low density cells as seen in Fig. 4. B, apical
endocytosis of low density cells seeded on high density ECM before or
after treatment with 1 mM DTT (n = 9); 1 mM NEM (n = 9); treatment with 1 mM DTT followed by treatment with 5 mM NEM and
extensive washing (n = 9); or treatment with 1 mM DMMA (n = 9). Vertical bars
represent the standard errors of the mean. n is the number
of independent experiments.
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What is the form of hensin that can induce the reversal of polarity?
Soluble monomeric hensin, in fractions 3 and 4, was concentrated and
applied to low density cells; it was unable to induce apical endocytosis in low density cells (Fig. 7A). To examine the
extent of multimerization needed to induce the change in phenotype, we concentrated the soluble fractions 7 and 8 and fraction 12 and exposed
low density cells to these multimeric fractions for 5 days and assayed
for apical endocytosis. These concentrated fractions did not induce
apical endocytosis (Fig. 7A) implying that such multimers
were either not polymeric enough, of different conformation, or else
required another protein to exert their effect. We point out that these
multimers were still soluble since they were identified in the media of
high density cells making it likely that only the polymeric form of
hensin is functionally active. Pretreatment of high density ECM by DTT
or N-ethylmaleimide or both sequentially to alkylate all
reducible disulfide bonds (and therefore to convert hensin to a
monomeric form) abolished the ability of high density ECM to induce
apical endocytosis (Fig. 7B). When high density ECM-containing filters were pretreated with DTT or NEM, hensin was
extracted to the same extent as from untreated filters. Hence, the lack
of effect on endocytosis was not due to loss of hensin from these
filters. Similarly, treatment of high density ECM by DMMA also
abolished its ability to induce apical endocytosis (Fig. 7B). These results demonstrate that only precipitated ECM
hensin is capable of inducing the change in phenotype.
High Density Cells Can Multimerize Extracellular Hensin--
The
mechanism by which different forms of hensin exist in the two
phenotypes could be due to a biosynthetic intracellular event, similar
to what occurs during the initial stages of collagen biosynthesis (19).
Alternatively, a cell-surface event in the high density phenotype
occurs which will lead to polymerization and precipitation, a
phenomenon described with fibronectin fibril formation. To distinguish
between these possibilities, we labeled low density cells with
[35S]methionine and concentrated the labeled hensin
(i.e. fractions 3 and 4). This was then added to unlabeled
low and high density cells. After a 24-h incubation, the media were
removed, concentrated, and separated on sucrose density gradients.
Hensin in all gradient fractions was immunoprecipitated and separated
on SDS-PAGE, and 35S-labeled hensin was subjected to
autoradiography and quantitated by densitometry. It is clear from Fig.
8 that high density cells were able to
induce polymerization of hensin, whereas low density cells did not.

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Fig. 8.
High density cells affect oligomerization
status of hensin. Low density cells were labeled with
35S for 12 h, and media were collected, concentrated,
and added to fresh, unlabeled cells seeded at low or high density.
After 24 h of incubation, the conditioned media were collected and
separated on 5-30% sucrose gradient, and hensin was
immunoprecipitated from each fraction. Densitometric analysis of hensin
is displayed in the figure below as a percentage of the total hensin on
the gradient.
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DISCUSSION |
The functional assay used in this study, induction of apical
endocytosis, is one of the cardinal events that occurs during the
conversion of polarity in vitro and in vivo (9,
10). As an assay for this plasticity, it is more quantitative than the
immunocytochemical localization of proteins, and the fact that low
density cells had no apical endocytosis provides an excellent signal to
noise ratio. High density cells (and
cells in situ) have
vigorous apical endocytosis, and this process occurs early after
seeding at high density and remains during the 2 weeks of observation.
Since the vacuolar H+-ATPase is packaged in sub-apical
endocytic vesicles which continuously form and then fuse with the
apical membrane, the induction of apical endocytosis is an excellent
surrogate marker for the presence of the H+-ATPase in the
apical membrane (20-22). Furthermore, we recently found that high
density cells have dramatically different apical cytoskeleton, such as
the appearance of a sub-apical actin network and the induction of
apical villin and cytokeratin 19 (11). These findings demonstrate that
the induction of apical endocytosis, an important phenomenon in its own
right, is also a legitimate marker for the reversal of polarity of the
H+-ATPase and the anion exchanger kAE1.
We initially found that the ECM of high density cells (defined as the 4 M guanidine extract after solubilizing cells with 1%
Triton) contained an activity that retargeted kAE1 to the basolateral membrane and induced apical endocytosis in low density cells; the
latter experiments were confirmed in the new studies shown in Fig.
7A. Purification using standard chromatographic methods resulted in a highly active fraction whose major protein component was
hensin (9). However, further purification of hensin required denaturation with 4 M urea and 0.1% SDS, a procedure that
resulted in a homogenously pure protein but that lacked the ability to induce apical endocytosis (13). Whether the loss of activity was due to
denaturation of hensin, to a loss of an associated protein, or whether
hensin is unrelated to the polarity reversal required additional
information. A fusion protein composed of two of the SRCR domains of
hensin was used to generate polyclonal antibodies. These sera inhibited
the ability of unfractionated high density ECM to induce apical
endocytosis (results that are also recapitulated in Fig.
7A), whereas preimmune sera had no effect (13). Extensive
morphological studies also show that anti-hensin antibodies also
prevented the development of the above mentioned changes in apical
cytoskeleton (11). Furthermore, seeding cells on purified ECM
components (fibronectin, laminin, or collagen IV) or on complex ECM
preparations (matrigel) did not induce the changes in apical
cytoskeleton, nor did specific antibodies against ECM proteins prevent
the change in phenotype (11). These results demonstrate that hensin is
necessary for the reversal of polarity, but whether it is sufficient by
itself to perform this activity or needs an additional protein will
require more studies.
The surprising finding of the current study is that hensin was
abundantly synthesized and secreted by low density cells, but it was
not retained in the ECM. Because this soluble hensin appeared to be a
monomer, it is likely that retention in the ECM requires oligomerization and or association with other ECM proteins. High density cells secreted soluble hensin into the medium and insoluble hensin that was localized in the ECM. The apparent molecular mass of
the soluble hensin was as high as a tetramer. However, ECM hensin is
much larger than a tetramer, and it precipitates in the ECM either
alone or in association with other ECM proteins (Fig. 6). That it could
not be purified to homogeneity without harsh conditions suggests that
it is likely enmeshed in a network of other ECM proteins. Importantly,
procedures that disrupted the multimeric formation inhibited the
activity of hensin (Figs. 6 and 7). Furthermore, it appeared that
multimerization to a lower order such as that which occurs in soluble
hensin failed to induce the activity. Hence, these results demonstrate
that only precipitated hensin can induce the change in phenotype.
What causes precipitation of hensin? We believe that studies on other
ECM proteins might suggest an answer. Fibronectin and collagen are
synthesized as soluble monomers, but their deposition in the ECM
requires oligomerization and fibril formation, a process that is
"catalyzed" by other proteins such as activated integrin receptors
or proteases (19, 23, 24). The case of fibronectin is particularly
instructive. Soluble fibronectin is unable to activate its receptor.
However, "activation" of the receptor by a variety of procedures
induces a high affinity state. This high affinity integrin receptor is
capable of binding fibronectin and inducing it to form fibrils (23,
24). Can high density seeding perform the same function on the putative
hensin receptor? Although this must await the identification of such a
receptor, the studies of Fig. 8 demonstrate that addition of monomeric
hensin produced by low density cells to cells seeded at high density
causes hensin to form multimers, even precipitates. Of course, these
studies do not exclude the role of other ECM proteins in helping to
precipitate hensin. However, they demonstrate that oligomerization of
hensin and its precipitation is not a biosynthetic event that occurs only in high density cells, similar to what occurs in collagen trimer
formation (19). Rather, the high density cells act from the outside to
multimerize it.
We recently found that the change in phenotype induced by high seeding
density includes the formation of exuberant microvilli, the
localization of villin and cytokeratin 19 to the apical cytoplasm, and
the assumption of a columnar shape by these epithelial cells (11).
These findings are remarkably similar to what occurs in the intestinal
epithelial cell when it differentiates from the crypt stem cell to a
villus absorptive cell (12, 25). It has been demonstrated that some ECM
component is involved in this terminal differentiation (26). Hensin is
expressed in most epithelia, but its expression in the intestine is
especially robust (13). We recently found that hensin is distributed in
crypt cells in a pattern identical to that of low density intercalated
cells, whereas in the villus cells its expression was like that of high density cells (13). These results suggest that hensin might be involved
in terminal differentiation of other epithelia. Terminal differentiation is a critical step in epithelial biology whose interruption often leads to the development of malignancies. It was
recently reported that chromosome 10q25-26 contains a region often
deleted in malignant brain tumors, and a cDNA encoded by that
region, DMBT1, was found to be deleted in 20% of malignant gliomas
(27). We recently discovered that DMBT1 and hensin are alternately
spliced forms of the same gene raising the possibility that hensin
might be a tumor suppressor (28).