| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Pathology, Division of Cellular and Molecular Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-smooth muscle actin,
loss of epithelial marker E-cadherin, transformation of
myofibroblastic morphology, and production of interstitial
matrix. Time-course studies revealed that loss of E-cadherin was an
early event that preceded other alterations during EMT. The transformed
cells secreted a large amount of matrix metalloproteinase-2 that
specifically degraded tubular basement membrane. They also exhibited an
enhanced motility and invasive capacity. These alterations in
epithelial phenotypes in vitro were essentially
recapitulated in a mouse model of renal fibrosis induced by unilateral
ureteral obstruction. Hence, these results indicate that
tubular epithelial to myofibroblast transition is an
orchestrated, highly regulated process involving four key steps
including: 1) loss of epithelial cell adhesion, 2) de
novo -smooth muscle actin expression and actin
reorganization, 3) disruption of tubular basement
membrane, and 4) enhanced cell migration and
invasion.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-smooth muscle actin (-SMA)-positive myofibroblasts remain
uncertain,7-9
emerging evidence suggests that they may
derive from tubular epithelial cells by an epithelial to mesenchymal transition (EMT) process under pathological conditions.10-12 However, the details regarding the conversion between these two distinct types of cells are poorly defined.
Because tubular epithelial cells and interstitial myofibroblasts
dramatically differ in their morphology and phenotypes, and are located
in separated tissue compartments within the kidneys, one can envision
that there have to be remarkable alterations in the expression of many
sets of genes to make this phenotypic conversion possible. Indeed,
previous studies have identified altered expression patterns of several
genes such as -SMA and fibroblast-specific protein-1
(Fsp1) during tubular epithelial to myofibroblast
transition (EMT).13,14
However, the cause-effect
relationship of these changes in EMT as well as the key events during
the entire EMT course at cellular level remain to be fascinating
unanswered questions.
Given the fact that several key obstacles have to be overcome to make epithelial to myofibroblast transition possible, we propose that tubular epithelial to myofibroblast transition at the cellular level is likely a step-wise process involving several crucial events that eventually lead to the completion of the entire course. It is conceivable that these tubular epithelial cells lose the key epithelial cell markers that make them epithelium in the first place, while acquiring de novo expression of myofibroblastic markers that define their newly adapted morphology and phenotypes. Likewise, these cells have to find a way to pass across the tubular basement membrane (TBM) that surrounds the renal tubule and finally enter their newly found home, the interstitial compartments of the kidneys.
In this study, we attempt to decipher the key events controlling the tubular epithelial to myofibroblast transition both in vitro and in vivo. Our data suggest that the entire EMT process consists of several key steps that depend on hyperactive transforming growth factor (TGF)-ß1 signaling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human proximal tubular epithelial cells (HKC-8) were kindly
provided by Dr. L. Racusen of Johns Hopkins University.15
Cells were cultured in Dulbecco’s modified Eagle’s medium-F12
medium supplemented with 5% fetal bovine serum (Life Technologies,
Inc., Grand Island, NY).16
For cytokine treatment, HKC
cells were seeded at 
70% confluence in complete medium containing
5% fetal bovine serum. Twenty-four hours later, the cells were changed
to serum-free medium, and recombinant TGF-ß1 (R & D Systems,
Minneapolis, MN) was added at a final concentration of 4 ng/ml except
where otherwise indicated. The cells and conditioned media were
collected at different time points for further characterization.
Animal Model
Male CD-1 mice weighing 20 to 22 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Unilateral ureteral obstruction (UUO) was performed using an established procedure.17 Briefly, under general anesthesia, complete ureteral obstruction was performed by double-ligating the left ureter using 4-0 silk after a midline abdominal incision. Sham-operated mice had their ureters exposed, manipulated but not ligated. Mice were sacrificed at different time points as indicated after surgery, and the kidneys were removed. One part of the kidneys was fixed in 10% phosphate-buffered formalin followed by paraffin embedding for histological and immunohistochemical studies. The remaining kidneys were snap-frozen in liquid nitrogen and stored at -80°C for protein extractions.
Western Immunoblot Analysis
HKC cells and cytokine-treated cells were lysed with sodium
dodecyl sulfate (SDS) sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol
blue). Kidney tissue was homogenized by a polytron homogenizer
(Brinkmann Instruments, Westbury, NY) and the supernatant was
collected after centrifugation at 13,000 x g at 4°C
for 20 minutes, as described previously.18
After protein
concentration was determined using a bicinchoninic acid protein assay
kit (Sigma Chemical Co., St. Louis, MO), the tissue lysate was mixed
with an equal amount 2x SDS sample buffer. Samples were heated at
100°C for 5 to 10 minutes before loading and separated on
precasted 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The
proteins were electrotransferred to a nitrocellulose membrane
(Amersham, Arlington Heights, IL) in transfer buffer containing 48
mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at
4°C for 1 hour. Nonspecific binding to the membrane was blocked for 1
hour at room temperature with 5% Carnation nonfat milk in TBS buffer
(20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The
membranes were incubated for 16 hours at 4°C with various primary
antibodies in TBS buffer containing 5% milk at the
dilutions specified by the manufacturers. The monoclonal -SMA
antibody was purchased from Sigma Chemical Co. The antibody for
E-cadherin was obtained from Transduction Laboratories (Lexington, KY).
The antibody against ß-actin was purchased from Santa Cruz
Biochemicals (Santa Cruz, CA). The anti-human matrix metalloproteinase
(MMP)-2 antibody was purchased from Oncogene Research Products
(Cambridge, MA). Binding of primary antibodies was followed by
incubation for 1 hour at room temperature with the secondary
horseradish peroxidase-conjugated IgG in 1% nonfat milk. The signals
were visualized by the enhanced chemiluminescence system (ECL,
Amersham), as described previously.18
Immunostaining
Indirect immunofluorescence staining was performed using an
established procedure on HKC cells cultured on coverslips. Briefly,
control or cytokine-treated HKC cells were washed with cold
phosphate-buffered saline (PBS) twice, and fixed with cold
methanol:acetone (1:1) for 10 minutes on ice. After extensive washing
with PBS containing 0.5% bovine serum albumin, the cells were blocked
with 20% normal donkey serum in PBS buffer for 30 minutes at room
temperature, and then incubated with specific primary antibodies
described above, except the rat monoclonal anti-E-cadherin (clone
DECMA-1) that was obtained from Sigma. The cells were then routinely
stained with fluorescein isothiocyanate-conjugated secondary antibodies
(Sigma). Cells were also stained with 4',6-diamidino-2-phenylindole,
HCl to visualize the nuclei. For visualizing F-actin, cells were
stained with tetramethylrhodamine isothiocyanate-conjugated phalloidin
(Sigma). Stained cells were mounted with anti-fade mounting
medium (Vector Laboratories, Burlingame, CA) and viewed on a Nikon
Eclipse E600 Epi-fluorescence microscope (Nikon, Melville, NY). For
immunostaining renal tissue, kidney sections from paraffin-embedded
tissues were prepared at 4-µm thickness using a routine
procedure.10
Immunohistochemical localization was
performed using the Vector M.O.M. immunodetection kit (Vector
Laboratories). The primary antibodies used were anti-E-cadherin and
anti--SMA (Sigma), anti-TGF-ß1 and anti-TGF-ß type I receptor
(Santa Cruz Biochemicals). As a negative control, the primary antibody
was replaced with either nonimmune mouse or rabbit IgG, corresponding
to species of the primary antibodies.
Gelatin Zymographic Analysis
Zymographic analysis of the MMP proteolytic activity in the
supernatant of cultured cells or kidney tissue homogenates was
performed according to the method described
previously.10,19
Briefly, an equal number of the HKC cells
were seeded on 6-well plates at a density of 4 x
105/well in Dulbecco’s modified Eagle’s
medium-F12 medium containing 5% fetal bovine serum. Twenty-four
hours later, culture medium was changed to 0.7 ml of serum-free medium,
and TGF-ß1 was added to the cultures. At different time points as
indicated, conditioned media were collected and centrifuged at
13,000 x g for 5 minutes to remove any cell debris.
The protein concentration was determined using a protein assay kit with
bovine serum albumin as a standard (Sigma). Kidney tissue homogenates
were prepared essentially according to the methods described by Kim and
colleagues.19
A constant amount of protein from the
conditioned media (15 µg) or kidney tissue homogenates (30 µg) was
loaded into 10% SDS-polyacrylamide gel containing 1 mg/ml gelatin
(Bio-Rad). After electrophoresis, SDS was removed from the gel by
incubation in 2.5% Triton X-100 at room temperature for 30 minutes
with gentle shaking. The gel was washed well with distilled
water to remove detergent and incubated at 37°C for 16 to 36 hours
in a developing buffer containing 50 mmol/L Tris-HCl, pH 7.6, 0.2 mol/L
NaCl, 5 mmol/L CaCl2, and 0.02% Brij 35. The gel
was then stained with a solution of 30% methanol, 10% glacial acetic
acid, and 0.5% Coomassie Blue G250, followed by destaining in the same
solution without dye. Proteinase activity was detected as unstained
bands on a blue background representing areas of gelatin digestion.
Determination of Basement Membrane Integrity by Bacterial Translocation
Bacterial translocation was performed for evaluating TBM integrity
using a two-compartment Boyden chamber with transwell filters
containing 3-µm diameter pores (Corning Co., Corning, NY). Matrigel
(Becton Dickinson Labware, Bedford, MA), a solubilized basement
membrane matrix consisting of laminin (56%), collagen IV (31%),
entactin (8%), and heparan sulfate proteoglycan (perlecan), was added
onto the transwell filters to form matrix gels at 37°C that
essentially reconstitute the TBM in vivo.20
Matrigel at a concentration of 22 µg/cm2
produced a matrix gel layer at 15-µm depth, which represents 100-fold
thickness of native TBM (150 nm).21
Preliminary studies
showed that the matrix gel on the transwell filters maintained its
structural integrity for >7 days at 37°C that completely blocked
bacterial translocation through the gel (data not shown). The transwell
filters with the Matrigel were then incubated with the conditioned
media (rich in MMP-2) from HKC cells treated with or without TGF-ß1
at 37°C for 4 days. Escherichia coli DH5 (Life
Technologies) was grown in Luria Broth (LB) medium at 37°C overnight
and bacterial concentration was estimated by reading at an optical
density of 600 nm with 1 optical density equivalent to
109
bacteria/ml.22
Approximately
108
bacteria in 100 µl were added to the upper
compartment of the Boyden chamber in a final volume of 400 µl.
Aliquots (20 µl) were removed at 0.5 and 2 hours, respectively, from
the bottom compartment of the chamber containing 1 ml of media.
Dilutions from each aliquot were plated on LB agar plates and
incubated at 37°C for 16 hours and colonies were counted. The entire
experiments were performed in triplicate for each time points per
treatment.
Boyden Chamber Motility Assay
Cell motility and migration were evaluated using Boyden chamber motogenicity assay with tissue culture-treated transwell filters (Costar).23 HKC cells (1 x 104) were seeded onto the filters (8-µm pore size, 0.33-cm2 growth area) in the top compartment of the chamber. After 2 or 5 days of incubation with or without TGF-ß1 at 37°C, filters were fixed with 3% paraformaldehyde in PBS, and stained with 0.1% Coomassie Blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that passed through the pores were counted in five nonoverlapping x20 fields and photographed with a Nikon microscope.
Matrigel Invasion Assay
Matrigel (1.43 mg/cm2) was added onto the transwell filters (8-µm pore size, 0.33-cm2 growth area) of the Boyden chamber to form matrix gels at 1.0-mm depth. HKC cells (1 x 104) in a volume of 100 µl were added onto the top of the gels. After 2 and 5 days of incubation with or without TGF-ß1 at 37°C, filters were fixed with 3% paraformaldehyde in PBS, and stained with 0.1% Coomassie Blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that invaded and migrated across the Matrigel and passed the transwell filter pores toward the lower surface of the filters were counted in five nonoverlapping x10 fields. The experiments were performed in triplicate cultures.
Determination of Tissue TGF-ß1 Levels by Enzyme-Linked Immunosorbent Assay
For measurement of tissue TGF-ß1 level, kidneys were homogenized in the extraction buffer containing 20 mmol/L Tris-HCl, pH 7.5, 2 mol/L NaCl, 0.1% Tween-80, 1 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L phenylmethyl sulfonyl fluoride, and the supernatant was recovered after centrifugation at 19,000 x g for 20 minutes at 4°C. Kidney tissue TGF-ß1 level was determined by using a commercial Quantikine TGF-ß1 enzyme-linked immunosorbent assay kit in accordance with the protocol specified by the manufacturer (R & D Systems). This kit measures the abundance of active TGF-ß1 protein that binds to its soluble type II receptor precoated onto a microplate. The concentration of tissue TGF-ß1 in kidneys was expressed as pg/mg total protein.
Statistical Analysis
All data examined were expressed as mean ± SE. For Western blot analysis, quantitation was performed by scanning and analyzing the intensity of the hybridization signals using NIH Imagine software. Statistical analysis of the data were performed by the Student-Newman-Keuls test using SigmaStat software (Jandel Scientific, San Rafael CA). A P value <0.05 was considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To demonstrate tubular epithelial to myofibroblast transition
in vitro, we examined the de novo expression of
-SMA, a phenotypic marker for myofibroblast cells, in human renal
tubular epithelial cells (HKC). As shown in Figure 1
, incubation of HKC cells with TGF-ß1
induced abundant expression of -SMA protein. Dose-dependence studies
revealed that TGF-ß1 was able to induce -SMA at a concentration as
low as 0.1 ng/ml, suggesting that this induction is readily achievable
in vivo under pathological conditions. The induction of
-SMA expression in tubular epithelial cells reached a plateau when
TGF-ß1 was >2 ng/ml.
|
shows the phenotypic conversion
of tubular epithelial cells after TGF-ß1 treatment. The transformed
cells displayed the presence of abundant -SMA-positive
microfilaments in the cytoplasm. Meanwhile, they totally lost the
staining of E-cadherin, an epithelial marker that is essential for the
structural integrity of renal epithelium (Figure 2)
. We observed
dramatic alteration in the organization of actin cytoskeleton. The
transformed cells underwent F-actin reorganization to form long stress
fibers (Figure 2)
. Consistent with these actin reorganizations that
often define cell morphology, the transformed cells lost the typical
cobblestone pattern of an epithelial monolayer, and displayed a
spindle-shape, fibroblast-like morphology (Figure 2)
. In addition,
TGF-ß1-treated cells expressed vimentin, a marker of mesenchymal
cells, and began to markedly produce fibronectin and collagen I (data
not shown). All together, these data suggest that tubular epithelial
cells, under appropriate stimulus, undergo a conversion process into
myofibroblasts in vitro.
|
To identify the early event essential for epithelial to
myofibroblast conversion, we investigated the time course of the gene
expression after TGF-ß1 treatment. As shown in Figure 3
, we found that loss of E-cadherin
expression was an early event that took place as early as 6
hours after TGF-ß1 treatment, whereas induction of de
novo expression of -SMA was a delayed response requiring 36
hours of incubation (Figure 3)
. Other changes in cell phenotype such as
induced vimentin and fibronectin expression as well as morphological
transformation also required longer periods of persistent incubation
with TGF-ß1 ranging from 2 to 5 days (data not shown). These results
establish that loss of E-cadherin expression probably is an early
event, which allows dissociation of structural integrity of renal
epithelia and collapse of epithelial polarity.
|
To gain insights into the mechanism underlying the destruction of
TBM in vivo, we examined the expression pattern of MMPs
during TGF-ß1-induced EMT. Zymographic analysis of conditioned media
exhibited that TGF-ß1 induced a marked increase in MMP-2 expression
and secretion in a dose-dependent manner (Figure 4A)
. TGF-ß1 not only induced pro-MMP-2
abundance, but also stimulated activation of pro-MMP-2, as demonstrated
by increased abundance of active MMP-2 in the conditioned media.
Time-course studies revealed that this induction was also a delayed
response that took place after 48 hours of incubation with TGF-ß1
(Figure 4B)
. TGF-ß1 also marginally increased MMP-9 activation, as
demonstrated by an increase in active MMP-9 abundance in zymographic
gels (Figure 4A)
. The induction of MMP-2 expression by TGF-ß1 in
tubular epithelial cells was independently confirmed by Western blot
analyses of the conditioned media (Figure 4)
.
|
, incubation of Matrigels with
conditioned media derived from TGF-ß1-treated cells markedly
disrupted the integrity of basement membrane matrix. Bacterial
translocation experiments revealed that there was little resistance for
bacteria to translocate from the upper to the bottom compartments of
the Boyden chamber across the matrix gels and 3-µm pores on the
transwell filters (Figure 5)
, suggesting that the integrity of
Matrigels is impaired, presumably because of the degradation by MMP-2
that is rich in the conditioned media. However, under the same
conditions, bacterial translocation was primarily blocked when the
Matrigels were incubated with conditioned media from control HKC cells
(Figure 5)
.
|
To understand how the transformed cells migrate and finally enter
the interstitial compartment in vivo, we studied the
motility of transformed cells by Boyden chamber assay. As shown in
Figure 6
, incubation of HKC cells with
TGF-ß1 for 2 days began to induce cell migration across the pores of
the transwell filters. Approximately 50% of the pores in transwell
filters were filled with cell extensions after 2 days of TGF-ß1
treatment, but no cells truly migrated through the pores of filters
toward the opposite side of the filters at this stage (Figure 6)
.
However, the control HKC cells had barely started to move toward the
pores of transwell filters after the same period of incubation. After 5
days of incubation, increased numbers of the cells in TGF-ß1-treated
groups actually migrated across the pores to the lower surface of the
filters, compared to that in control groups (Figure 6)
.
|
, incubation of HKC cells with TGF-ß1
for 5 days markedly promoted cell invasion into and migration across
the Matrigel. Most pores in transwell filters were filled with cell
extensions after 5 days of incubation with TGF-ß1, which resulted
from the cells invading and migrating across the Matrigels (Figure 7)
.
Under the same conditions, control HKC cells, without TGF-ß1
treatment, displayed a much less invasive property in this assay
(Figure 7)
. These data suggest that tubular epithelial cells on
stimulation by TGF-ß1 acquire new properties that are able to disrupt
TBM matrix and invade and migrate through the Matrigel, which
presumably allow them to move across the TBM and ultimately to enter
the interstitial compartments of the kidney in vivo.
|
To demonstrate tubular epithelial to myofibroblast conversion
in vivo under pathological conditions, we examined the
kidneys of mice with chronic renal disease induced by UUO. As shown in
Figure 8, a
rapid and marked induction of
both TGF-ß1 and its type I receptor (TßR-I) was observed in the
obstructed kidneys as early as 1 day after UUO, as demonstrated
quantitatively by enzyme-linked immunosorbent assay and Western blot
analyses. Immunohistochemical localization studies revealed that the
expression of TGF-ß1 and TßR-I was specifically induced in renal
tubular epithelia of the obstructed kidneys (Figure 8)
. This rapid,
tubule-specific induction of TGF-ß1 axis clearly precedes the
phenotypic changes observed in the obstructed kidneys in this model
(see below), suggesting that hyperactive TGF-ß1 signaling in
vivo specifically targets renal tubules and its induction is early
enough for initiating epithelial to myofibroblast transition under
pathological conditions.
|
-SMA (Figure 9)
. There was a reciprocally inverse
correlation between the loss of E-cadherin and de novo
expression of -SMA in the obstructed kidneys. These alterations in
cell phenotypes after UUO were coinciding with an increase in
TBM-degrading enzymes in these kidneys (Figure 10)
. Zymographic analysis of whole
kidney lysates revealed that MMP-2 and MMP-9 expressions were
dramatically increased in a time-dependent manner (Figure 10)
. All
together, these data essentially recapitulate our in vitro
findings and suggest that tubular epithelial to myofibroblast
conversion occurs in vivo as well, and presumably plays an
important role in the pathogenesis of renal interstitial fibrosis.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, these four key events include: 1)
loss of epithelial adhesion properties, 2) de novo
expression of -SMA and actin reorganization, 3) disruption of the
TBM, and 4) enhanced cell migration and invasion. TGF-ß1, a
well-characterized pro-fibrogenic cytokine,27,28
is
capable of inducing tubular epithelial cells to undergo all four
steps, and thereby, leads to the completion of the entire EMT course.
Our data establish that EMT is an orchestrated, step-wise process that
depends on hyperactive TGF-ß1 signaling and provide novel insights
into the mechanism underlying the myofibroblast activation under
pathological conditions.
|
-SMA
and Fsp1.13,14
However, it remains to be one of
the greatest challenges to distinguish those changes playing a key role
in EMT from those just associated with it. Because TGF-ß1 rapidly
suppresses E-cadherin expression in cultured tubular epithelial cells,
it is conceivable that loss of epithelial adhesion properties is an
early important step that precedes all other major events such as
induction of -SMA and MMP-2 expression. Because E-cadherin, the
well-characterized adhesion receptor found within adherens-type
junctions, plays an essential role in maintaining the structural
integrity of renal epithelia and in epithelial
polarization,31,32
its loss consequently allows
destabilization of the structural integrity of renal epithelium and
makes cells dissociate from their neighbors and lose polarity.
E-cadherin is linked to the actin filament network by catenins, a
family of intracellular adhesive junction proteins. The importance of
E-cadherin for development of normal epithelium has been established by
knock-out of its gene in mice33
and by its role in
embryonic epitheliogenesis during early nephrogenesis.34
Recent results further demonstrate that suppression of E-cadherin
expression alone, by the transcription factor Snail, induces EMT in
carcinoma cells.35,36
Because tubular epithelial cells and myofibroblasts locate in separate
tissue compartments in vivo, disruption of TBM will be of
fundamental importance in clearing the path for transformed cells to
migrate toward interstitium. In light of the fact that MMP-2
specifically cleave native type IV collagen and
laminin,24,37
the principal proteins found in the TBM, our
results on induction of MMP-2 during TGF-ß1-initiated EMT provide
significant insights into the mechanism underlying the destruction of
TBM in vivo (Figures 4 and 5)
. In accordance with this,
incubation of Matrigel, which essentially reconstitutes TBM as shown by
the similarity in its structure, composition, physical property, and
ability to retain functional characteristics typical of TBMs in
vivo,20
with conditioned media from the transformed
cells results in drastic destruction of its structural and functional
integrity as evidenced by the bacterial translocation assay (Figure 5)
.
Of note, induction of MMP-2 expression occurs at 48 hours after
TGF-ß1 incubation in vitro (Figure 4)
and at 3 days after
UUO in vivo (Figure 10)
, suggesting that destruction of TBM
is a delayed event that follows loss of epithelial adhesion as well as
-SMA de novo expression during the entire course of
tubular epithelial to myofibroblast transition. It is of interest to
note that because TGF-ß1 does not increase MMP-2 abundance in
cultured rat renal interstitial fibroblast (NRK-49F) cells (data not
shown), this induction of MMP-2 expressions in vivo is
contributed, at least in a large part, by tubular epithelial cells
under pathological conditions.
Because the transformed cells have to enter interstitial compartments,
it is essential for them to acquire the motile and invasive capacity to
eventually migrate into peritubular interstitium. The reorganization of
actin cytoskeleton, and induction of -SMA, may provide a structural
foundation not only for defining the morphology of the transformed
cells,38,39
but also for them to migrate, invade, and even
acquire the capacity for contractility. The observation that the
transformed cells are more motile suggests that the enhanced motility
could readily allow them to migrate through the TBM, which is already
destructed by elevated MMP-2 as discussed above, toward the
interstitial compartment. The transformed cells could, in reality,
combine the efforts of simultaneous destruction of TBM and migration.
Such a notion is experimentally confirmed by the Matrigel invasion
assay, in which transformed cells grown on top of Matrigel gels have
the ability to destroy and migrate through a reconstituted TBM matrix.
Of note, TGF-ß1 promotion of HKC cell motility and invasion is also a
late event that occurs after long periods of incubation. This suggests
that enhanced cell motility could be a consequence resulting from the
tubular epithelial to myofibroblast conversion. In addition,
myofibroblasts are morphologically intermediate between fibroblasts and
smooth muscle cells.4-6
Like fibroblasts, they produce
interstitial matrix components including collagens I and
III and fibronectin; and like smooth muscle cells, they retain -SMA
expression and have the ability to contract.40,41
The
possibility of these transformed cells to attain contractility, as
evidenced by the well-assembled -SMA microfilament fibers, implies
that contraction could potentially be another powerful force leading
the transformed cells toward the interstitium.
Major key events during EMT, such as loss of epithelial adhesion,
de novo expression of -SMA, and induction of
TBM-degrading enzymes, are recapitulated in the diseased kidneys in an
animal model of renal interstitial fibrosis. The fact that rapid,
tubule-specific induction of TGF-ß1 axis in the obstructed kidneys
suggests that, similar to in vitro situation, TGF-ß1 is
also responsible for initiating epithelial to myofibroblast transition
in vivo. However, suppression of E-cadherin expression
in vivo does not significantly precede other alterations
(Figure 9)
. This discrepancy between in vitro and in
vivo studies is probably because of the nature of heterogeneity in
cell population in the diseased kidneys. Unlike cultured epithelial
cells with homogeneous population, the response of tubular cells
in vivo is more complex so that loss of E-cadherin at an
early time point in a small percentage of the cell population may not
be readily detected by Western blot analyses of whole kidney lysate.
Consistent with this notion, loss of E-cadherin staining is observed in
renal epithelia in the areas that otherwise are relatively normal
(Figure 9)
, suggesting that disruption of epithelial adhesion perhaps
is an early event in vivo as well. Nonetheless, we cannot
exclude the possibility that in vivo once tubular epithelial
cells are initiated to undergo EMT, they may be programmed to progress
by simultaneously inducing suppression of E-cadherin, -SMA
expression, and TBM destruction.
TGF-ß1, as a sole factor, initiates and completes the entire EMT
course that consists of four key steps. This extraordinary ability of
TGF-ß1 leads one to re-think its roles and mechanisms in progressive
renal fibrosis. TGF-ß1 is widely considered as a key modulator of
organ fibrosis after a wide variety of tissue
injuries.27,28,42
Although TGF-ß1 stimulation of
fibroblasts to become activated is well documented, little is known
about the effects of TGF-ß1 on tubular epithelial cells in renal
fibrogenesis. Ironically, it is in the tubular epithelium where
TGF-ß1 receptors are rapidly and specifically up-regulated in
diseased kidneys43
(Figure 8)
, suggesting that tubular
epithelial cells are the natural targets of TGF-ß1 under pathological
conditions in vivo. Our current results indicate that the
pro-fibrogenic role of TGF-ß1 is mediated, at least in part, by
promoting myofibroblast activation via inducing tubular epithelial to
myofibroblast transition.
In summary, the results of our study suggest that tubular epithelial to myofibroblast transition is an orchestrated, highly regulated, step-wise process that depends on hyperactive TGF-ß1 signaling. Hence, disruption of any of these key steps could potentially offer unique opportunities to block the EMT process and, thereby, to inhibit myofibroblast activation and prevent renal interstitial fibrogenesis.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the National Institutes of Health (grants R01 DK-54922 and K02 DK-02611 to Y. L.) and in part by a Pathology Postdoctoral Research Training Grant from the Department of Pathology at the University of Pittsburgh School of Medicine (to J. Y.).
Accepted for publication July 11, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  B. Shan, T.-p. Yao, H. T. Nguyen, Y. Zhuo, D. R. Levy, R. C. Klingsberg, H. Tao, M. L. Palmer, K. N. Holder, and J. A. Lasky Requirement of HDAC6 for Transforming Growth Factor-{beta}1-induced Epithelial-Mesenchymal Transition J. Biol. Chem., July 25, 2008; 283(30): 21065 - 21073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Morrison, R. L. Viney, M. A. Saleem, and M. R. Ladomery New insights into the function of the Wilms tumor suppressor gene WT1 in podocytes Am J Physiol Renal Physiol, July 1, 2008; 295(1): F12 - F17. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Giannopoulou, C. Dai, X. Tan, X. Wen, G. K. Michalopoulos, and Y. Liu Hepatocyte Growth Factor Exerts Its Anti-Inflammatory Action by Disrupting Nuclear Factor-{kappa}B Signaling Am. J. Pathol., July 1, 2008; 173(1): 30 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Mao, Z. Li, Y. Zhou, Z. Li, S. Zhuang, X. An, B. Zhang, W. Chen, J. Nie, Z. Wang, et al. HSP72 attenuates renal tubular cell apoptosis and interstitial fibrosis in obstructive nephropathy Am J Physiol Renal Physiol, July 1, 2008; 295(1): F202 - F214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Diaz Encarnacion, G. M. Warner, C. E. Gray, J. Cheng, H. K. H. Keryakos, K. A. Nath, and J. P. Grande Signaling pathways modulated by fish oil in salt-sensitive hypertension Am J Physiol Renal Physiol, June 1, 2008; 294(6): F1323 - F1335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Ivanova, M. J. Butt, and D. G. Matsell Mesenchymal transition in kidney collecting duct epithelial cells Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1238 - F1248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Elberg, L. Chen, D. Elberg, M. D. Chan, C. J. Logan, and M. A. Turman MKL1 mediates TGF-{beta}1-induced {alpha}-smooth muscle actin expression in human renal epithelial cells Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1116 - F1128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Tan, W. He, X. Lin, L. P. Kiss, and Y. Liu Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1076 - F1083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. Laurent, R. J. McAnulty, M. Hill, and R. Chambers Escape from the Matrix: Multiple Mechanisms for Fibroblast Activation in Pulmonary Fibrosis Proceedings of the ATS, April 15, 2008; 5(3): 311 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Li, K. S. Hering-Smith, E. E. Simon, and V. Batuman Myeloma light chains induce epithelial-mesenchymal transition in human renal proximal tubule epithelial cells Nephrol. Dial. Transplant., March 1, 2008; 23(3): 860 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zeng, Y. Yao, M. Han, X. Zhao, X.-C. Liu, J. Wei, Y. Luo, J. Zhang, J. Zhou, S. Wang, et al. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt J. Am. Soc. Nephrol., February 1, 2008; 19(2): 380 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, Y. S. Kang, C. Dai, L. P. Kiss, X. Wen, and Y. Liu Epithelial-to-Mesenchymal Transition Is a Potential Pathway Leading to Podocyte Dysfunction and Proteinuria Am. J. Pathol., February 1, 2008; 172(2): 299 - 308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. He, H. Kubo, K. Ishizawa, A. E. Hegab, Y. Yamamoto, H. Yamamoto, and M. Yamaya The role of the receptor for advanced glycation end-products in lung fibrosis Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1427 - L1436. [Abstract] [Full Text] |
