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First published online 5 August 2003
doi: 10.1242/jcs.00725

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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



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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.

 


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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).

 


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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.

 


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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.

 


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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).

 




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