1 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine,
University of Toronto, Toronto, Ontario, M5S 1A8 Canada
2 Department of Anatomy and Cell Biology, Faculty of Medicine, University of
Toronto, Toronto, Ontario, M5S 1A8 Canada
* Author for correspondence (e-mail: paul.hamel{at}utoronto.ca)
Accepted 26 October 2001
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Summary |
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Key words: Pax3, Mesenchymal-epithelial transition, Cell aggregation, Cell adhesion, c-met
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Introduction |
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Aborted organogenesis at the point of mesenchymal condensation or cell
aggregation is a feature common to mice with mutations in distinct Pax genes
(Dahl et al., 1997). The
Pax-family genes encode a class of transcription factors that are essential
for normal embryonic development. Originally identified as regulators of
pattern formation during Drosophila embryogenesis, nine different Pax
genes have been identified in mammals, Paxl to Pax9
(Walther et al., 1991
), all of
which encode proteins containing a DNA-binding motif termed the paired box
(Noll, 1993
). Analyses of
animals harboring naturally occurring or targeted mutations of different Pax
genes revealed their fundamental requirement for orchestrating proper
morphological development of various tissues and organs
(Dahl et al., 1997
). Some of
the many examples include Pax3 mutant mice, where maintenance of
epithelial aggregation in somitic cells of the dermomyotome is lost
(Daston et al., 1996
), and
Pax2 mutant mice, in which aborted kidney development occurs due to
the failure of the metanephric mesenchyme to condense and undergo epithelial
transformation (Torres et al.,
1995
). For these mutant animals, a role for Pax proteins in
regulating cell aggregation/mesenchymal condensation and/or
mesenchymal-to-epithelial transformation has been implied.
During mouse embryogenesis, Pax3 is expressed in several developing tissues
including the brain, dorsally throughout the neural tube, in neural crest
cells, the dermomyotome and in migratory somitic muscle precursors
(Goulding et al., 1991).
Mutations to Pax3 in mice results in the splotch (sp)
phenotype (Epstein et al.,
1991
). Homozygous mutant sp embryos exhibit a number of
developmental defects including impaired neural tube closure, absence of limb
muscles, persistent truncus arteriosus and defects to many neural
crest-derived structures (Auerbach,
1954
; Franz, 1989
;
Franz et al., 1993
). In
humans, heterozygous mutations to Pax3 cause Waardenburg syndrome,
which is characterized by pigmentation, and hearing and facio-skeletal
anomalies (Baldwin et al.,
1992
; Tassabehji et al.,
1992
). Recent studies have begun to provide insights into the
cellular and molecular processes that Pax genes may regulate during
embryogenesis. During muscle development, for example, Pax3 appears to
regulate the expression of myogenic determination factors
(Maroto et al., 1997
;
Tajbakhsh et al., 1997
) as
well as migration of muscle precursors via regulation of c-met expression
(Daston et al., 1996
;
Epstein et al., 1996
;
Yang et al., 1996
). However,
the cellular and molecular mechanisms by which Pax3 specifically regulates
morphogenesis remains elusive.
We show that ectopic Pax3 expression in osteogenic Saos-2 cells results in the formation of cell aggregates with epithelial characteristics. Ectopic Pax3 expression in Saos-2 cells leads to increased Ca2+-dependent cadherin-mediated intercellular adhesion and formation of polarized epithelium. Furthermore, we show that, although Pax3 induces phenotypic epithelialization of these mesenchymal cells, it also induces these cells to become competent to respond to a factor, specifically hepatocyte growth factor/scatter factor (HGF/SF), that reverts the epithelial phenotype. Our results reveal a novel activity for Pax3 and suggest a mechanism by which Pax genes may regulate pattern formation during development.
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Materials and Methods |
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Cell culture and metabolic labeling
Both human Saos-2 osterosarcoma and Rh30 rhabdomyosarcoma cells were grown
in DMEM supplemented with 10% fetal calf serum (Sigma, Oakville, ON, Canada).
Saos-2 cells do not express Pax3, determined by western blot analysis of
whole-cell lysates (O.W., unpublished). The Rh30 rhabdomyosarcoma expresses a
Pax3/FKHR fusion protein, which has been shown previously to exhibit
activities distinct from that of Pax3
(Epstein et al., 1998).
Metabolic labeling and immunoprecipitations were as previously described
(Hinck et al., 1994
).
Construction of adenoviruses and infection procedures
Adenoviruses were generated as previously described
(He et al., 1998). Briefly,
cDNA encoding either ß-galactosidase or an aminoterminal flag-epitope
tagged mouse Pax3 was subcloned into the pAdtrack vector. pAdtrack constructs
were linearized with PmeI and co-transformed with pAdeasy into BJ5183
bacterial cells. REcombinant viral DNA was isolated from BJ5183 cells and used
to transfect 293 cells to generate replication-deficient infectious adenoviral
particles. Viruses were harvested from 293 cells and titered on human C33A
cells by counting live green fluorescent protein (GFP)-positive cells at 24
hours postinfection. Viral titers were typically in the range of
1x108-1x109 expression forming units/ml. For
infection of Saos-2 or Rh30 cells, typically 1x106 cells were
plated on 60 mm plates the day before infection. The following day, cells were
infected at an m.o.i of 10 in a total of 4 ml of normal media. Media was
replenished at 2-3 day intervals.
Immunofluorescence and microscopy
Cells grown on glass coverslips were rinsed in PBS before being fixed in
freshly prepared 4% paraformaldehyde for 10 minutes at room temperature. For
ß-tubulin staining, cells were fixed for 10 minutes in 50% methanol/50%
acetone at room temperature. Coverslips were rinsed in PBS and cells were then
exposed to a solution of 0.2% Triton X-100 and 3% bovine serum albumin (BSA)
in PBS for 30 minutes for permeablization and to block nonspecific binding.
Cells were then incubated for 60 minutes at room temperature with the
appropriate primary antibody, diluted in 3% BSA in PBS. Cells were then rinsed
briefly 4-5 times with PBS and subsequently incubated with the appropriate
Texas Red-conjugated secondary antibody in 3% BSA in PBS for 50 minutes. After
four washes with PBS, coverslips were then mounted with vinol mountant
(Opas et al., 1996). For
F-Actin staining, cells were incubated with Rhodamine-conjugated phalloidin in
3% BSA in PBS for 50 minutes following permeablization, rinsed with PBS and
then mounted. GFP images were captured on a Zeiss axiophot microscope equipped
with a CCD camera. Confocal images were obtained on a Zeiss LSM microscope.
Unless otherwise indicated, confocal images represent projections of 10-15
optical sections acquired at 0.4 µm intervals. For
Fig. 7 (I-L), optical sections
were acquired at 0.8 µm intervals. Phase-contrast images were obtained with
a Nikon inverted microscope onto Kodak Plus-X pan 125ASA film. Morphometric
analysis was performed using Scion Image beta 3b, an adaptation of NIH Image
for PC (Scion Corporation, Fredrick, MD) and Image-1 (Universal Imaging, West
Chester, Pennsylvania) software. Scanning electron micrographs were obtained
on a Hitachi 570 microscope at 15 kV.
|
RNA isolation and RT-PCR
RNA was isolated using Trizol (Gibco, Burlington, ON, Canada) according to
the manufacturer's instructions. DNase treatment of the RNA and cDNA synthesis
was done essentially as described previously
(Munsterberg et al., 1995).
Exceptions were that 2 µg of RNA was used for cDNA synthesis, and the
reverse transcriptase utilized was Superscript II RT (Gibco). For PCR
amplification, 1 µl cDNA was used in a final volume of 50 µl with 1 unit
of Taq polymerase (Gibco). PCR products were amplified for 27 cycles, which
was determined to be in the linear range for all three products; 15 µl of
each PCR reaction were analyzed on a 2% agarose gel. Primers for c-met,
designed to be degenerate for detection of both human and mouse transcripts,
were as follows: 5'-GTG T/CTG GAA CAC CCA GAT TGT T-3', 5'-
CAA AGA AA/G TGA TGA ACC GGT CC-3' (nucleotides 265-587). Primers for
mouse Pax3 were 5'- CAG GAG ACA GGC TCC ATC CGA-3', 5'-CCT
TTC TAG ATC CGC CTC CTC-3' (nucleotides 271-522). Primers for GAPDH were
as described (Wang et al.,
1997
). The expected product sizes are as follows: c-met, 322bp;
Pax3, 251bp; GAPDH, 880 bp. PCR products were confirmed by restriction
digests. PCR products were quantified using NIH Image software.
HGF experiment
Conditioned media containing HGF/SF was obtained by transfection of COS
cells with a cDNA encoding human HGF/SF. Control conditioned media was
obtained by transfection with an empty pcDNA3 vector. Conditioned media was
harvested at 3 days posttransfection and HGF/SF titer determined in a MDCK
scatter assay as described previously
(Royal et al., 2000). Saos-2
cells were plated in 12-well plates and infected with control or
Ad-Pax3flag viruses at a multiplicity of infection (m.o.i.) of 10.
At 3 days postinfection, by which time Ad-Pax3flag-infected cells
had formed aggregates, cells were exposed to six scatter units of HGF
conditioned media or the equivalent volume of control conditioned media in a
total volume of 1 ml. Live cells were analyzed by phase contrast microscopy at
various times following HGF treatment for up to 24 hours.
Ca2+ switch assay
The Ca2+ switch assay was performed as described
(Gumbiner et al., 1988).
Briefly, Saos-2 cells were infected with control or Ad-Pax3flag
viruses in the presence of normal Ca2+-containing media. At 3 days
postinfection the media was replaced with low Ca2+-containing
medium. Live cells were subsequently examined at various times up to 24 hours
by phase contrast microscopy.
Western immunoblots and cell fractionation
For cell fractionation, cells were washed with cold PBS and then scraped
into lysis buffer (10 mM Tris.HCl (pH 8.0), 120 mM NaCl, 1% NP-40) plus a
cocktail of protease inhibitors. Lysates were vortexed and incubated at
4°C for 15 minutes followed by centrifugation at 17,000 g
for 15 minutes at 4°C. The supernatant (soluble fraction) was recovered
and the pellet, representing the insoluble material, was resuspended by
sonication in lysis buffer containing 1% SDS. The SDS content was diluted to
0.1% with lysis buffer. Total protein content was determined by Bio-Rad
protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada). For c-met,
flag-tag and actin immunoblots, cell lysates were prepared and used for
western blotting as described previously
(Wiggan et al., 1998).
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Results |
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Analysis of Saos-2 cells at three days postinfection by scanning electron microscopy revealed striking morphological changes in Ad-Pax3flag-infected relative to the control infected cells (Fig. 2). Control infected cells were loosely associated and had a flattened fibroblast-like appearance (Fig. 2A,B). Membrane blebbing was frequently observed on both control infected and Ad-Pax3flag-infected cells. In aggregates of Ad-Pax3flag-infected cells, cells were intimately associated, having almost indiscernible cell-cell boundaries (Fig. 2C,D). In these aggregates, cells exhibited extensive membrane ruffling over their entire dorsal surface. Other distinct morphological features evident in Ad-Pax3flag-infected cells relative to control infected cells included the presence of cilia of varying lengths and an apparent increase in height in these cells. Analysis of the height of individual cells by confocal microscopy revealed an average 60% increase in the height of AdPax3-infected cells relative to control infected cells (6.1±1.2 µm, n=30 vs. 3.7±0.6 µm, n=30). Together, these data show that ectopic Pax3flag expression induces morphologic alterations in cultured cells, which is consistent with a mesenchymal-to-epithelial phenotypic transformation.
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Epithelial junction formation of Pax3-expressing cells
The morphological changes induced by Pax3 predicted significant alterations
in intercellular adhesion properties of these cells. We first assessed whether
the Pax3-induced aggregation of Saos-2 cells was cadherin dependent by
determining whether aggregation was Ca2+ dependent. Saos-2 cells,
infected with Ad-Pax3flag, were induced to form aggregates by
maintaining them in the presence of normal Ca2+-containing medium
for three days (Fig. 3A). When
infected cultures were then switched to low-Ca2+-containing media,
the Pax3-induced aggregates began dissociating within two hours.
Fig. 3B illustrates that 24
hours after the switch to low-Ca2+-containing media, aggregated
cells had completely dispersed, supporting the notion that the Pax3-induced
cell aggregation involved the induction of Ca2+-dependent,
cadherin-mediated cell-cell adhesion.
|
Formation of epithelial type cell-cell contacts in
Ad-Pax3flag-infected Saos-2 cells was further characterized by
examining the distribution of cadherins and cadherin-associated proteins at
three days postinfection by indirect immunofluorescence. Transcripts for both
an alternatively spliced version of N-cadherin
(Cheng et al., 1998;
Ferrari et al., 2000
) and for
a type II-classical cadherin expressed principally in mesenchymal tissues,
cadherin-11/OB-cadherin (Cheng et al.,
1998
), have been detected in Saos-2 cells. Using a pan-cadherin
antibody, which recognizes a common C-terminal motif present in classical
cadherins, and which most likely detects a N-cadherin variant in Saos-2 cells
(see below), signal was localized in Ad-ßgal-infected cells to sites of
intercellular cell contact in a discontinuous, punctate or serrate pattern
(Fig. 4D). This staining
pattern resembles that described previously for spot-adherens junctions
described in nonepithelial cells (Yonemura
et al., 1995
). In Pax3flag-expressing cells, although
the pan-cadherin signal also localized to sites of cell contact
(Fig. 4E), the staining pattern
was distinct from control Ad-ßgal-infected cells. Specifically,
Pax3flag-expressing cells had a continuous band of cadherin
staining at sites of cell-cell contact, reminiscent of that observed at
adherens junctions of epithelial cells
(Yonemura et al., 1995
).
Moreover, cell-cell contacts were more extensive in Pax3-infected cells
relative to control cells.
|
Using a cadherin-11-specific antibody, we observed a distinct staining
pattern from that observed with pan-cadherin antibodies. In control infected
cells, cadherin-11 displayed a finger-like distribution at sites of cell-cell
contact (Fig. 4A). A similar
distribution of cadherin-11 was observed at sites of cell-cell contact in
loosely associated Ad-Pax3flag-infected cells
(Fig. 4B). By contrast,
aggregates of Pax3flag-infected cells displayed a strong reduction
in cadherin-11 staining at sites of cell-cell contact
(Fig. 4C). Cells in aggregates
generally displayed low levels of diffuse vesicular cadherin-11 staining.
Junctional and/or lateral membrane staining of cells at the margins of
aggregates was frequently observed. Dual labeling of cells with both
-pan-cadherin and
-cadherin-11 antibodies revealed that the
junctional signals detected by each antibody had different spatial
distributions, cadherin-11 signals being typically localized more apically
than those detected by pan-cadherin antibodies (data not shown).
We also characterized the subcellular distribution of - and
ß-catenin; both are cadherin-associated proteins important for adherens
junction formation and cytoskeletal communication. In Ad-ßgal-infected
cells, the pattern of
-catenin (Fig.
4F) and ß-catenin (Fig.
4G) was the same as that of pan-cadherin. Similarly, in aggregates
of Pax3flag-expressing cells,
-catenin
(Fig. 4H) and ß-catenin
(Fig. 4I) formed an enriched
continuous belt at sites of cell contact, a pattern identical to that of
pan-cadherin and consistent with that found in epithelial cell adherens
junctions.
We next examined the mechanism by which Pax3 induced increased cell
adhesion in Saos-2 cells. A previous study indicated that expression of
Wnt-signaling ligands may be regulated by Pax3 and other related Pax genes in
vivo (Mansouri and Gruss,
1998). Wnts have been shown to regulate cell adhesion by
regulating steady-state levels and subcellular distribution of cadherins or
associated catenins, or both (Bradley et
al., 1993
; Hinck et al.,
1994
). Thus, we assessed by means of a pulse-chase assay whether
expression of Pax3flag affected the rate of turnover of
ß-catenin. As shown in Fig.
5A, no significant difference was detected in the turnover rate of
ß-catenin in Ad-Pax3flag-infected cells relative to control
cells.
|
Increased cell adhesion is associated with redistribution of cadherins and
catenins from a cytoplasmic, detergent-soluble pool to a
cytoskeleton-associated, detergent-insoluble pool
(Bradley et al., 1993). Thus,
we determined the subcellular distribution of cadherins and catenins by
biochemical fractionation followed by western blotting
(Fig. 5B). Anti-pan-cadherin
antibodies detected a single band of approximately 140 kDa in cell extracts of
Saos-2 cells. Anti-E- and P-cadherin antibodies failed to recognize this 140
kDa band; neither did these antibodies detect these cadherins by
immunohistochemical analysis (data not shown). Although not detected by
western analysis or cell staining using commercially available N-cadherin
antibodies, we predict that this 140 kDa cadherin is a variant of N-cadherin
based on its mass (Volk and Geiger,
1984
) and the presence of N-cadherin transcripts in Saos-2 cells.
The top panel in Fig. 5B
illustrates the reduction (36±6%) of the cadherin detected by
-pan-cadherin antibodies in the soluble fraction of
Ad-Pax3flag-infected cells, and its reciprocal increase in the
insoluble fraction relative to control Ad-ßgal-infected cells. Western
blots with antibodies to cadherin-11 detected a single band of approximately
120 kDa (Fig. 5B, second
panel). In contrast to the type I classical cadherin detected using the
pan-cadherin antibody, Pax3flag-expression caused a
three-to-fourfold decrease in cadherin-11 in the detergent-soluble fraction
but no commensurate increase in the insoluble fraction
(Fig. 5B, second panel). Like
the pan-cadherin product, ß-catenin was detected in both the soluble and
insoluble fractions of control and Ad-Pax3flag-infected cells.
Expression of Pax3flag also resulted in altered distribution of
ß-catenin, as 28±1% of total ß-catenin was found in the
insoluble fraction of AdPax3flag-infected cells vs. 21±1% in
control infected cells. A small but reproducible decrease in the apparent mass
of ß-catenin in the insoluble fraction of Pax3-expressing Saos-2 cells
was also observed. Thus, ectopic Pax3 expression induces the formation of
epithelial-type cell junctions, in part, by inducing altered subcellular
distribution of cadherins and catenins.
Pax3 induces formation of epithelial apico-basal cell polarity and
cytoskeletal rearrangement
One hallmark of epithelial cells is their distinct apico-basal cell
polarity, these cells exhibiting distinct apical and basal structural and
membrane domains (Davies and Garrod,
1997). We determined whether phenotypic epithelioid
Pax3flag-expressing Saos-2 cells developed apico-basal polarity by
characterizing the distribution of the tight junction-associated protein,
ZO-1. Optical sections through control or Ad-Pax3flag-infected
cells revealed the presence of ZO-1 at lateral membrane cell-cell contact
sites (Fig. 6). In control
infected cells, ZO-1 signal is detected throughout the entire lateral contact
sites along the apico-basal axis (Fig.
6A-D,M). By contrast, in both single-layered
(Fig. 6E-H,N) and multilayered
aggregates (Fig. 6I-L) of
Pax3flag-expressing cells, ZO-1 became concentrated at the apex of
lateral cell-cell junctions. These data confirm that Pax3 induces a
morphological epithelial conversion of Saos-2 cells resulting in the formation
of tight junctions and cells that have acquired significant apico-basal cell
polarity.
|
The Pax3flag-induced morphological changes in Saos-2 cells led us to examine whether there were alterations to the cytoskeletal architecture. Immunofluorescence staining at three days postinfection revealed that Ad-ßgal-infected cells possessed many F-actin stress fibers present throughout the cell at all levels along the apico-basal axis (Fig. 7A-D). By contrast, in aggregates of tightly associated Pax3-expressing cells, actin filaments formed distinct structures along the apico-basal axis. In these cells, thick actin bundles at the cell cortex was observed basally (Fig. 7E). Also basally, very few stress fibers were present, with actin localized to cell-cell junctions. At the level of the nucleus, strong actin staining was observed at cell-cell junctions and peripherally around the nucleus (Fig. 7F). Apically, at the submembrane cortex, actin formed wavy-like bundles that were associated with the formation of dorsal membrane ruffles in these cells (Fig. 7G).
We also examined the distribution of the cytoskeletal protein vinculin. In
ßgal-expressing cells, vinculin was enriched at sites of focal adhesions
(Fig. 7I). In aggregates of
Pax3flag-expressing cells there was a significant reduction in the
number of focal adhesions. In these cells, vinculin concentrated at sites of
cell-cell contact, at the tips of filopodia and diffusely throughout the
cytoplasm (Fig. 7J). The
presence of vinculin at sites of cell-cell contact in aggregates of
Pax3flag-expressing cells is consistent with the formation of
epithelial-type adherens junctions
(Yonemura et al., 1995).
Dramatic rearrangements to the microtubule-based cytoskeleton occurs during
the formation of polarized epithelial cells
(Bacallao et al., 1989;
Grindstaff et al., 1998
). We
therefore examined microtubule organization by confocal immunohistochemical
analysis with antibodies to ß-tubulin. In control infected Saos-2 cells,
microtubules emanate radially towards the cell periphery and run parallel to
the axis of the cell substratum at all levels along the apico-basal axis
(Fig. 8A-D). In aggregates of
Pax3-expressing cells, a dramatic reorganization of the microtubules had
occurred. Basal to the nucleus, microtubules formed a mat of criss-crossed
parallel microtubules, with punctate dot-like staining also observed
(Fig. 8E,I). At the level of
the nucleus, ß-tubulin staining was punctate, appearing as bright dots
localized peripherally around the nucleus and along the lateral edges of cells
(Fig. 8F,J). These punctate
dots are indicative of microtubules oriented parallel to the apico-basal axis
(Bacallao et al., 1989
).
Apically, above the level of the nucleus, microtubules also formed a dense
web-like mat with many bright punctate structures
(Fig. 8G,K). This organization
for microtubules was observed only in aggregated
Pax3flag-expressing Saos-2 cells. Furthermore, this arrangement is
restricted to polarized epithelial cells
(Bacallao et al., 1989
) and
provides further evidence for a mesenchymal-to-epithelial transformation
induced by Pax3 in these cells.
|
Epithelial-to-mesenchymal phenotypic reversion in response to
HGF/SF
In vertebrates, limb muscles are derived from a population of somitic cells
that migrate from the lateral dermomyotome to the developing limb bud
(Cossu et al., 1996).
Migration requires the Pax3-dependent expression in these cells of a receptor,
c-met (Bladt et al., 1995
;
Daston et al., 1996
;
Epstein et al., 1996
), and
expression of its ligand, HGF/SF, from cells in the proximal regions of the
adjacent limb bud (Dietrich et al.,
1999
; Schmidt et al.,
1995
). HGF/SF induces dissociation of the c-met-expressing cells
at lateral tips of the epithelial dermomyotome, facilitating their migration
to the limb bud (Brand-Saberi et al.,
1996a
; Heymann et al.,
1996
). We determined, therefore, whether
Pax3flag-induced morphological changes in Saos-2 cells were
accompanied by induction of c-met and whether these cells could respond to
morphogenic signaling by HGF/SF. As the RT-PCR signal and western blot reveal
(Fig. 9A), persistent levels of
exogenous Pax3flag expression caused a 1.5-to-2-fold increase of
endogenous c-met transcript by 24 hours. A sevenfold increase in c-met message
was detected by 72 hours. These changes in transcript levels were translated
into a twofold increase of c-met protein within 48 hours and greater than
fourfold by 72 hours (Fig.
9B).
|
We determined next whether the Pax3-dependent induction of endogenous c-met allowed Saos-2 cells to respond to HGF/SF. Six units of human HGF/SF were added to aggregated Ad-Pax3flag- or control Ad-ßgal-infected cells. Fig. 9D shows the complete dissociation and phenotypic epithelial-to-mesenchymal reversion of Pax3flag-expressing cell aggregates within 24 hours of HGF/SF treatment (compare Fig. 9C with D). Reversion to a mesenchymal phenotype is supported by shape changes, from a cuboidal morphology to a more flattened stellate morphology, and an increase of the average cell surface area of these HGF-treated cells from 306 µm2 to 1234 µm2, which was not significantly different to that of control cells. Moreover, after HGF/SF treatment, many Pax3flag-expressing cells assumed an elongated motile appearance. This effect of HGF/SF was not due to the normal levels of c-met expressed in Saos-2 cells as treatment of uninfected (not shown) or Ad-ßgal-infected cultures (Fig. 9E,F) with HGF/SF displayed no significant alteration of cell morphology.
Taken together, our data reveal a novel activity of Pax3 where it induces cell aggregation and a phenotypic mesenchymal-to-epithelial transition in phenotypically mesenchymal cell lines. However, Pax3 simultaneously establishes a novel cell state where these epithelialized cells can now respond to a subsequent HGF/SF-induced epithelial-to-mesenchymal transition.
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Discussion |
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Our finding that Pax3 regulates cell adhesion and epithelial cell
morphogenesis in vitro provides a mechanism for the developmental defects that
arise when Pax3 activity or that of other Pax-family proteins is defective or
absent in vivo. During vertebrate embryonic development, for example, the
epithelial somite differentiates into an epithelial dermomyotome and a
mesenchymal sclerotome (Brand-Saberi et
al., 1996b). Pax3 is normally expressed throughout the entire
epithelial dermomyotome at E9.5 of murine embryonic development. In homozygous
Pax3-deficient Splotch (sp) mice, the ventrolateral aspect
of the dermomyotome looses its epithelial morphology at this developmental
stage (Daston et al., 1996
).
The medial portion of the dermomyotome in these animals retains an epithelial
morphology. This latter portion corresponds to the domain, where expression of
a closely related Pax gene, Pax7, is maintained, indicating potential
redundant activities for Pax3 and Pax7 in the maintenance of epithelial
morphology of the dermomyotome (Daston et
al., 1996
).
Previous studies have further implicated a role for Pax3 in regulation of
intercellular cell adhesion during vertebrate neurulation. During neural tube
closure in normal murine development, neuroepithelial cells are tightly
associated, exhibiting very little extracellular spaces. Significant
intercellular spaces between neuroepithelial cells were observed, however, in
the unfused neural tube of both Pax3 sp and spd
embryos (Morris and O'Shea,
1983; Yang and Trasler,
1991
). Thus, during neural tube development, Pax3 promotes and/or
maintains cadherin-mediated intercellular adhesion, as well as the epithelial
architecture of neuroepithelial cells. The mechanisms by which Pax3 regulates
cell adhesion and phenotypic mesenchymal-epithelial transformation are not yet
clear. Wnt ligands regulate both cell adhesion
(Bradley et al., 1993
;
Hinck et al., 1994
) and
mesenchymal-epithelial transitions
(Kispert et al., 1998
;
Stark et al., 1994
). In
addition, the expression of distinct Wnt ligands has been shown to be
regulated by Pax proteins (Kim et al.,
2001
; Mansouri and Gruss,
1998
). Our data showed an induction in junctional cadherin and
catenins following ectopic Pax3 expression. This increase was not a
consequence of increased cadherin or catenin protein levels, nor was it due to
stabilization of the ß-catenin, as no significant change in the protein
half life of ß-catenin in response to Pax3 expression was observed.
Rather, increased junctional cadherin and catenins was a result of
redistribution of the detergent-soluble cytosolic pool of these proteins.
These results are consistent with a role for Wnt activity in mediating
increased cell adhesion induced by Pax3. Additionally, examination of the
subcellular distribution and activity of both canonical and non-canonical
Wnt-signaling components has provided evidence for the activation of a
non-canonical Wnt-signaling cascade during Pax3 induced cell aggregation and
phenotypic mesenchymal-epithelial transition
(Wiggan and Hamel, 2002
).
Interestingly, this non-canonical Wnt signaling cascade, which entails
activation of Jun-N-terminal kinase (JNK), has been implicated to regulate
changes to cytoskeletal architecture and cell shape
(Sokol, 2000
). The dramatic
Pax3-induced changes to the cytoarchitecture described herein, coupled with
our finding that several components of this non-canonical Wnt-signaling
cascade localize to the actin cytoskeleton, further suggests a role for Wnt
activity downstream of Pax3 in mediating aspects of phenotypic
mesenchymal-epithelial transition in Saos-2 cells. It remains to be
determined, however, whether Pax3 regulates cell adhesion and
mesenchymal-epithelial transition by inducing the expression of specific Wnt
ligands.
The function of cadherin-11 in mesenchymal tissues is poorly understood.
The fact that cadherin-11 is present at cell-cell junctions in loosely
associated cells but its protein levels decrease during the formation of
tightly aggregated polarized cells suggests that this cadherin may be involved
in the initial aggregation of cells, but may be antagonistic to the
maintenance of epithelial-type cell-cell junctions. Interestingly, during
similar mesenchymal-epithelial transformations that occur during both somite
and kidney development, cadherin-11 is expressed in the condensed mesenchyme
but is not detected in their epithelial derivatives
(Hoffmann and Balling, 1995;
Kimura et al., 1995
;
Simonneau et al., 1995
).
Taken together with previous studies, our evidence for Pax3-induced
formation of condensed cell aggregates in two different phenotypically
mesenchymal cell lines defines an apparent common function for the Pax-family
proteins. Mesenchymal condensation is initiated at the early stages of tooth,
hair and kidney formation, chondrogenesis, osteogenesis and in the formation
of many other organs (Thesleff et al.,
1995). Mesenchymal condensation is also a pivotal early step in
the mesenchymal-epithelial transitions that occur during somitogenesis and in
nephrogenesis (Davies and Garrod,
1997
). Our observation that ectopic Pax3 induces the formation of
condensed cell aggregates and mesenchymal-epithelial transition suggests that
Pax proteins may directly control the mesenchymal condensation process in the
genesis of specific organs and tissues during development. Our studies
indicate further that the initiation of mesenchymal condensations induced by
Pax genes may occur, at least in part, through regulation of cell size and
intercellular cell adhesion. We note that Pax3 does not induce cell
aggregation in all cell types, for example fibroblasts (3T3), to the same
extent as in Saos-2 and Rh30. We hypothesize that the ability of Pax3 to
induce cell aggregation may depend, in part, on preexisting expression and/or
sufficient levels of, for example, specific cadherins and catenins. In
addition, we showed previously that the activity of Pax3 could be regulated by
the pRB-family proteins (Wiggan et al.,
1998
). Our data here showed a significant effect of Pax3
expression on the pRB-deficient osteosarcoma line, Saos-2. By contrast, a
negligible effect of Pax3 expression on the osteosarcoma line, U2OS, which
expresses a functional pRB protein, was observed (O.W., unpublished).
Furthermore, inactivation in Saos-2 cells of the pRB-related factors, p107 and
p130, by expression of the human papilloma virus protein E7, enhanced the
ability of Pax3 to alter the Saos-2 phenotype (O.W., unpublished).
Finally, it is interesting to note that ectopic Pax3 expression did not
alter cell fate along a myogenic lineage in Saos-2 cells. A previous study
indicated that ectopic Pax3 expression induces myogenesis in various embryonic
tissues (Maroto et al., 1997).
Pax3 did not induce myogenesis in Saos-2 cells as judged by myogenin staining
(data not shown). Thus, the ability of Pax3 to induce myogenesis appears to be
distinct from its ability to regulate epithelial morphogenesis, the former
potentially requiring the activity of distinct cell-specific cofactors.
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