Early role of Fsp1 in epithelial-mesenchymal
transformation
Hirokazu
Okada,
Theodore M.
Danoff,
Raghuram
Kalluri, and
Eric
G.
Neilson
Penn Center for Molecular Studies of Kidney Disease,
Renal-Electrolyte and Hypertension Division, University of
Pennsylvania, Philadelphia, Pennsylvania 19104-6144
 |
ABSTRACT |
A seamless plasticity exists among cells shifting
between epithelial and mesenchymal phenotypes during early development
and again later, in adult tissues, following wound repair or organ remodeling in response to injury.
Fsp1, a gene encoding a
fibroblast-specific protein associated with mesenchymal cell morphology
and motility, is expressed during epithelial-mesenchymal
transformations (EMT) in vivo. In the current study, we identified
several cytokines that induce Fsp1 in cultured epithelial cells. A
combination of these factors, however, was most efficacious at
completing the process of EMT. The optimal combination identified were
two of the cytokines classically associated with fibrosis, i.e.,
transforming growth factor-
1 (TGF-
1) and epidermal growth factor
(EGF). To confirm that it was the induction of Fsp1 by these cytokines
mediating EMT, we used antisense oligomers to block Fsp1 production and subsequently measured cell motility and markers of EMT phenotype. The
antisense oligomers suppressed Fsp1 expression and epithelial transformation; therefore, we conclude that the appearance of Fsp1 is
an important early event in the pathway toward EMT.
transforming growth factor-
1; epidermal growth factor; antisense; fibrosis; fibroblast; cell motility
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INTRODUCTION |
VERTEBRATES ACHIEVE THEIR structural complexity during
early development, in part, by undergoing primary
epithelial-to-mesenchymal transformations (EMT). EMT at this stage
permits primitive cells of the skeleton, connective tissue, and organ
anlagen to redistribute in the body plan as a phenotype capable of
movement (33, 34). Many of these repositioned mesenchymal cells are
subsequently induced back into secondary epithelium, where they enter
cell fate maps for pattern formation, spatial position, and stationary organ structure. Mature secondary epithelium assembles into functional units in these organ tissues using extracellular contact junctions between neighboring cells as well as attachments to underlying basement
membrane (18). These specialized cell attachments help determine cell
polarity and transport vectors built out from a rigging of highly
organized cytoskeletal fibers and signaling networks. Mesenchyme not
utilized to reform epithelium is attenuated by apoptosis (40, 52, 72).
The molecular programs that guide these events, to the extent they are
known, are typically restrictive, decisional, tissue specific, and in
most cases reflect changes in transcription modulated by morphogenic
cues (13, 43, 48, 75, 80).
Adult fibroblasts appear late in vertebrate development, probably the
result of EMT from secondary epithelium (50, 76), and remain quiescent
in the interstitial and perivascular spaces of organ and connective
tissues. The process of local conversion and stimulation of new
fibroblasts can be accelerated during wound healing or tissue
inflammation (33, 34, 36, 57, 77, 88). However, these repair responses
at maturity typically disturb the structure of complex epithelial units
by inundating that microenvironment with excessive connective tissue.
During these responses, it appears that new fibroblasts formed by EMT
(77) retain a permanent mesenchymal state, as long as inciting stimuli
persist (16, 70, 85). When this happens, fibrogenesis in adult tissues
can be relentless.
EMT has also been observed in cell culture systems (3, 35, 36).
Epithelium in culture can lose polarity, adherence to adjacent cells
and basal lamina, convert into elongated fusiform shapes, and gain
mesenchymal properties including motility (36). Growth factors (9, 24,
38, 61, 71, 86), oncogenes (6, 7, 74), and cell surface adhesion
molecules (6, 46, 94) have been proposed as modulators of EMT; however, the sequential coordination of events has not been established.
Recently, we cloned Fsp1, a murine fibroblast-specific protein (77)
that belongs to the calmodulin-S100-troponin C superfamily of
intracellular calcium-binding proteins (77). The members of this family
have been implicated in microtubule dynamics (17, 21, 53, 68),
cytoskeletal-membrane interactions (4, 21, 25, 31, 59, 64), calcium
signal transduction (21, 28), cell-cycle regulation (49), and cellular
growth and differentiation (11, 15, 41, 58, 59). Although the precise
functions of Fsp1 and its homologs are not entirely clear, their
interaction with nonmuscle myosin II (23), nonmuscle tropomyosin (83), actin (27, 82, 87), or tubulin (53, 67), as well as the inducibility of
a migratory or metastatic phenotype when transfected into nonmetastatic
cells in vitro (14, 22, 30, 68), suggests that Fsp1 may be associated
with mesenchymal cell shape and motility. Tubular epithelium
transfected with cDNA encoding Fsp1
exhibited several properties of EMT, including a reduction
in cell adhesion, cytokeratin, and new expression of vimentin (77). In
this report, we describe the cytokine inducers of Fsp1 that promote
EMT.
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MATERIALS AND METHODS |
Cell culture. Renal proximal tubular
epithelial cells (MCT cells), NIH-3T3 fibroblasts (3T3), and
tubulointerstitial fibroblasts (TFB cells) have been maintained in
culture in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal calf serum (FCS), 100 U/ml enicillin, and 100 µg/ml
streptomycin (1, 32). In EMT experiments, the medium was replaced with
serum-free K-1 medium (50:50 Ham's F-12/DMEM with 5 µg/ml
transferrin, 5 µg/ml insulin, and 5 × 10
8 M hydrocortisone)
containing various concentrations of cytokines. For the assay of
secreted collagens, K-1 medium was additionally supplemented with
ascorbic acid (50 µg/ml) and
1-aminopropionitrile (50 µg/ml).
Microscopic examination was performed during each experiment to assess
the morphological changes of MCT cells prior to sample analysis.
Reagents. Recombinant human
transforming growth factor (TGF)-
, tumor necrosis factor-
(TNF-
), TGF-
1, epidermal growth factor (EGF), platelet-derived
growth factor-
1 (PDGF-
1), basic fibroblast growth factor (basic
FGF), hepatocyte growth factor (HGF), interleukin-2
(IL-2), Mullerian inhibitory factor (MIF), RANTES, murine IL-1
,
IL-4, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from R & D Systems (Minneapolis, MN), and
phorbol 12-myristate 13-acetate was purchased from Sigma (St. Louis, MO). The following antibodies were used: rabbit anti-Fsp1 (77); rabbit anti-vimentin (77); TROMA-1 and -3, rat monoclonal anti-cytokeratins (45); mouse monoclonal anti-
-smooth muscle actin
(anti-
-SMA; Sigma); rat monoclonal anti-mouse
syndecan-1 (Pharmingen, San Diego, CA); rat anti-ZO-1 (Chemicon,
Temecula, CA); alkaline phosphatase (ALP)-conjugated goat anti-rabbit
immunoglobulin G (IgG), anti-rat IgG (Sigma); fluorescein
isothiocyanate (FITC)-conjugated F(ab')2 goat anti-rabbit
IgG, anti-mouse IgG, and anti-rat IgG (Zymed). Anti-type I collagen and
anti-NC1 domain of type IV collagen were raised in rabbit to acid
solubilized rat tail collagen and bovine NC1 domain of type IV
collagen, respectively (44). FITC-conjugated phalloidin (Sigma) was
used to detect F-actin. Cell culture plates coated with rat tail type I
collagen, Engelbreth-Holm-Swarm type IV collagen, mouse
laminin, human fibronectin, and Matrigel basement membrane matrix were
obtained from Collaborative Research (Bedford, MA).
Direct enzyme-linked immunosorbent
assay. Cells (5 × 104 cells/well) were plated in
12-well plates and grown in DMEM with 10% FCS overnight. Then the
medium was replaced with serum-free K-1 media containing various
concentrations of cytokines. After 48 h culture, cells were harvested
by trypsin/EDTA, spun down and suspended in phosphate-buffered saline
(PBS). The number of cells was counted in a hemocytometer. A quantity
of 5 × 104 cells were
pelleted, then lysed in 500 µl of 6 M guanidine
hydrochloride, buffered to pH 7.5 with 50 mM
tris(hydroxymethyl)aminomethane (Tris) hydrochloride; 96-well
multititer enzyme-linked immunosorbent assay (ELISA) plates were coated
in triplicate with 100 µl of cell lysate (44). The
plates were incubated overnight at room temperature. After coating, the
plates were washed three times with 0.15 M NaCl and 0.05% Tween 20 washing solution. After washing, the plates were blocked with 2%
bovine serum albumin (BSA) and 0.1% Tween 20 in PBS incubating buffer
for 30 min at 37°C. After blocking, the plates were again washed
with the washing solution and then incubated with 1:1,000 dilution of
anti-Fsp1 or in some experiments with 1:500 dilution of anti-vimentin
or anti-cytokeratin in the incubation buffer. Preimmune serum or IgG
was used as control. The plates were incubated for 1 h at room
temperature. After primary antibody incubation, the
plates were again washed and subsequently incubated with 1:1,000
dilution of ALP-conjugated secondary antibodies in the incubation
buffer for 1 h at room temperature. Subsequently, the plates were again
washed thoroughly, and substrate, disodium p-nitrophenyl phosphate (5 µg/ml),
was added. After color development, the absorbance was measured with an
ELISA autoreader at 450 nm.
For assay of the secreted collagens,
105 cells/well were plated in
six-well plates and grown overnight in DMEM with 10% FCS. At that
point the medium was replaced with serum-free K-1 medium supplemented
with ascorbic acid and
1-aminopropionitrile containing various
concentrations of cytokines. After 72 h, the supernatants and the cells
were harvested separately, and the number of cells was counted on
hemocytometer. A volume of 200 µl of the supernatants/well in
triplicate was applied into the ELISA plates and dried under negative
pressure for 48 h at room temperature. The direct ELISA assay was
carried out with 1:1,000 dilution of anti-type I collagen or type IV
collagen as described above. The results were normalized for the cell
numbers.
Immunocytochemistry. Cells were grown
on Lab-Tek slides (Nunc) and stimulated with cytokines for 48-72
h. The medium was removed, and the cell layer was rinsed with 1 mM
CaCl2 and 0.5 mM
MgCl2 in PBS. For
the immunostaining of Fsp1, cytokeratins, vimentin, and
-SMA, the
cells were fixed and permeabilized with acetone-methanol for 20 min at
20°C. For the staining of
syndecan-1, ZO-1, and the detection of F-actin, the cells
were fixed with freshly prepared 4% paraformaldehyde in 1 mM
CaCl2 and 0.5 mM
MgCl2 in PBS for 30 min at room
temperature and permeabilized with 0.5% Triton X-100 in PBS for 4 min.
The cells were rehydrated with PBS, blocked with 5% BSA in PBS for 1 h, and incubated with a primary antibody for 1 h at room temperature.
After washing with PBS, bound antibodies were detected using
FITC-conjugated secondary antibodies described above and analyzed by
fluorescence microscopy (77). Negative controls were performed using
nonimmune serum or IgG instead of first antibodies. F-actin was
detected directly using FITC-conjugated phalloidin.
Flow cytometric analysis.
Cytokine-treated cells were lifted from the surface of the culture
plate by gentle pipetting of monolayers incubated in 1 mM
EDTA-Tris-buffered saline (25 mM Tris · HCl, pH 7.6, and 150 mM NaCl) on ice. The cells were spun down and
washed with ice-cold washing buffer, 1% BSA in 0.5 mM EDTA-PBS. This washing step was repeated four times. A
quantity of 106 cells were
suspended in 50 µl of staining buffer, 1% BSA-PBS, and incubated
with 1:100 dilution of anti-syndecan-1 for 20 min on ice. The cells
were then washed two times with washing buffer. Subsequently, the cells
were resuspended in 50 µl of staining buffer and incubated with 1:500
dilution of FITC-conjugated goat anti-rat IgG for 20 min on ice. The
cells were then washed two times with washing buffer and fixed with 1%
paraformaldehyde in PBS. The fixed cell samples were stored at 4°C
under shade until assay, and the assay was carried out within 1 wk.
FACScan analysis (Becton-Dickinson) was performed on
104 cells using CellQuest software
(32). Controls were performed using isotype-matched rat IgG and
FITC-conjugated antibodies.
Inhibition of in vitro EMT by Fsp1 antisense
oligodeoxynucleotides. Phosphorothioate-capped
oligodeoxynucleotides (oligomers) were synthesized on an automated
synthesizer (Applied Biosystems, Foster City, CA). After deprotection,
oligomers were dissolved in water, extracted with
phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and
redissolved in water. The Fsp1 sense oligomers sequence comprised
5' CACGGTTACCATGGCAAGAC 3', and antisense oligomers sequence comprised 5' GTCTTGCCATGGTAACCGTG 3'; these
sequences were chosen as likely to corrupt ribosomal docking by their
location near the initial site of translation. Another oligomers used
as a mismatch oligomers was a degenerate antisense sequence (5'
GTCNTGNCATGGNAANCGNG 3'). Oligomers were introduced into cells by
permeabilization with streptolysin O (5). Briefly, 2 × 105 cells were suspended in 0.5 ml
of permeabilization buffer [137 mM NaCl, 100 mM
piperazine-N,N'-bis(2-ethanesulfonic
acid); pH 7.4, 5.6 mM glucose, 2.7 mM KCl, 2.7 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1 mM sodium ATP, 0.1% BSA] containing 0.2 U/ml
streptolysin O (Sigma) and 60 µM oligomers. After 5 min incubation at
room temperature, 5 ml of DMEM with 10% FCS was added. The cells were
immediately pelted, seeded into two wells of six-well plates, and grown
overnight in DMEM with 10% FCS. At that point the medium was replaced
with serum-free K-1 medium supplemented with EGF and TGF-
1. In case
of collagen synthesis assay, ascorbic acid and
1-aminopropionitrile
were also added. After 36 h, morphological alterations were checked, and the cell layer was partly scratched by a sterile razor blade. After
subsequent 12 h, cell migration across the scratched area was
evaluated. Supernatant and cells were collected, and biochemical assay
for Fsp1, cytokeratins and collagens were carried out described above.
Statistics. Results are
presented as means ± SE. The analysis of variance
(Scheffé t-test) was performed
where appropriate; significance of results was indicated when
P < 0.05.
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RESULTS |
Cytokines and extracellular matrix molecules affect
expression of Fsp1 protein in MCT epithelium. All
experiments were carried out using syngeneic TFB and tubular epithelium
(MCT) as cell lines (1, 32). Expression of Fsp1 mRNA and protein in TFB
or other fibroblasts is abundant under basal culture conditions but
absent in MCT epithelium by Western and Northern blotting (77).
Numerous cytokines at varying concentrations were cocultured with MCT
cells so that the expression of Fsp1 protein could be analyzed by
ELISA; only peak doses are reported. In Fig.
1A,
where the largest incremental response, if any, of each cytokine at
peak concentration is illustrated, EGF (10 ng/ml), TGF-
(1 ng/ml),
and TGF-
1 (3 ng/ml) robustly increased Fsp1 expression in MCT cells
at 48 h. Northern analysis confirmed these results at the RNA level
(data not shown). The effects observed with EGF or TGF-
were not
likely the result of cell proliferation, because HGF was
not effective, despite the strong promotion of cell proliferation
(proliferation data not shown). GM-CSF (0.1 ng/ml), basic FGF (1 ng/ml), PDGF-
1 (1 ng/ml), IL-6 (1 ng/ml), IL-1
(1 ng/ml), and
phorbol 12-myristate 13-acetate (30 ng/ml) also were coincubated with
MCT cells, but no significant Fsp1 response was observed (data not
shown).

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Fig. 1.
De novo expression of Fsp1 by renal proximal tubular epithelial cells
(MCT cells). A: Fsp1 protein level of
MCT treated with humoral factors determined by direct enzyme-linked
immunosorbent assay (ELISA). Values are relative to tubulointerstitial
fibroblast (TFB) cells. Transforming growth factor- 1 (TGF- 1),
TGF- , and epidermal growth factor (EGF) induce de novo expression of
Fsp1. B: Fsp1 protein level of MCT
grown on different extracellular matrix molecules determined by direct
ELISA. MCT grown on type I collagen express Fsp1.
C: Combined effects of potent humoral
factors and type I collagen determined by direct ELISA. HGF, hepatocyte
growth factor. * Statistically significant,
P < 0.05.
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MCT cells were also tested for an Fsp1 response after culturing on
various extracellular matrix (ECM) molecules. Type I
collagen was most effective in elevating Fsp1 expression by ELISA in
MCT cells (Fig. 1B). The effects of
EGF, TGF-
1, and type I collagen were also tested to observe the
additive action of these Fsp1 inducers. The effects of cytokines EGF or
TGF-
1 were not much different than the effects of type I collagen
alone on the Fsp1 expression in MCT epithelium, and the inductive
effect was not intensified when combined in coculture (Fig.
1C); the combination of type I
collagen and EGF, however, seemed slightly more effective.
EGF and TGF-
1 synergistically
induce EMT in MCT epithelium.
Although the addition of EGF (10 ng/ml) or TGF-
1 (3 ng/ml) alone to
MCT cells in culture increased Fsp1, EMT-related morphological changes
on light microscopy were not complete, as treated cells only showed a
somewhat elongated appearance compared with controls (Fig.
2A vs.
B and
C). However, cotreatment of MCT
cultures with EGF (10 ng/ml) and TGF-
1 (3 ng/ml) together produced a
more obvious and consistent alteration in the shape of MCT cells,
resulting in elongated, spindle-shapes characteristic of fibroblasts
(Fig. 2D). All these alteration in
morphology, even partial changes, accompanied the de novo expression of
Fsp1 (Fig. 3,
A-D).
The EMT effect of peak doses of EGF (10 ng/ml) or TGF-
1 (3 ng/ml) did not produce more Fsp1, probably because the cytokine doses had been
titrated to produce near maximal effects when used alone.

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Fig. 2.
Fsp1-inducible cytokines affect MCT morphology.
A: control MCT grown on plastic
surface without any treatment. They grow as monotonous, cuboidal cell
sheet. B: MCT treated with EGF show
slightly elongated, less cuboidal appearance.
C: MCT treated with TGF- 1 become
fusiform in shape, losing cell-cell adhesion.
D: TGF- 1 in combination with EGF
induces drastic morphological change in MCT, suggesting more complete
epithelial-to-mesenchymal transformation (EMT). Magnification for
A-D,
×70.
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Fig. 3.
De novo expression of Fsp1 of MCT detected by immunocytochemistry.
A: control MCT are negative for Fsp1.
B: MCT treated with EGF are strongly
positive for Fsp1. C and
D: MCT treated with TGF- 1 alone
(C) and in combination with EGF
(D) are also positive for Fsp1.
Magnification for
A-D,
×160.
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To further characterize the EMT of MCT cells following treatment with
EGF and TGF-
1, we examined whether changes in other phenotypic
markers besides cell shape correlated with morphological transformation; the two cytokines, EGF and TGF-
1, were used
together, because alone they did not consistently produce predicted
changes. After exposure to cytokine, we stained MCT cells for the
expression of cytokeratins, ZO-1, and syndecan-1 as epithelial markers
using immunocytochemistry, and vimentin,
-SMA, and the intracellular distribution pattern of F-actin as mesenchymal markers.
The expression of cytokeratins were observed in control MCT cells and
cells treated with EGF, but MCT cells treated with EGF and TGF-
1
were negative for cytokeratins (Fig. 4,
A and
B). MCT cultures treated with
TGF-
1 alone contained cytokeratin-positive cells, and
these findings on immunocytochemistry were confirmed by direct ELISA
(Fig.
5A).
Control MCT cells were positive for ZO-1 and syndecan-1 (Fig. 4,
C and
E), which appeared as
circumferential staining at the boundaries between neighboring cells.
However, following treatment with TGF-
1 and EGF, staining for ZO-1
and syndecan-1 largely decreased in parallel with the loss of formation of cuboidal sheets of MCT cells during EMT (Fig. 4,
D and
F). The phenotypic transformation of
MCT cells following treatment with TGF-
1 alone, again, was uneven
and incomplete; residual immunostaining of ZO-1 and syndecan-1, for
example, partially remained at the end of the culture
period (data not shown). Quantitative analysis of
syndecan-1 expression was also performed, and fluorescence-activated cell sorting (FACS) analysis indicates that the total cell surface expression of syndecan-1 was not changed by these treatment (data not
shown).

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Fig. 4.
Transdifferentiated MCT treated with EGF and TGF- 1 show reduced
epithelial markers, increased mesenchymal markers, and reorganized
F-actin bundles. A: control MCT are
positive for cytokeratins in the cytoplasmic pattern.
B: MCT treated with EGF and TGF- 1
lose cytokeratin expression. C:
control MCT are positive for ZO-1 at their cell-cell boundaries.
D: transdifferentiated MCT by EGF and
TGF- 1 are negative for ZO-1. E:
control MCT are positive for syndecan-1 at their cell-cell boundaries.
F: treatment with EGF and TGF- 1
abolish syndecan-1 expression of MCT.
G: control MCT are negative for
vimentin. H: MCT treated with EGF and
TGF- 1 are positive for vimentin. I:
control MCT are negative for -smooth muscle actin ( -SMA).
J: treatment with EGF and TGF- 1
induce -SMA in MCT. K: F-actin
distributes at the cell-cell boundaries of control MCT.
L: F-actin bundles reorganize into the
stress fiber pattern in MCT treated with EGF and TGF- 1.
Magnification for
A-L,
×150.
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Fig. 5.
Quantitative assay of phenotypic markers by direct ELISA.
A: Effects of EMT related cytokines on
cytokeratin expression of MCT. Treatment with EGF and TGF- 1
significantly reduce epithelial cytokeratin expression in MCT during
EMT. B: TGF- 1 alone and in
combination with EGF increases mesenchymal vimentin expression in MCT.
* Statistically significant, P < 0.05.
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Meanwhile, the expression of vimentin and
-SMA in MCT cells
increased following treatment with TGF-
1 in combination with EGF
(Fig. 4, G to
J). Direct ELISA assays were also
performed to analyze the level of expression of vimentin in MCT cells.
These studies demonstrated that the level of expression of vimentin increased during EMT (Fig. 5B). In
addition, F-actin is generally connected with zonula adherens in
epithelial cells and was detectable as a continuous line at the
cell-cell boundaries in the untreated MCT cells (Fig.
4K). Following treatment with
TGF-
1 and EGF, F-actin was reorganized during EMT into
longitudinal stress fibers (Fig.
4L). All the data regarding changes
in phenotypic markers are summarized in Table
1.
Alteration in collagen synthesis during
EMT. Fibroblasts are a major source of interstitial ECM
(50), particularly collagen types I and III and to a much lesser extent
type IV collagen; the collagen secretory profiles of cultured
fibroblasts derived from different tissues are heterogeneous (76). In
the present study, we examined the changes in the intracellular content
of types I and IV collagen in MCT cells following EMT and compared the
results to TFB fibroblasts. In Fig. 6, the
collagen ratio of type I to type IV increased in the presence of
TGF-
1; in data not shown, both increased, but type I increased more,
whereas with EGF, type I decreased slightly relative to unchanged type IV content. Quantitatively, untreated MCT cells secrete more type IV
than type I collagen (1, 32).

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Fig. 6.
Quantitative assay of collagen synthesis by direct ELISA. Relative
secretion of types I and IV collagen in MCT cells was determined after
stimulation of cultures by EGF and TGF- 1 alone or in combination.
Combination of EGF and TGF- 1 best shifts the ratio of collagens
toward the profile observed in TFB cells. * Statistically
significant, P < 0.05.
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Fsp1 antisense oligomers can inhibit cell motility and
phenotype. Fsp1 antisense oligomers suppressed de novo
expression of Fsp1 protein in MCT cells treated with EGF and TGF-
1,
but sense and mismatch oligomers were without effect (Fig.
7). Trypan blue exclusion assay showed no
measurable toxicity (data not shown). EGF and TGF-
1 also induced EMT
morphology, decreased cytokeratin proteins, and increased type I
collagen synthesis in MCT cells treated with sense or mismatch
oligomers but not with antisense (Fig. 8).
Finally, since mesenchymal cells are typically motile while epithelium
is not, we examined the effect of these Fsp1 oligomers in a scratch
motility study. In this experiment, control MCT epithelium did not move
into the cell-free region of the slide during a 12-h
interval, regardless of the presence of any oligomers (Fig.
9, A and
B). MCT cells induced toward EMT
with EGF and TGF-
1 migrated a visible distance over 12 h
even in the presence of sense oligomers (Fig. 9,
C and
D) or mismatch oligomers (data not
shown); however, migration was substantially attenuated in the presence
of Fsp1 antisense oligomers (Fig. 9, E
and F).

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Fig. 7.
Effects of Fsp1 antisense oligomers on Fsp1 protein expression.
Fsp1 protein expression was determined with cell lysates by direct
ELISA assay. Significant increases in Fsp1 expression by EGF and
TGF- 1 treatment were observed in control MCT epithelium, MCT cells
treated with Fsp1 sense oligomers, and MCT cells treated with mismatch
oligomers. In contrast, de novo expression of Fsp1 was blocked by
antisense oligomers. * Statistically significant,
P < 0.05.
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Fig. 8.
Effects of Fsp1 antisense oligomers on cytokeratin and collagen type I
expression in MCT cells induced by coculture with EGF and TGF- 1.
Relative expression of cytokeratin and collagen type I was determined
by direct ELISA assay in a representative experiment.
A: expected levels of native
cytokeratin expression were observed in control MCT epithelium, MCT
cells treated with Fsp1 sense, antisense, or mismatch oligomers. In
contrast, native expression of cytokeratin was inhibited by treatment
with EGF and TGF- 1 and EGF and TGF- 1 plus sense or mismatched
oligomers but not by EGF and TGF- 1 plus antisense oligomers.
B: expected increases in collagen type
I expression following treatment with EGF and TGF- 1 were observed in
MCT epithelium and in MCT cells cotreated either with Fsp1 sense
oligomers or mismatch oligomers. In contrast, more expression of
collagen type I by treatment with EGF and TGF- 1 could be blocked by
coincubation with antisense but not sense or mismatched oligomers.
* Statistically significant, P < 0.05.
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Fig. 9.
Effects of Fsp1 antisense oligomers on cell motility induced by EGF and
TGF- 1 treatment. A and
B: MCT epithelium treated with Fsp1
sense oligomers barely moved in 12 h
(A, 0 h;
B-12 h).
C and
D: MCT cells transformed with EGF and
TGF- 1 and treated with Fsp1 sense oligomers moved from their
starting place (C, 0 h;
D, 12 h).
E and
F: movement of MCT cells transformed
with TGF- 1 and EGF was inhibited when the cells were treated with
Fsp1 antisense oligomers (E, 0 h;
F, 12 h). For
A-F,
all magnifications are ×100.
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 |
DISCUSSION |
We have begun to identify the conversion drivers of EMT in epithelium
obtained from adult organ tissue. One of the markers of fibroblast
formation is the expression of Fsp1, a 10-kDa cytoskeletal protein
belonging to the calmodulin-S100-troponin C superfamily of
intracellular calcium binding proteins associated with cytoskeletal fibers, cell motility, and a mesenchymal phenotype (93). We initiated
the current study to better describe the early role of Fsp1 in EMT.
The process of EMT begins with destabilization of the differentiated
state of candidate epithelium. The loss of epithelial adhesion
properties is an early permissive event (46, 55) that is followed by
the engagement of promoter factors that signal the transformation to
completion (36). Inducers of EMT have been examined in other systems.
EGF (60) and TGF-
1 (61), for example, have shown some special
promise. Other growth factors like TGF-
(24), MIF
(86), acidic FGF (9), and a commercial serum substitute (10) have also
been shown to induce EMT. These initiators of normal transformation
trigger second-order signals that involve selected oncogenes, like
v-src, (7)
v-ras,
v-mos (6), and
c-fos (74); uncontrolled
expression of these proteins facilitates oncogenesis (73) and may
relate to the mesenchymal characteristics of some tumors. Furthermore,
type I collagen gel can induce cultured epithelium to lose cell
polarity and become fusiform in shape (95), and we have previously
shown that renal epithelial cells submerged in type I collagen 3D gels
lost cytokeratin expression while acquiring Fsp1 (77).
From among many combinations of cytokines tested in our experiments, no
one cytokine alone induced all characteristics of EMT in MCT
epithelium. EGF alone did promote proliferation and an elongated shape
in MCT cells, probably due to de novo expression of Fsp1. In other
systems, immortalized mammary epithelial cell lines can transiently
lose epithelial markers, desmoplakins, and become motile by EGF
treatment (60). In our studies, however, the transformation was
incomplete, as EGF-treated MCT cells were still rich in epithelial
markers (20). The actions of EGF on MCT cells also seemed different
from those of HGF, another potent epithelial cell growth factor that
stimulates epithelial tubulogenesis and branching morphogenesis in
vitro (78). HGF, in particular, failed to increase expression of Fsp1
in MCT cells. All this is consistent with the in vitro observation that
EGF can facilitate the phenotypic drift toward stromal cells (89), and,
if anything, HGF encourages the opposite effect (45).
TGF-
1 also appears to be one of the principal initiating factors for
EMT (38, 61, 71). MCT epithelium treated with TGF-
1 became fusiform
in shape and rich in mesenchymal markers including Fsp1. Even though
several mesenchymal markers appear in MCT epithelium treated with
TGF-
1, the response toward EMT also did not go to completion because
the cells still expressed abundant epithelial markers, like
cytokeratins and ZO-1, and continued synthesize type IV collagen.
It clearly induces EMT in mammary epithelium (61), but in the case of
renal epithelium, EMT initiated by TGF-
1 alone is not entirely
sufficient (12, 38, 39, 61).
Because single cytokine effects were incomplete inducers of EMT, we
also examined cytokine combinations. Combined treatment with EGF and
TGF-
1 could fully effect EMT and together were strong inducers of Fsp1 protein and a mesenchymal phenotype, suggesting in
renal epithelium that EGF and TGF-
1 compensate for each other and
synergistically promote the transformation of epithelium.
Some interactions between EGF and TGF-
1 on epithelial biology have
been published (38, 39, 42, 47), but they mostly focus on their
conflicting effects on epithelial cell growth. The mechanism of the
antiproliferative effect of TGF-
1 on EGF relates to inhibition of
c-myc transcription (69), replacement of EGF receptor with lower affinity receptors, p53-mediated
G1 cell cycle arrest (42), or
induction of ECM molecules that can affect target cell responses (90).
The effect of TGF-
1 on Fsp1 in our experiments, for example, can be
connected through new synthesis of type I collagen, which in turn
stimulates the production of Fsp1. Both EGF and TGF-
1 can also
induce the appearance of new forms of the adenosine
3',5'-cyclic monophosphate response element binding
proteins (CREB) in renal epithelial cells (2), which may explain the
additive effect of those factors, if CREB is involved in
transcriptional regulation of EMT-related molecules. TGF-
1 also
increases EGF receptor mRNA in rat kidney fibroblasts and synergizes
with EGF to stimulate growth in soft agar, a characteristic of the
transformed phenotype (37). Finally, the antiproliferative effects of
TGF-
on epithelium in culture is associated with apoptotic cell
death, and this tendency is obviated by the presence of EGF (79). These
observations suggest that collective interactions between these two
cytokines may confer a stabilizing benefit or survival advantage to
cells changing phenotype in a cytokine-rich environment. The threshold
quantification of this effect is difficult, however, without absolute
biological standards of measure.
MCT cells induced to undergo EMT with EGF and TGF-
1 also demonstrate
an increase in vimentin and
-SMA, a reorganization of F-actin into
stress fibers, and an altered collagen synthesis profile showing more
type I collagen synthesis than type IV collagen. In addition to the
morphological change, immunochemistry and FACS analysis for syndecan-1,
a substrate adhesion molecule, indicated a reversion of expression from
a cell-cell boundary to global cell-surface boundary,
possibly as a result of loss in cell polarity.
In our study, we observed that de novo expression of Fsp1 preceded the
EMT phenotype and capacity for movement in MCT epithelium, as it could
be blocked with Fsp1 antisense oligomers. This finding supports our
previous observation that overexpression of cDNA encoding Fsp1 in
epithelium could effect a mesenchymal phenotype (77).
Acquiring the capacity for motility is one cardinal feature of a
metastatic phenotype (50, 54), and the expression of Fsp1 appears
emblematic of that process as well (29, 30, 68, 81). Fsp1 protein
participates in motile events probably by the interaction with
nonmuscle myosin II (23), nonmuscle tropomyosin (83), or actin (27, 82,
87) by which protrusion of lamellipods, detachment of the
cell rear, and translocation of the cell body forward occur (54).
Finally, several lines of evidence also point to TGF-
1 as a key
modulator of organ fibrosis following tissue injury (8, 19, 56, 66, 76)
with supportive contributions from TGF-
(51), TNF-
(62), PDGF
(84), and GM-CSF (91). The focus of this notion has emphasized the role
of TGF-
1 in promoting the infiltration and activation of
inflammatory cells and stimulating fibroblasts to proliferate or
produce fibrogenic proteins. Little has been said, however, of the
origin of these fibroblasts (76). Early de novo expression of vimentin
(63) or Fsp1 (66, 77) has been reported in resident epithelial cells of
nephritic kidneys. EMT initiators identified in this study, like EGF
and TGF-
1, are also quite common in the nephritic kidneys (26, 92).
Single cells or loosely organized small cell clusters still positive for epithelial markers can be found in the widened interstitium of the
advanced or end-stage kidney (65).
These observations collectively suggest, as a hypothesis, that renal
epithelium sitting on basement membrane damaged during the inflammatory
phase of tubulointerstitial nephritis and exposed to TGF-
1 and EGF
in that microenvironment begin expressing Fsp1 and
"mesenchymalize" into fibroblasts. These fibroblasts further flood the interstitium with fibrotic collagen types I and III, and this
in turn, sustains the transformation of more epithelium leading to
tubular atrophy and progressive renal failure.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-07006, DK-30280, DK-41110,
DK-02334, and DK-45191 and by research administrative and educational
funds from the DCI RED Fund. Hirokazu Okada was a
recipient of a fellowship from Eli-Lilly Japan, and received financial
support from Takeda Science Foundation.
 |
FOOTNOTES |
Address for reprint requests: E. G. Neilson, C. Mahlon Kline Professor
of Medicine, 700 Clinical Research Bldg., Univ. of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6144.
Received 8 January 1997; accepted in final form 12 June 1997.
 |
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