1 Keratinocyte Laboratory, CR-UK London Research Institute, 44 Lincoln's Inn
Fields, London WC2A 3PX, UK
2 Pfizer Global Research and Development, Sandwich Data Centre, Sandwich CT13
9NJ, UK
Author for correspondence (e-mail:
fiona.watt{at}cancer.org.uk)
Accepted 24 June 2003
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Summary |
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Key words: Keratinocyte, Differentiation, Carcinogenesis, Skin, Smad
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Introduction |
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Whereas integrin expression is normally confined to the basal layer of the
epidermis, expression is frequently perturbed in keratinocyte tumours. The
alteration that is most heavily implicated in epithelial carcinogenesis is
upregulated expression of the 6ß4 integrin
(Rabinovitz and Mercurio,
1996
). Suprabasal expression of
6ß4 in keratinocytes
that are not adjacent to the tumour stroma correlates with poor prognosis in
human squamous cell carcinomas (SCCs)
(Rabinovitz and Mercurio,
1996
; van Waes et al.,
1995
). In mouse skin, suprabasal expression of
6ß4 is
observed in those benign papillomas with a high risk of conversion to SCCs
(Tennenbaum et al., 1993
).
Mechanistic studies of 6ß4 have emphasized its role in
promoting invasion by stimulating epithelial cell motility
(Mercurio et al., 2001
). In
invasive carcinomas,
6ß4 can be found associated with the actin
cytoskeleton in the absence of hemidesmosomes and it is becoming clear that
6ß4 is capable of ligand-independent signal transduction
(Mercurio et al., 2001
).
Mobilization of
6ß4 from hemidesmosomes occurs in response to
chemotactic factors and is correlated with phosphorylation of the ß4
cytoplasmic domain (Mainiero et al.,
1996
).
6ß4 cooperates with growth factor receptors to
activate phosphoinositide 3-kinase (PI3-K), and PI3-K activation is necessary
for
6ß4-mediated invasiveness
(Mercurio et al., 2001
). Human
keratinocytes lacking
6ß4 are resistant to the tumourigenic
effects of Ras and NF-
B (Dajee et
al., 2003
).
Whereas there is strong evidence that 6ß4 promotes invasion of
carcinoma cells, little is known about how integrin overexpression influences
the early stages of tumourigenesis. It is also unclear how inappropriate
expression of
6ß4 in the differentiated compartment of a tumour
could influence the growth and metastatic potential of undifferentiated cells
in the basal layer. To address these questions, we have generated transgenic
mice in which
6ß4 is expressed in the suprabasal layers of the
epidermis under the control of the involucrin promoter
(Carroll et al., 1995
). We
demonstrate that suprabasal
6ß4 has a profound and positive
influence on the susceptibility of keratinocytes to forming tumours, relieving
the growth-inhibitory effects of TGFß via a mechanism that requires
E-cadherin-mediated cell-cell adhesion and PI3-K activity.
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Materials and Methods |
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The Inv6 transgenics, described previously
(Romero et al., 1999
), and the
Invß4 mice were derived in an F1 hybrid (C57Bl/6xCBA) mouse strain,
then backcrossed for at least seven generations onto a homogeneous FVB/N
background. The Invß4
mice were generated directly in the FVB/N
strain. Inv
6ß4 and Inv
6ß4
double-transgenic and
wild-type (wt) experimental mice were generated by crossing heterozygous
6 single transgenics to heterozygous ß4 or ß4
single
transgenics. Animal husbandry was as described previously
(Owens and Watt, 2001
).
Tumour studies
Seven-week-old female Inv6ß4, Inv
6ß4
and wt
littermate mice (25 animals/group) were shaved once on the dorsal surface with
electric clippers. After one week, all animals that did not show signs of hair
regrowth received one topical application of 100 nmol (25 µg)
7,12-Dimethylbenz[a]anthracene (DMBA; Acros Organics) in 200 µl acetone or
200 µl acetone alone. One week later, mice received twice weekly
applications of 6 nmol (3.7 µg)
12-O-tetradecanoylphorbol-13-acetate (TPA; LC Laboratories) in 200
µl acetone or 200 µl acetone alone for 15 weeks.
Benign and malignant skin tumours were recorded once weekly for up to 52
weeks after the start of promotion. The statistical significance of
differences in papilloma and SCC formation between transgenic and wt mice was
determined with a Student's t-test. To confirm weekly papilloma and
SCC counts, tumour sections were graded as described previously
(Owens and Watt, 2001). The
experiment comparing Inv
6ß4 and wt mice was performed twice, as
was the experiment comparing Inv
6ß4, Inv
6ß4
and
wt mice.
Immunohistochemistry
Endogenous and transgenic integrin expression was examined in frozen skin,
tumour and lymph node sections, essentially as described previously
(Owens and Watt, 2001), using
antibodies to human
6 (MP4F10), human ß4 (3E1; Life Technologies)
or an antibody that detects mouse
6ß4 (CD49f; Serotec).
5-bromo-2'-deoxyuridine (BrdU) incorporation was examined in
formalin-fixed sections (Owens and Watt,
2001
) from mice that received an i.p. injection of 100 mg/kg BrdU
1 hour prior to sacrifice. For keratin 14 and Smad staining, formalin-fixed
sections were microwaved in Citra Plus antigen retrieval solution (Biogenex)
for 8 minutes (keratin14) or 21 minutes (Smad). Slides were cooled, blocked in
10% normal goat serum and probed overnight at 4°C with rabbit anti-keratin
14 (Babco), anti-Smad2/3 (Transduction Laboratories) or anti-phosphoSmad2
(kind gift of C. Heldin, Ludwig Institute, Uppsala, Sweden).
TGFß responsiveness of cultured keratinocytes
Spontaneously immortalized mouse keratinocyte lines were derived from
Inv6ß4 and wt adult mouse epidermis and maintained with a feeder
layer as described previously (Romero et
al., 1999
). In some experiments, cells were transduced with
retroviral vectors encoding a dominant-negative E-cadherin
(H-2Kd-E-cad) or a control construct in which the
ß-catenin-binding site in the cadherin cytoplasmic domain was deleted
(H-2Kd-E-cad
C25) (Zhu
and Watt, 1996
).
To measure TGFß responsiveness, transgenic and wt mouse keratinocytes
were plated onto glass coverslips and grown to form confluent monolayers or
for up to 5 days post-confluence. Cells were treated with 2 ng/ml TGFß1
(Pepro Tech EC) for 1 hour to induce Smad2/3 nuclear translocation. Cells were
labelled with the anti-Smad2/3 antibodies (Transduction Laboratories)
(Pierreux et al., 2000) or
anti-human
6 integrin antibodies
(Romero et al., 1999
)
described above.
Stratified cultures were also reconstituted as follows. Transgenic and wt
mouse keratinocytes were induced to differentiate in suspension
(Romero et al., 1999) and then
seeded onto confluent monolayers of transgenic or wt mouse keratinocytes and
allowed to attach for 18 hours. Cultures were treated with 2 ng/ml TGFß1
for 1 hour and stained for Smad2/3
(Pierreux et al., 2000
) or
involucrin (Owens and Watt,
2001
).
For inhibitor studies, cells were pre-incubated with 10 µM U0126 MEK
inhibitor (Promega), 50 nM LY 294002 PI3-K inhibitor (Sigma) or
dimethylsulphoxide for one hour before treatment with TGFß1. In some
cases, medium conditioned for 6 or 24 hours by 5-day postconfluent stratified
Inv6ß4 or wt keratinocyte cultures was incubated with monolayer
cultures of keratinocytes.
To measure BrdU incorporation, keratinocytes were serum starved for 24 hours, transferred to complete medium ±2 ng/ml TGFß1 for 19 hours and then pulsed with 80 µg/ml BrdU for 1 hour. Cells were fixed in 3.7% formaldehyde and permeabilized in 2 M HCl/0.5% Triton X-100 followed by treatment with 0.1 M NH4Cl, then immunolabelled with a monoclonal BrdU antibody (Becton Dickinson).
Immunoblot analysis
Confluent monolayers and 5-day post-confluent stratified cultures were
treated with 2 ng/ml TGFß1 or PBS for 1 hour, washed with cold PBS and
scraped from the dish in RIPA lysis buffer. After 10 minutes incubation on
ice, the lysates were centrifuged at 13,000 rpm at 4°C. The supernatants
were recovered and subjected to electrophoresis on 10% Tris-glycine pre-cast
polyacrylamide gels (Zaxis). Proteins were transferred to Immobilon-P PVDF
membranes (Millipore) and probed with antibodies against total Smad2/3
(Transduction Laboratories), phosphorylated Smad2 (courtesy of C. Heldin),
TGFßRI (Santa Cruz Biotech.), Ser473 phosphorylated Akt (Cell
Signalling), total Akt (Upstate Biotech.) or actin (Sigma). Bands were
visualized by incubation with horseradish peroxidase (HRP)-linked secondary
antibodies (Amersham) followed by chemiluminescence in Western Lightning
(Perkin Elmer).
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Results |
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|
Suprabasal expression of 6ß4 integrin increases epidermal
sensitivity to chemical carcinogenesis
None of the mice expressing suprabasal 6ß4 displayed any gross
skin phenotype or developed any spontaneous tumours. In addition, the
histological appearance of wt and transgenic skin was indistinguishable
(acetone treatment; Fig. 1B,E).
A single application of the phorbol ester tumour-promoter TPA resulted in a
similar increase in the number of epidermal nucleated cell layers and degree
of dermal inflammatory infiltrate in transgenic and wt skin
(Fig. 1C,F). However,
visualizing S-phase cells by BrdU incorporation revealed that the presence of
6ß4 in the differentiated epidermal layers resulted in a greater
increase in proliferation in the basal (transgene-negative) epidermal layers
in response to TPA (Fig. 1D,G).
This indicates that the presence of suprabasal
6ß4 did not induce
differentiated cells to proliferate, but enhanced the proliferative response
of basal cells to TPA.
To determine whether the transgenics had altered sensitivity to tumour
formation, Inv6ß4 and wt mice were subjected to a classical
two-stage carcinogenesis protocol in which DMBA induces Ha-Ras mutations and
repeated TPA treatments cause tumour promotion
(Owens and Watt, 2001
).
Inv
6ß4 mice developed 3-4 times as many benign papillomas as wt
mice (Inv
6ß4: 13.1 papillomas/mouse; wt: 4.0 papillomas/mouse;
P=0.001) (Fig. 2A).
100% of Inv
6ß4 mice developed papillomas, compared with 80% of wt
animals. No skin tumours were observed in wt or Inv
6ß4 mice either
initiated with acetone vehicle and promoted with TPA or initiated with DMBA
and promoted with acetone vehicle (data not shown).
|
To determine whether the papillomas in Inv6ß4 mice were
phenotypically distinct from those of wt animals, their histopathology and
growth fraction were compared. As illustrated in
Fig. 2C, the two groups of
papillomas tended to be well differentiated and could not be distinguished by
the extent of dysplasia and cellular atypia. There were no differences in the
number or location of BrdU-positive S-phase cells in wt and transgenic
papillomas (data not shown).
Inv6ß4 mice went on to develop 3-4 times more SCCs than wt mice
(Inv
6ß4: 2.16 SCCs/mouse; wt: 0.65 SCCs/mouse; P=0.0001)
(Fig. 2B). The proportion of
Inv
6ß4 mice that developed SCCs was also greater than that of wt
mice (Inv
6ß4: 100%; wt: 40%). Both wt and transgenic SCCs were
well differentiated and no spindle cell carcinomas were observed
(Fig. 2C and data not
shown).
The total number of SCCs observed in Inv6ß4 mice was probably
an underestimate because these mice had a higher morbidity rate than wt
animals (Fig. 2B). By 27 weeks,
a time when the frequency of SCCs was still rising, 50% of the transgenics
were dead, compared with only 4% of wt mice (data not shown). The reason for
the increased morbidity was that Inv
6ß4 mice had a higher
frequency of metastases. As shown in Table
1, 68% of Inv
6ß4 mice developed metastases compared
with 8% of wt mice. In addition, Inv
6ß4 mice developed 6-7 times
as many metastases per mouse, on average, than wt mice
(Table 1).
|
Whereas the higher frequency of SCCs in Inv6ß4 mice is likely
to result from the higher frequency of papillomas
(Fig. 2A,B), it does not
explain the increased number of metastases. As shown in
Table 1, the metastasis
(secondary tumour) to SCC (primary tumour) ratio was 4 times higher in
Inv
6ß4 mice, indicating that Inv
6ß4 SCCs were 4 times
more likely to spread than those from wt animals. Only those mice that
developed SCCs were found to contain secondary tumours.
Histological sections of Inv6ß4 metastases were examined to
confirm the presence of keratinocytes (Fig.
2D). All of the metastases stained positive for keratin 14 and the
6 transgene. Expression of the
6 transgene indicates that the
metastatic keratinocytes were still capable of some degree of terminal
differentiation.
The tumour response in Inv6ß4 mice does not depend on the
ß4 cytoplasmic domain
Signalling via the cytoplasmic domain of the ß4 integrin subunit is
critical for the pro-invasive and migratory effects of 6ß4 on
tumour cells (Mercurio et al.,
2001
). To test whether it was also required for the tumourigenic
effects of suprabasal
6ß4, we generated Inv
6ß4
(tailless) double-transgenic mice. The ß4
subunit is truncated
immediately after Lys734, the boundary between the transmembrane and
intracellular domains (Mainiero et al.,
1997
). Like the full-length ß4 subunit, ß4
was
expressed on the surface of suprabasal keratinocytes
(Fig. 1A). The ß4
founder line 2417C was used for all subsequent experiments.
Remarkably, deletion of the ß4 subunit cytoplasmic tail did not
suppress, but rather enhanced, the proliferative effects of suprabasal
6ß4 expression. Acetone-treated Inv
6ß4
skin was
mildly hyperplastic and contained a higher dermal infiltrate than wt or
Inv
6ß4 skin (Fig.
1H). The number of nucleated epidermal cell layers dramatically
increased after a single application of TPA and was considerably greater than
in Inv
6ß4 or wt skin (Fig.
1I). Greater than 90% of basal keratinocytes in TPA-treated
Inv
6ß4
epidermis were BrdU-positive
(Fig. 1J) compared with
approximately 15% of basal keratinocytes in TPA-treated wt epidermis
(Fig. 1D) and 40% in
Inv
6ß4 epidermis (Fig.
1G). Suprabasal BrdU-labelled cells were rarely observed in
Inv
6ß4
epidermis (Fig.
1J).
Inv6ß4
mice developed even more papillomas than
Inv
6ß4 mice (Fig.
2A), averaging 27.8 papillomas/mouse compared with 13.1
papillomas/mouse in Inv
6ß4 mice (P=0.009).
Inv
6ß4
mice also developed significantly more SCCs than wt
mice (Fig. 2B)
(Inv
6ß4
: 2.33 SCCs/mouse; wt: 0.65 SCCs/mouse;
P=0.0001). The numbers of SCCs
(Fig. 2B) and metastases (data
not shown) in Inv
6ß4 and Inv
6ß4
mice were not
significantly different. Unlike Inv
6ß4 and wt mice,
Inv
6ß4
animals developed tumours with DMBA treatment alone
(4.33 papillomas/mouse; 0.67 SCCs/mouse). Therefore, the ß4 cytoplasmic
tail was not required for the tumourigenic effect of suprabasal
6ß4, and removal of the cytoplasmic domain actually enhanced
tumour formation.
Suprabasal 6ß4 expression disrupts TGFß signalling
in vivo and in culture
Since suprabasal 6ß4 expression correlated with increased
proliferation of keratinocytes in the underlying basal layer
(Fig. 1D,G,J), we postulated
that it might alter growth factor signalling or responsiveness. TGFß
negatively regulates keratinocyte proliferation and the early stages of
epidermal tumour promotion (Derynck et al.,
2001
; Wakefield and Roberts,
2002
) and also mediates the effects of the
vß6 and
vß8 integrins on epithelial homeostasis
(Mu et al., 2002
;
Morris et al., 2003
).
TGFß signalling is mediated by receptor activation of the Smad proteins
Smad2 and Smad3 (Moustakas et al.,
2001
). Phosphorylated Smad2/3 forms a complex with Smad4 and
translocates from the cytoplasm to the nucleus to activate gene transcription
(Moustakas et al., 2001
). To
determine whether TGFß signalling was perturbed in
Inv
6ß4
mice, we stained sections of skin with an antibody
to phosphoSmad2 (Fig. 2E). In
wt epidermis treated once with TPA or acetone vehicle, the majority of basal
and suprabasal nuclei were positively stained. By contrast, very few nuclei
stained positive in acetone- or TPA-treated transgenic epidermis.
Reduced Smad2/3 activity is known to be associated with progression of skin
papillomas to SCCs (He et al.,
2001). The number of phosphoSmad2-positive nuclei was indeed
significantly lower in wt papillomas and SCCs than in wt epidermis
(Fig. 2E). Nevertheless, the
number of positive nuclei was further reduced in the transgenic tumours
(Fig. 2E).
We next developed in vitro models to examine the mechanism of disruption of
TGFß signalling (Fig.
3A,B). Keratinocytes from wt and transgenic epidermis were grown
either to confluent monolayers in which very few differentiated cells were
present, or to 5-day post-confluent cultures that contained stratified,
involucrin-positive suprabasal cells. In stratified Inv6ß4
cultures, the involucrin-positive cells were also transgene-positive
(Romero et al., 1999
). The
effects of the transgene on TGFß responsiveness could then be monitored
by translocation of receptor-activated Smad2/3 from the cytoplasm to the
nucleus (Wakefield and Roberts,
2002
) in monolayers and postconfluent, stratified cultures of
transgenic and wt keratinocytes (Fig.
3A).
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In untreated wt and transgenic monolayers
(Fig. 3C,D) or stratified
cultures (Fig. 3E,F), Smad2/3
was found almost exclusively in the cytoplasm. Upon treatment with TGFß1,
Smad2/3 translocated to the nucleus in wt and transgenic monolayers
(Fig. 3G,H). Nuclear Smad2/3
was also observed in the basal layer of post-confluent stratified cultures of
wt cells (Fig. 3I). However, it
did not occur in basal cells of Inv6ß4 post-confluent stratified
cultures (Fig. 3J). The
presence or absence of the transgene was confirmed by immunofluorescence
staining with an antibody to the human
6 integrin subunit
(Fig. 3K,L and data not shown).
These experiments suggest that suprabasal expression of
6ß4
suppresses the responsiveness of basal keratinocytes to TGFß.
No differences in the total levels of Smad2/3 or TGFßRI were observed
in wt and transgenic keratinocytes, whether grown as monolayers or stratified
cultures and whether treated with TGFß or untreated
(Fig. 3M). Activation of Smad2,
measured with antibodies specific for phosphoSmad2, increased on TGFß
treatment and was greater in monolayers than stratified cultures, possibly
reflecting the greater proportion of basal cells
(Fig. 3M). However, no major
differences in the level of phosphorylated (activated) Smad2 were seen in
transgenic versus wt cultures (Fig.
3M). We conclude that suprabasal 6ß4 does not inhibit
TGFß-mediated Smad2 phosphorylation
(Fig. 3M) but prevents
translocation of phosphoSmad2 to the nucleus
(Fig. 3J). This is consistent
with in vivo immunolocalization data obtained with an antibody to total (data
not shown), as opposed to phosphorylated
(Fig. 2E), Smad2.
Disruption of TGFß signalling by 6ß4 requires
cell-cell contact and PI3-K activity
To investigate the mechanism by which suprabasal keratinocytes expressing
6ß4 inhibited the TGFß responsiveness of basal keratinocytes,
monolayers of wt or Inv
6ß4 keratinocytes were combined with
suprabasal cells that had been induced to undergo terminal differentiation in
suspension (Romero et al.,
1999
) (Fig. 3B).
When wt or transgenic monolayers were combined with wt suprabasal cells,
Smad2/3 underwent nuclear translocation in response to TGFß
(Fig. 4A,B,E,F). When
suprabasal transgene-positive cells were attached to wt or transgenic
monolayers, Smad2/3 translocation was inhibited
(Fig. 4C,D,G,H). These results
support the conclusion from post-confluent cultures
(Fig. 3) that suprabasal
6ß4 inhibits Smad2/3 translocation.
|
Wild-type keratinocytes incubated with conditioned medium from
post-confluent stratified Inv6ß4 keratinocytes remained completely
responsive to TGFß, suggesting that cell-cell contact was required for
the effect (Fig. 4I,J). This
was confirmed by examining incompletely stratified cultures of
Inv
6ß4 keratinocytes treated with TGFß
(Fig. 4K,L). In basal cells
underneath transgene- and involucrin-positive suprabasal cells, there was no
Smad2/3 translocation; however, adjacent basal cells that were not in contact
with suprabasal cells had nuclear Smad2/3.
Keratinocyte intercellular adhesion is mediated by adherens junctions and
desmosomes. When adherens junction formation is inhibited by overexpression of
a dominant-negative E-cadherin construct (dnEcad; consisting of the
transmembrane and cytoplasmic domains of E-cadherin coupled to the
extracellular domain of an irrelevant protein), intercellular adhesion is
inhibited in keratinocyte monolayers; however post-confluent cells are able to
stratify through intercellular adhesion mediated by desmosomal junctions
(Zhu and Watt, 1996).
Expression of dnEcad relieved suprabasal
6ß4-mediated inhibition
of Smad2/3 translocation in response to TGFß, whereas a control construct
in which the ß-catenin-binding site of E-cadherin is deleted
(dnEcad
C25) did not (Fig.
5A). The proportion of Smad2/3-positive nuclei was significantly
increased in TGFß-treated Inv
6ß4 cells expressing dnEcad
compared with untransduced Inv
6ß4 cells
(Fig. 5A).
|
The ability of the 6ß4 integrin to promote carcinoma invasion
depends on activation of PI3-K (Shaw et
al., 1997
), and
6ß4 can also signal to the Ras-MAPK
(mitogen-activated protein kinase) pathway
(Mainiero et al., 1997
). In
addition, Smad2/3 nuclear translocation can be inhibited by MAP kinase
phosphorylation and may also involve PI3-K activity
(Kretzschmar et al., 1999
). We
therefore tested the effects of PI3-K and ERK/MAPK kinase (MEK) inhibitors in
our assays. As shown in Fig.
5B, the percentage of Smad2/3-positive nuclei was not increased in
Inv
6ß4 cells pre-treated with the MEK inhibitor U0126 compared
with DMSO controls, whereas pre-treatment of cells with the PI3-K inhibitor LY
294002 prior to addition of TGFß increased the number of Smad2/3-positive
nuclei by 4-5-fold. In view of the effect of the PI3-K inhibitor, we examined
whether PI3-K activity was elevated in Inv
6ß4 keratinocytes, using
phosphorylation of Akt as a read-out
(Cantley, 2002
)
(Fig. 5C). The basal level of
phosphoAkt was higher in Inv
6ß4 than wt cells and could be
completely abolished by pre-incubation with LY 294002.
Collectively, these results show that the repression of TGFß-induced
Smad2/3 translocation by suprabasal 6ß4 expression is not due to
release of diffusible factors from suprabasal cells but is dependent on
cadherin-mediated cell-cell adhesion and requires PI3-K activity.
Suprabasal 6ß4 expression relieves the growth-inhibitory
response of keratinocytes to TGFß
TGFß inhibits proliferation of primary keratinocytes and
overexpression of TGFß in transgenic mouse epidermis reduces papilloma
formation during chemical carcinogenesis
(Derynck et al., 2001;
Shipley et al., 1986
).
TPA-induced proliferation of basal keratinocytes is enhanced in
Inv
6ß4 epidermis (Fig.
1D,G,J). These observations suggested that suprabasal
6ß4 might overcome TGFß-mediated growth inhibition. We tested
this by measuring BrdU incorporation in cultured keratinocytes in the presence
or absence of suprabasal
6ß4 and TGFß
(Fig. 5D). Monolayers of wt or
Inv
6ß4 keratinocytes responded to TGFß1 with similar
reductions (40-50%) in BrdU uptake (Fig.
5D, left panel). When wt suprabasal keratinocytes were combined
with wt basal keratinocytes, the same suppression of BrdU uptake was observed
(Fig. 5D, right panel).
However, when suprabasal Inv
6ß4 keratinocytes were attached to wt
monolayers, the number of BrdU-positive cells was not decreased by TGFß1
treatment. Furthermore, addition of suprabasal transgene-positive cells in the
absence of TGFß stimulated BrdU incorporation relative to the addition of
wt cells (Fig. 5D, right
panel).
Our results indicate that suprabasal expression of 6ß4 can
enhance proliferation of the underlying basal cells both in vivo and in
culture, and can overcome TGFß-mediated growth inhibition in culture.
This might provide the explanation for the increased susceptibility of
Inv
6ß4 epidermis to chemical carcinogenesis.
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Discussion |
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Suprabasal expression of the 6ß4 integrin did not result in
spontaneous epidermal tumours, but greatly increased the susceptibility of the
epidermis to chemical carcinogenesis. Nuclear phosphoSmad2 levels were
markedly decreased in both control and TPA-treated transgenic epidermis,
suggesting that the elevated proliferation of keratinocytes in the basal layer
relative to wt epidermis could be due to lack of TGFß-mediated growth
inhibition. It is striking that suprabasal expression of
6ß4 led
exclusively to increased proliferation of basal keratinocytes, whereas
suprabasal expression of
2ß1 or
3ß1 leads to
suprabasal BrdU incorporation in TPA-treated epidermis
(Owens and Watt, 2001
).
Suprabasal
6ß4 relieved TGFß-mediated growth inhibition in
cultured keratinocytes and the effect was at the level of nuclear
translocation of phosphorylated Smads. We have thus demonstrated a link
between
6ß4 overexpression and loss of TGFß signalling in the
progression of chemically induced tumours.
Since 6ß4 signalling in carcinoma invasion is dependent on the
ß4 cytoplasmic domain (Mercurio et
al., 2001
), it is surprising that the phenotype of
Inv
6ß4
mice was more severe than that of Inv
6ß4
mice. Mice with the truncated ß4 subunit had spontaneous epidermal
hyperproliferation, an increased incidence of papillomas on treatment with
DMBA and TPA, and even developed papillomas when treated with DMBA alone.
There is good evidence for signalling via integrin
subunit cytoplasmic
domains and for modulation of integrin signalling through interactions between
and ß cytoplasmic domains
(Hughes and Pfaff, 1998
;
Hynes, 2002
). One possibility
is therefore that the ability of
6ß4 to inhibit TGFß
responsiveness depends on the
6 cytoplasmic domain and that, whereas
the ß4 cytoplasmic domain is permissive, its loss leads to an enhancement
of the
6 signal (Han et al.,
2001
; Zhang et al.,
2001
).
The major adhesive contacts between basal and suprabasal keratinocytes are
adherens junctions and desmosomes, with E-cadherin being the main adhesive
receptor in the adherens junctions. Cell-cell attachment was required for
suprabasal 6ß4-mediated disruption of TGFß signalling.
TGFß responsiveness was restored to Inv
6ß4 cells by
overexpressing the E-cadherin cytoplasmic domain, which inhibits adherens
junction formation but not desmosome formation
(Zhu and Watt, 1996
).
E-cadherin is linked to the actin cytoskeleton via ß-catenin, and
deletion of the ß-catenin-binding site prevented the E-cadherin
cytoplasmic domain from inhibiting cell-cell adhesion and interfering with
suprabasal
6ß4 (Fig.
5A). We conclude that cadherin-mediated intercellular adhesion is
required for the suppression of TGFß signalling by
6ß4.
Two observations suggest that the effect of suprabasal 6ß4 is
mediated by PI3-kinase. PI3-K inhibitors relieved the block in
TGFß-mediated Smad translocation, and phosphoAkt levels were higher in
transgenic than wt keratinocytes, indicative of increased PI3-K activity
(Fig. 5B,C). PI3-K activation
is required for the pro-invasive and survival effects of
6ß4
(Mercurio et al., 2001
;
Shaw et al., 1997
); however,
this depends on a tyrosine residue, Y1494, which is deleted in
Inv
6ß4 mice (Shaw,
2001
). Instead, the increase in PI3-K activity in transgenic
keratinocytes could potentially involve cross-talk between
6ß4 and
E-cadherin (Hodivala and Watt,
1994
; Arregui et al.,
2000
), since homotypic E-cadherin interactions can activate PI3-K
(Kovacs et al., 2002
).
The inhibition of TGFß signalling was at the level of translocation of
phosphorylated Smad2/3 into the nucleus
(Fig. 3). In contrast to an
earlier report (Dong et al.,
2000), we found no evidence that sequestration of Smads in the
cytoplasm required intact microtubules (data not shown). Instead, we favour a
model in which reorganization of the actin cytoskeleton by TGFß plays a
role in Smad translocation. TGFß can rapidly rearrange actin filaments by
a Smad-independent process that involves the Rho GTPases Cdc42 and RhoA, and
it has previously been proposed that TGFß activation of small GTPases is
required for Smad translocation (Edlund et
al., 2002
). In keratinocytes, suprabasal
6ß4 was
linked to actin filaments (data not shown) and actin-associated
6ß4 is known to activate RhoA
(O'Connor et al., 2000
).
E-cadherin-mediated cell-cell adhesion activates Rac1, which is upstream of
PI3-K (Nakagawa et al., 2001
).
We propose that, in the presence of suprabasal
6ß4 and E-cadherin,
TGFß is unable to effect the actin reorganization required for Smad
translocation. dnEcad may restore the subcellular location of components of
the PI3-K signalling cascade (Khayat et
al., 2000
) by reduced activation of Rho GTPases.
Whereas previous work has emphasized the positive effect of 6ß4
on the behaviour of carcinoma cells
(Rabinovitz and Mercurio,
1996
; Mercurio et al.,
2001
), our experiments establish that changes in expression of
this integrin in otherwise normal epithelium can have a major impact on the
initiation and course of the disease. We have uncovered a novel mechanism by
which aberrant integrin expression enhances the tumour microenvironment by
altering growth regulation of neighbouring cells.
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Acknowledgments |
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References |
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