Possible Links between Epidermal Hyperproliferation and Down-regulation of AJ Proteins in Snail Tg Mice
Given that the E-cadherin promoter is a direct target for Snail-mediated repression in vitro [4,12,13], and that E-cadherin was down-regulated in Snail-expressing hair buds, we examined the status of E-cadherin and other AJ proteins within regions of hyperproliferative epidermis where Tg Snail was present (Figure 4A). In these regions, immunofluorescence staining of E-cadherin and α-catenin were markedly diminished. In contrast, the intensity of antibody staining for two other AJ proteins, β-catenin and Ajuba, was still strong. Interestingly, however, despite appreciable immunofluorescence, localization of β-catenin and Ajuba appeared to be largely cytoplasmic rather than at cell-cell borders (Figure 4A insets).
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. Architectural differences in the epidermis made Western blot analyses somewhat difficult to gauge. However, in regions such as ear skin, where the highest levels of Snail protein were expressed, the effects were accentuated. In both back skin and ear skin, overall levels of E-cadherin and α-catenin were reduced, under conditions where β-catenin and Ajuba levels remained unchanged relative to controls (Figure 4B). Taken together, these data were consistent with our results obtained from immunofluorescence microscopy.
A priori, the decrease in α-catenin levels could be due to either direct transcriptional repression by Snail or perturbations in AJ formation caused by the decrease in E-cadherin gene expression. To distinguish between these possibilities, we tested whether α-catenin levels could be restored by exogenous expression of E-cadherin in Snail-expressing keratinocytes. As shown in Figure 4C, transiently transfected keratinocytes expressing HA-tagged Snail displayed a loss of E-cadherin and α-catenin at cell-cell borders. Coexpression of exogenous HA-tagged E-cadherin not only enabled cell-cell border localization of E-cadherin protein, but also rescued the cell-cell border staining of α-catenin (Figure 4C). The ability to restore α-catenin expression and localization under these conditions argues against the notion that Snail transcriptionally represses α-catenin. Rather, the findings are consistent with a previous report that E-cadherin is required for the translation of α-catenin mRNA [22].
Despite the reductions in AJ markers, Tg skin still displayed sealed membranes and intercellular junctions that were largely intact, as judged by ultrastructural analyses (unpublished data). In this respect, the skin epithelium resembled that of the hair bud, where the down-regulation in junction proteins is permissive for cell-cell remodeling without abrogating intercellular adhesion.
The similarities between Snail Tg epidermis and hair buds extended to the hyperproliferative state, leading us to wonder whether the down-regulation of AJ proteins might contribute to this condition. Given the increase in pMAPK staining in Snail Tg epidermis (see Figure 2G), we used pMAPK levels as our assay to test whether the loss of E-cadherin contributed to the Snail-mediated increase in proliferation. Consistent with our in vivo observations, transfected keratinocytes expressing Snail exhibited a substantial increase in pMAPK levels relative to control cells (Figure 4D). Coexpression of E-cadherin with Snail appeared to abrogate this effect. Together, these findings raised the possibility that an AJ-associated protein that is normally sequestered at the plasma membrane may participate in a proliferation signaling pathway when AJs are deconstructed.
Numerous studies have correlated a down-regulation of E-cadherin with a translocation of β-catenin to the nucleus and a transactivation of genes that are regulated by the LEF-1/T cell factor (TCF) family of DNA binding proteins [23,24,25]. The presence of nuclear cyclin D in hyperproliferative Snail Tg epidermis was particularly intriguing since prior studies have reported cyclin D gene as a direct target of TCF/β-catenin transcription [26]. This said, we did not detect nuclear β-catenin in our Tg epidermis, and mating the Snail Tg mice against the TOPGal reporter mouse [20] gave no signs of ectopic LEF-1/Tcf/β-catenin activity (unpublished data).
We next turned to the presence of cytoplasmic Ajuba for a possible mechanistic link to the proliferative increase in our Snail Tg epidermis. In addition to its documented ability to bind α-catenin [10], Ajuba can also associate with growth factor receptor-bound protein-2 (Grb-2)/son of sevenless (Sos), the nucleotide exchange factor for Ras, which is upstream from activation of MAPK [9]. Given the increase in pMAPK staining in Tg skin, we examined the possibility that Ajuba might have changed its binding partner in Snail-expressing epidermis. Interestingly, Ajuba was readily detected in anti-Grb-2 immunoprecipitates of protein lysates from skins of Snail Tg mice but not from the corresponding wild-type (WT) animals (Figure 4E). When these experiments were repeated with α-catenin-null epidermis, a similar Grb-2-Ajuba association was detected, and again, this interaction was not detected in the protein extracts from control littermate skin (Figure 4E). Together, these data demonstrate that the reduction in α-catenin levels, either by Snail-mediated down-regulation of E-cadherin or by α-catenin conditional targeting, allows Ajuba to interact with Grb-2/Sos.
If the competition between Grb-2/Sos and α-catenin for Ajuba is functionally relevant to the hyperproliferative state of a keratinocyte, then overexpression of Ajuba would be expected to bypass the competition and promote activation of the Ras-MAPK pathway in WT keratinocytes. Indeed, when serum-starved keratinocytes were transiently transfected with an Ajuba expression vector, the levels of pMAPK were not only elevated but also comparable to those transfected with the K14-HASnail transgene (Figure 4F). This activation was abolished when cells were treated with a small peptide inhibitor that specifically interrupts the Grb-2/Sos interaction (Figure 4F; see lanes marked “inh”) [27].
Ajuba's pre-LIM domain is the segment that associates with Grb-2's Src-homology 3 domain [9]. When this domain was overexpressed in serum-starved keratinocytes, a comparable elevation in pMAPK was observed (Figure 4F). As expected, the small peptide inhibitor that interrupts the Grb-2/Sos association blocked the effects. These data suggested that by elevating cytosolic Ajuba levels, Ajuba's pre-LIM domain may associate with Grb-2/Sos in a manner that stimulates its nucleotide exchange activity and leads to activation of the Ras-MAPK pathway. Although this pathway provides one mechanism by which Snail expression and proliferation may be coupled in skin epithelium, proliferative circuitries involving AJs are known to be complex and often interwoven. Future studies will be needed to systematically dissect these putative intricacies at a molecular level.