Suprabasal {alpha}6ß4 integrin expression in epidermis results in enhanced tumourigenesis and disruption of TGFß signalling

David M. Owens1, M. Rosario Romero1,*, Clare Gardner2 and Fiona M. Watt1,{ddagger}

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

{ddagger} Author for correspondence (e-mail: fiona.watt{at}cancer.org.uk)

Accepted 24 June 2003


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inappropriate {alpha}6ß4 integrin expression correlates with a high risk of tumour progression in stratified squamous epithelia. Targeted expression of {alpha}6ß4 in the suprabasal layers of transgenic mouse epidermis dramatically increased the frequency of papillomas, carcinomas and metastases induced by chemical carcinogenesis, independent of the ß4 cytoplasmic domain. Suprabasal {alpha}6ß4 also perturbed transforming growth factor ß (TGFß) signalling as demonstrated by decreased nuclear Smad2 in transgenic epidermis and tumours. In cultured keratinocytes, suprabasal {alpha}6ß4 relieved TGFß-mediated growth inhibition and blocked nuclear translocation of activated Smad2/3. Responsiveness to TGFß could be restored by inhibiting cadherin-mediated cell-cell adhesion or phosphoinositide 3-kinase (PI3-K) activity, but not by inhibiting mitogen-activated protein kinase (MAPK) activity. These data suggest that suprabasal {alpha}6ß4 promotes tumourigenesis by preventing TGFß from suppressing clonal expansion of initiated cells in the epidermal basal layer.

Key words: Keratinocyte, Differentiation, Carcinogenesis, Skin, Smad


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epidermis is a constantly renewing tissue that is maintained by a highly coordinated balance between keratinocyte proliferation and terminal differentiation. Extracellular matrix receptors of the integrin family play an important role in regulating normal epidermal homeostasis (Watt, 2002Go). The major keratinocyte integrins are {alpha}2ß1 (collagen receptor), {alpha}3ß1 (laminin receptor) and {alpha}6ß4 (laminin receptor) (van der Flier and Sonnenberg, 2001Go; Watt, 2002Go). The ß1 integrins have a pericellular distribution in the basal layer of the epidermis and are associated with the actin cytoskeleton. The {alpha}6ß4 integrin is concentrated at the basement membrane zone and is associated with hemidesmosomes and the intermediate filament system.

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 {alpha}6ß4 integrin (Rabinovitz and Mercurio, 1996Go). Suprabasal expression of {alpha}6ß4 in keratinocytes that are not adjacent to the tumour stroma correlates with poor prognosis in human squamous cell carcinomas (SCCs) (Rabinovitz and Mercurio, 1996Go; van Waes et al., 1995Go). In mouse skin, suprabasal expression of {alpha}6ß4 is observed in those benign papillomas with a high risk of conversion to SCCs (Tennenbaum et al., 1993Go).

Mechanistic studies of {alpha}6ß4 have emphasized its role in promoting invasion by stimulating epithelial cell motility (Mercurio et al., 2001Go). In invasive carcinomas, {alpha}6ß4 can be found associated with the actin cytoskeleton in the absence of hemidesmosomes and it is becoming clear that {alpha}6ß4 is capable of ligand-independent signal transduction (Mercurio et al., 2001Go). Mobilization of {alpha}6ß4 from hemidesmosomes occurs in response to chemotactic factors and is correlated with phosphorylation of the ß4 cytoplasmic domain (Mainiero et al., 1996Go). {alpha}6ß4 cooperates with growth factor receptors to activate phosphoinositide 3-kinase (PI3-K), and PI3-K activation is necessary for {alpha}6ß4-mediated invasiveness (Mercurio et al., 2001Go). Human keratinocytes lacking {alpha}6ß4 are resistant to the tumourigenic effects of Ras and NF-{kappa}B (Dajee et al., 2003Go).

Whereas there is strong evidence that {alpha}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 {alpha}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 {alpha}6ß4 is expressed in the suprabasal layers of the epidermis under the control of the involucrin promoter (Carroll et al., 1995Go). We demonstrate that suprabasal {alpha}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.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of transgenic mice
Human full-length {alpha}6 and ß4 (Giancotti et al., 1992Go) and truncated ß4 (ß4{Delta} 1-2329) (Mainiero et al., 1997Go) integrin subunit cDNAs, kindly provided by F. Giancotti (Sloan Kettering Cancer Center, New York), were subcloned into a previously reported expression cassette containing the involucrin promoter (Carroll et al., 1995Go). All constructs were microinjected into fertilized mouse oocytes and at least three transgene-positive founders per construct were identified as previously described (Carroll et al., 1995Go).

The Inv{alpha}6 transgenics, described previously (Romero et al., 1999Go), 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{Delta} mice were generated directly in the FVB/N strain. Inv{alpha}6ß4 and Inv{alpha}6ß4{Delta} double-transgenic and wild-type (wt) experimental mice were generated by crossing heterozygous {alpha}6 single transgenics to heterozygous ß4 or ß4{Delta} single transgenics. Animal husbandry was as described previously (Owens and Watt, 2001Go).

Tumour studies
Seven-week-old female Inv{alpha}6ß4, Inv{alpha}6ß4{Delta} 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, 2001Go). The experiment comparing Inv{alpha}6ß4 and wt mice was performed twice, as was the experiment comparing Inv{alpha}6ß4, Inv{alpha}6ß4{Delta} 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, 2001Go), using antibodies to human {alpha}6 (MP4F10), human ß4 (3E1; Life Technologies) or an antibody that detects mouse {alpha}6ß4 (CD49f; Serotec). 5-bromo-2'-deoxyuridine (BrdU) incorporation was examined in formalin-fixed sections (Owens and Watt, 2001Go) 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 Inv{alpha}6ß4 and wt adult mouse epidermis and maintained with a feeder layer as described previously (Romero et al., 1999Go). 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{Delta}C25) (Zhu and Watt, 1996Go).

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., 2000Go) or anti-human {alpha}6 integrin antibodies (Romero et al., 1999Go) described above.

Stratified cultures were also reconstituted as follows. Transgenic and wt mouse keratinocytes were induced to differentiate in suspension (Romero et al., 1999Go) 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., 2000Go) or involucrin (Owens and Watt, 2001Go).

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 Inv{alpha}6ß4 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).


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of transgenic mice
Transgenic mice expressing the single {alpha}6 and ß4 integrin subunits under the control of the involucrin promoter (Inv{alpha}6 and Invß4) were crossed to create mice expressing suprabasal {alpha}6ß4 (Inv{alpha}6ß4). Endogenous {alpha}6ß4 expression was confined to the basolateral surface of basal keratinocytes in wt and transgenic mouse epidermis (Fig. 1A and data not shown). Transgenic {alpha}6ß4, detected with antibodies specific for the human integrin subunits, was expressed in the suprabasal epidermal layers (Fig. 1A). The {alpha}6 founder line 1374C+D and the ß4 founder line 1376A were used for all subsequent experiments.



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Fig. 1. Characterization of transgenic mice. (A) Detection of endogenous and transgenic {alpha}6ß4 by immunofluorescence staining with indicated antibodies; bm, basement membrane; hf, hair follicle. Bar, 50 µm. (B-J) Skin histology of Inv{alpha}6ß4 (E-G), Inv{alpha}6ß4{Delta} (H-G) and wt (B-D) littermate mice 24 hours after a single application of 6 nmol TPA or acetone. Sections in D, G and J were labelled with antibody to BrdU. Bar, 100 µm.

 

Suprabasal expression of {alpha}6ß4 integrin increases epidermal sensitivity to chemical carcinogenesis
None of the mice expressing suprabasal {alpha}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 {alpha}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 {alpha}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, Inv{alpha}6ß4 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, 2001Go). Inv{alpha}6ß4 mice developed 3-4 times as many benign papillomas as wt mice (Inv{alpha}6ß4: 13.1 papillomas/mouse; wt: 4.0 papillomas/mouse; P=0.001) (Fig. 2A). 100% of Inv{alpha}6ß4 mice developed papillomas, compared with 80% of wt animals. No skin tumours were observed in wt or Inv{alpha}6ß4 mice either initiated with acetone vehicle and promoted with TPA or initiated with DMBA and promoted with acetone vehicle (data not shown).



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Fig. 2. Papilloma and squamous cell carcinoma formation. (A-B) Frequency of papillomas (A) and SCCs (B); ++++, all mice in group deceased. (C) Histopathological analysis of papillomas (pap) and SCCs. (D) Metastases from Inv{alpha}6ß4 mice. (C,D left-hand panel) Stained with haematoxylin and eosin; (D) other panels labelled with antibodies to the {alpha}6 transgene or keratin 14. Bar, 100 µm. (E) Immunolocalization of phosphoSmad2 in wt and Inv{alpha}6ß4{Delta}-transgenic epidermis, papillomas (pap) and SCCs; ace, acetone treated; TPA, 24 hours after one dose of TPA. Bars, 50 µm (epidermis), 100 µm (pap, SCC).

 

To determine whether the papillomas in Inv{alpha}6ß4 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).

Inv{alpha}6ß4 mice went on to develop 3-4 times more SCCs than wt mice (Inv{alpha}6ß4: 2.16 SCCs/mouse; wt: 0.65 SCCs/mouse; P=0.0001) (Fig. 2B). The proportion of Inv{alpha}6ß4 mice that developed SCCs was also greater than that of wt mice (Inv{alpha}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 Inv{alpha}6ß4 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{alpha}6ß4 mice had a higher frequency of metastases. As shown in Table 1, 68% of Inv{alpha}6ß4 mice developed metastases compared with 8% of wt mice. In addition, Inv{alpha}6ß4 mice developed 6-7 times as many metastases per mouse, on average, than wt mice (Table 1).


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Table 1. Increase in metastasis in Inv{alpha}6ß4 transgenic mice compared with wild-type mice

 

Whereas the higher frequency of SCCs in Inv{alpha}6ß4 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{alpha}6ß4 mice, indicating that Inv{alpha}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 Inv{alpha}6ß4 metastases were examined to confirm the presence of keratinocytes (Fig. 2D). All of the metastases stained positive for keratin 14 and the {alpha}6 transgene. Expression of the {alpha}6 transgene indicates that the metastatic keratinocytes were still capable of some degree of terminal differentiation.

The tumour response in Inv{alpha}6ß4 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 {alpha}6ß4 on tumour cells (Mercurio et al., 2001Go). To test whether it was also required for the tumourigenic effects of suprabasal {alpha}6ß4, we generated Inv{alpha}6ß4{Delta} (tailless) double-transgenic mice. The ß4{Delta} subunit is truncated immediately after Lys734, the boundary between the transmembrane and intracellular domains (Mainiero et al., 1997Go). Like the full-length ß4 subunit, ß4{Delta} was expressed on the surface of suprabasal keratinocytes (Fig. 1A). The ß4{Delta} 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 {alpha}6ß4 expression. Acetone-treated Inv{alpha}6ß4{Delta} skin was mildly hyperplastic and contained a higher dermal infiltrate than wt or Inv{alpha}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{alpha}6ß4 or wt skin (Fig. 1I). Greater than 90% of basal keratinocytes in TPA-treated Inv{alpha}6ß4{Delta} epidermis were BrdU-positive (Fig. 1J) compared with approximately 15% of basal keratinocytes in TPA-treated wt epidermis (Fig. 1D) and 40% in Inv{alpha}6ß4 epidermis (Fig. 1G). Suprabasal BrdU-labelled cells were rarely observed in Inv{alpha}6ß4{Delta} epidermis (Fig. 1J).

Inv{alpha}6ß4{Delta} mice developed even more papillomas than Inv{alpha}6ß4 mice (Fig. 2A), averaging 27.8 papillomas/mouse compared with 13.1 papillomas/mouse in Inv{alpha}6ß4 mice (P=0.009). Inv{alpha}6ß4{Delta} mice also developed significantly more SCCs than wt mice (Fig. 2B) (Inv{alpha}6ß4{Delta}: 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{alpha}6ß4 and Inv{alpha}6ß4{Delta} mice were not significantly different. Unlike Inv{alpha}6ß4 and wt mice, Inv{alpha}6ß4{Delta} 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 {alpha}6ß4, and removal of the cytoplasmic domain actually enhanced tumour formation.

Suprabasal {alpha}6ß4 expression disrupts TGFß signalling in vivo and in culture
Since suprabasal {alpha}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., 2001Go; Wakefield and Roberts, 2002Go) and also mediates the effects of the {alpha}vß6 and {alpha}vß8 integrins on epithelial homeostasis (Mu et al., 2002Go; Morris et al., 2003Go). TGFß signalling is mediated by receptor activation of the Smad proteins Smad2 and Smad3 (Moustakas et al., 2001Go). Phosphorylated Smad2/3 forms a complex with Smad4 and translocates from the cytoplasm to the nucleus to activate gene transcription (Moustakas et al., 2001Go). To determine whether TGFß signalling was perturbed in Inv{alpha}6ß4{Delta} 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., 2001Go). 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 Inv{alpha}6ß4 cultures, the involucrin-positive cells were also transgene-positive (Romero et al., 1999Go). 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, 2002Go) in monolayers and postconfluent, stratified cultures of transgenic and wt keratinocytes (Fig. 3A).



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Fig. 3. Assays for TGFß responsiveness in vitro. (A,B) Schematics of in vitro assays. (A) Keratinocyte monolayers and stratified cultures used in panels C-M; PC, postconfluence. (B) Monolayers combined with keratinocytes that had been induced to differentiate in suspension. (C-J) Immunolocalization of Smad2/3. (K,L) Immunolabelling for {alpha}6 transgene. Bar, 50 µm. (M) Western blots of untreated (-) or TGFß1-treated (+) cells probed with antibodies to total Smad2/3, activated Smad2 (pSmad2), TGFßRI or, as a loading control, actin; M, monolayer, S, stratified cultures.

 

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 Inv{alpha}6ß4 post-confluent stratified cultures (Fig. 3J). The presence or absence of the transgene was confirmed by immunofluorescence staining with an antibody to the human {alpha}6 integrin subunit (Fig. 3K,L and data not shown). These experiments suggest that suprabasal expression of {alpha}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 {alpha}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 {alpha}6ß4 requires cell-cell contact and PI3-K activity
To investigate the mechanism by which suprabasal keratinocytes expressing {alpha}6ß4 inhibited the TGFß responsiveness of basal keratinocytes, monolayers of wt or Inv{alpha}6ß4 keratinocytes were combined with suprabasal cells that had been induced to undergo terminal differentiation in suspension (Romero et al., 1999Go) (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 {alpha}6ß4 inhibits Smad2/3 translocation.



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Fig. 4. Effect of suprabasal {alpha}6ß4 on Smad2/3 translocation requires cell-cell contact. (A-J,L) Immunolocalization of Smad2/3; (K) immunolocalization of involucrin. K and L show the same field; the dashed line separates the stratified area (left side) containing involucrin-positive suprabasal cells from the area consisting of basal cells only (right side). (A-H) Suspended cells were attached to basal cells to form suprabasal layers, as shown schematically in Fig. 3B; (I,J) wt monolayers treated with Inv{alpha}6ß4 keratinocyte-conditioned medium. Bar, 50 µm.

 

Wild-type keratinocytes incubated with conditioned medium from post-confluent stratified Inv{alpha}6ß4 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{alpha}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, 1996Go). Expression of dnEcad relieved suprabasal {alpha}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{Delta}C25) did not (Fig. 5A). The proportion of Smad2/3-positive nuclei was significantly increased in TGFß-treated Inv{alpha}6ß4 cells expressing dnEcad compared with untransduced Inv{alpha}6ß4 cells (Fig. 5A).



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Fig. 5. Roles of E-cadherin and PI3-K in TGFß responsiveness and effect of suprabasal {alpha}6ß4 on basal cell proliferation. (A) Stratified wt and untransduced Inv{alpha}6ß4 keratinocytes were compared with Inv{alpha}6ß4 cells transduced with dnEcad or dnEcad{Delta}C25 retroviral vectors following treatment with 2 ng/ml TGFß1 for 1 hour. *Significantly different from untransduced Inv{alpha}6ß4 keratinocytes, P<0.05. (B) Differentiated Inv{alpha}6ß4 cells were plated onto monolayers of wt cells overnight, incubated with the inhibitors shown or DMSO alone, then treated with 2 ng/ml TGFß1 for 1 hour. (C) Western blot of total and activated (pAkt) Akt levels in wt and transgenic stratified keratinocytes, ±LY 294002. Actin is the loading control. (D) BrdU incorporation in keratinocyte monolayers (basal, left-hand panel) or in stratified cultures formed by combining suspended wt or Inv{alpha}6ß4 cells with wt monolayers (suprabasal, right-hand panel). Cells were serum starved for 24 hours, then treated with complete medium - (black bars) or + (grey bars) 2 ng/ml TGFß1 for 20 hours. (A,B,D) Each data set represents the average count of % Smad2/3-positive (A,B) or BrdU-positive (D) nuclei versus total Hoescht-stained nuclei from three separate fields (monolayers: 100-200 cells/field; recombined and post-confluent cultures: 400-500 cells/field).

 

The ability of the {alpha}6ß4 integrin to promote carcinoma invasion depends on activation of PI3-K (Shaw et al., 1997Go), and {alpha}6ß4 can also signal to the Ras-MAPK (mitogen-activated protein kinase) pathway (Mainiero et al., 1997Go). In addition, Smad2/3 nuclear translocation can be inhibited by MAP kinase phosphorylation and may also involve PI3-K activity (Kretzschmar et al., 1999Go). 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{alpha}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{alpha}6ß4 keratinocytes, using phosphorylation of Akt as a read-out (Cantley, 2002Go) (Fig. 5C). The basal level of phosphoAkt was higher in Inv{alpha}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 {alpha}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 {alpha}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., 2001Go; Shipley et al., 1986Go). TPA-induced proliferation of basal keratinocytes is enhanced in Inv{alpha}6ß4 epidermis (Fig. 1D,G,J). These observations suggested that suprabasal {alpha}6ß4 might overcome TGFß-mediated growth inhibition. We tested this by measuring BrdU incorporation in cultured keratinocytes in the presence or absence of suprabasal {alpha}6ß4 and TGFß (Fig. 5D). Monolayers of wt or Inv{alpha}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{alpha}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 {alpha}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{alpha}6ß4 epidermis to chemical carcinogenesis.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Carcinogenesis is a complex multi-step process that requires the accumulation of multiple genetic and epigenetic events (Hahn and Weinberg, 2002Go). There are many factors that can influence this process, such as dose and frequency of exposure to mutagens, target cell phenotype and changes in the tumour cell microenvironment. As long-term residents of the basal layer, epidermal stem cells have the capacity to sustain multiple mutations and can thereby found a tumour (Owens and Watt, 2003Go). However, suprabasal or differentiated cells have the potential to influence the course of the disease by exerting a positive or negative influence over the underlying basal cells that either encourages or restricts the proliferation of mutant clones (Owens and Watt, 2003Go).

Suprabasal expression of the {alpha}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 {alpha}6ß4 led exclusively to increased proliferation of basal keratinocytes, whereas suprabasal expression of {alpha}2ß1 or {alpha}3ß1 leads to suprabasal BrdU incorporation in TPA-treated epidermis (Owens and Watt, 2001Go). Suprabasal {alpha}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 {alpha}6ß4 overexpression and loss of TGFß signalling in the progression of chemically induced tumours.

Since {alpha}6ß4 signalling in carcinoma invasion is dependent on the ß4 cytoplasmic domain (Mercurio et al., 2001Go), it is surprising that the phenotype of Inv{alpha}6ß4{Delta} mice was more severe than that of Inv{alpha}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 {alpha} subunit cytoplasmic domains and for modulation of integrin signalling through interactions between {alpha} and ß cytoplasmic domains (Hughes and Pfaff, 1998Go; Hynes, 2002Go). One possibility is therefore that the ability of {alpha}6ß4 to inhibit TGFß responsiveness depends on the {alpha}6 cytoplasmic domain and that, whereas the ß4 cytoplasmic domain is permissive, its loss leads to an enhancement of the {alpha}6 signal (Han et al., 2001Go; Zhang et al., 2001Go).

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 {alpha}6ß4-mediated disruption of TGFß signalling. TGFß responsiveness was restored to Inv{alpha}6ß4 cells by overexpressing the E-cadherin cytoplasmic domain, which inhibits adherens junction formation but not desmosome formation (Zhu and Watt, 1996Go). 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 {alpha}6ß4 (Fig. 5A). We conclude that cadherin-mediated intercellular adhesion is required for the suppression of TGFß signalling by {alpha}6ß4.

Two observations suggest that the effect of suprabasal {alpha}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 {alpha}6ß4 (Mercurio et al., 2001Go; Shaw et al., 1997Go); however, this depends on a tyrosine residue, Y1494, which is deleted in Inv{alpha}6ß4 mice (Shaw, 2001Go). Instead, the increase in PI3-K activity in transgenic keratinocytes could potentially involve cross-talk between {alpha}6ß4 and E-cadherin (Hodivala and Watt, 1994Go; Arregui et al., 2000Go), since homotypic E-cadherin interactions can activate PI3-K (Kovacs et al., 2002Go).

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., 2000Go), 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., 2002Go). In keratinocytes, suprabasal {alpha}6ß4 was linked to actin filaments (data not shown) and actin-associated {alpha}6ß4 is known to activate RhoA (O'Connor et al., 2000Go). E-cadherin-mediated cell-cell adhesion activates Rac1, which is upstream of PI3-K (Nakagawa et al., 2001Go). We propose that, in the presence of suprabasal {alpha}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., 2000Go) by reduced activation of Rho GTPases.

Whereas previous work has emphasized the positive effect of {alpha}6ß4 on the behaviour of carcinoma cells (Rabinovitz and Mercurio, 1996Go; Mercurio et al., 2001Go), 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.


    Acknowledgments
 
We thank R. Rudling, B. Cross and J. Groeninger for their assistance in conducting the mouse tumour studies and C. Hill for her advice. We are grateful to C. Heldin and F. Giancotti for reagents. D.M.O. was supported by a National Research Service Award from the NCI (CA75638). F.M.W. was supported by Cancer Research UK and a European Union Quality of Life Network grant.


    Footnotes
 
* Present address: MRC Mammalian Genetics Unit, Harwell OX11 ORD, UK Back


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 Discussion
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