Figure 4  Snail-Mediated Remodeling of AJs Contributes to Hyperproliferation
(A) Immunofluorescence of skin sections from P30 Wt and Tg mice. Shown are affected areas of Tg skin; in areas where Snail protein was not expressed, stainings were normal. Antibodies used are against AJ proteins and include E-cadherin (E-cad), the transmembrane core protein; β-catenin (β-cat), which binds E-cadherin at AJs and which can also participate as a transcription cofactor when associated with LEF-1/TCF proteins in the nucleus; α-catenin (α-cat) which binds to both β-catenin and Ajuba, a close relative of zyxin; and Ajuba, which can associate with proteins that bind to the actin cytoskeleton, as well as with Grb-2, a mediator of the GTP nucleotide-exchange protein Sos, involved in activation of the Ras-MAPK signaling cascade. In Snail-expressing Tg regions, there was a reduced staining with anti-E-cad and anti-α-cat and a more diffuse staining with anti-Ajuba. Insets in the panels for β-catenin and Ajuba staining are magnified views of the boxed areas. Arrows mark membrane localization of the protein and asterisks mark cells with elevated levels of cytoplasmic β-catenin or Ajuba.
(B) Western blot analyses of protein extracts from P30 Wt and Tg back and ear skins. Antibodies are as in (A) except anti-P-cad, which detects P-cadherin, whose expression in the hair follicle was not affected, and anti-tubulin, which detects tubulin, a control for equal protein loadings. Note that the reductions seen in E-cadherin and α-catenin are likely to be underestimates of the actual differences in affected regions, since the Tg skin expressed Snail mosaically.
(C) In the presence of elevated Snail, α-catenin levels can be restored by overexpression of E-cadherin. Keratinocytes were transfected with either HA-tagged Snail (Snail[HA]; images on the left) or Snail(HA) and Ecad(HA) (images on the right). 2 d after transfection, cells were switched from low-calcium growth medium to high-calcium medium for 6 h to induce AJ formation. Cells were stained with antibodies as indicated on the panels. Arrowheads point to sites of intercellular contact between a Snail-transfected keratinocyte and its neighboring untransfected cell.
(D) Reintroduction of E-cadherin in keratinocytes expressing Snail returns pMAPK to basal levels. Keratinocytes were transfected with control vector (K14), or Snail(HA), or Snail(HA) + E-cad(HA). After 2 d, cells were serum starved for 4 h and whole cell lysates were made and Western blotted with antibodies to pMAPK, HA to recognize the HA-tagged Snail and E-cadherin protein, 20or tubulin as a loading control.
(E) Ajuba interacts with Grb-2 under conditions where α-catenin levels are reduced. Protein extracts were made from skins of P30 Wt and K14-Snail Tg P30 mice (blots on the left) and of newborn Wt and K14-Cre/α-catenin (fl/fl) conditionally null animals (blots on the right) [7]. Equal amounts of protein extracts were treated with anti-Grb-2 antibody (+) or control isotype antibody (–), and following centrifugation, immunoprecipitates were subjected to SDS-PAGE and Western blot analysis with anti-Ajuba and anti-Grb-2 antibodies. Note the presence of Ajuba only under conditions where levels of α-catenin and other AJ proteins were aberrantly low or absent.
(F) Transgene expression of excess Ajuba or the Grb-2-interacting domain (pre-LIM) of Ajuba in keratinocytes results in the activation of the Ras-MAPK pathway. Primary newborn mouse keratinocytes were transfected with either the empty K14 expression vector (K14), or the expression vector driving Snail, full length Ajuba, or the pre-LIM domain of Ajuba in the absence or presence of a peptide inhibitor (inh) that disrupts the interaction between Grb-2 and Sos. 48 h posttransfection, protein extracts were prepared and subjected to SDS-PAGE and Western blot analyses with antibodies against pMAPK, total MAPK, Ajuba (also recognizing the smaller, pre-LIM domain), and Snail.