Cell–cell adhesion and communication are coordinated events that help maintain epithelial tissue homeostasis. The present study defined the ability of EphA2 to dampen contact-dependent Erk1/2-MAPK signaling in primary human keratinocytes without affecting cell proliferation and identified the importance of this receptor tyrosine kinase in enhancing desmoglein 1–dependent adhesion and differentiation in cells derived from stratified epithelial tissues.
EphA2 is abundantly expressed in primary keratinocytes and becomes concentrated at cell–cell contacts where it is activated by ephrin ligands. Our studies were focused on the consequences of EphA2 ligand targeting and support a model whereby this receptor responds to ephrins present on adjacent cells to fortify differentiation-associated adhesion complexes that facilitate keratinocyte stratification. Under steady-state conditions, keratinocytes in the basal layer of the epidermis are tightly associated with one another and EphA2/ephrin-A1 signaling complexes are likely present at cell–cell contacts (Guo et al., 2006
). Because EphA2 knockdown weakened adhesion in epidermal sheets, it seems likely that it plays a general role in maintaining tissue integrity even in the basal layer. The signaling mechanisms controlling the ability of basal keratinocytes to enter into suprabasal layers and adopt a more differentiated phenotype remain somewhat unclear. Our data suggest that one of the early signals that triggers keratinocyte differentiation may be increased forward signaling through EphA2 that elicits the expression of differentiation-associated desmosomal cadherins. Ephrin-A1-Fc peptidomimetics were used to enhance EphA2 forward signaling in our study, leading to receptor destabilization and down-regulation. It is possible that robust EphA2 activation and membrane destabilization may also normally occur in response to increased presentation of GPI-linked ephrin-A1 in the basal layer, possibly involving receptor internalization and subsequent down-regulation to promote differentiation in suprabasal keratinocytes.
Similar to peptide-mediated down-regulation of EphA2, the formation of native Eph receptor/ephrin signaling complexes often leads to their endocytosis and/or proteolysis in poorly adhesive cells, such as neurons (Miao and Wang, 2009
; Pasquale, 2010
). As previously shown in cell types with more robust intercellular junctions (Zantek et al., 1999
; Orsulic and Kemler, 2000
; Hess et al., 2006
; Miura et al., 2009
), cadherin-mediated adhesion stabilized EphA2 at keratinocyte borders. At present, it remains unclear whether EphA2 is preferentially stabilized on the cell surface or more efficiently recycled back to junctions after its activation by ephrin ligands. Interestingly, the lipid phosphatase SHIP2 interacts with EphA2 and regulates its endocytosis and degradation via inhibition of a phosphatidylinositol 3-kinase–dependent receptor internalization pathway that relies on Rac1 activity (Zhuang et al., 2007
). It is therefore possible that the dampening of Rac1 induced by long-term stabilization of E-cadherin–based cell–cell contacts allows SHIP2 to more efficiently stabilize EphA2 at the cell surface (Braga et al., 2000
; Perez et al., 2008
). Alternatively, EphA2 may be preferentially recruited into an endosomal recycling pathway in keratinocytes instead of being targeted for proteosomal and/or lysosomal degradation by modulation of c-Cbl–dependent ubiquitinylation and subsequent interactions with the ankyrin domain containing protein, Odin (Wang et al., 2002
; Kim et al., 2010
). In the future, it will be interesting to determine whether the extent of ligand activation of EphA2 modulates its association with these trafficking effectors in epidermal cells, possibly leading to its destabilization and loss in more differentiated keratinocytes.
Ephrins and Eph receptors have been shown to support the epithelial phenotype. For example, EphB forward signaling and ephrin-B reverse signaling enhance adherens junctions and tight junctions, respectively (Noren et al., 2006
; Cortina et al., 2007
; Lee et al., 2008
; Chiu et al., 2009
). Although ligand activation of EphA2 can disrupt adherens junctions by increasing RhoA activity when the receptor is overexpressed (Fang et al., 2008
), these complexes also promote cadherin-dependent compaction through the recruitment of Nck and Git1, which leads to the suppression of the GTPase, Arf 6 (Miura et al., 2009
). Moreover, EphA2 has been shown to regulate epithelial branching morphogenesis in the kidney and mammary gland, suggesting a dynamic interplay between this receptor tyrosine kinase and cell adhesion complexes (Miao et al., 2003
; Vaught et al., 2009
). Finally, EPHA2
mutations in humans or targeted deletion of this receptor or the ephrin-A5 ligand in mice leads to cataract development that has been associated with defects in N-cadherin–based junctions of lens fiber cells (Cooper et al., 2008
; Jun et al., 2009
); epithelial tissues, including the epidermis, appear to be relatively intact in this and independent lines of mice that lack EphA2 (Naruse-Nakajima et al., 2001
; Guo et al., 2006
), perhaps due to compensation by other EphA receptor subtypes. In the present study, we provide the first functional evidence for EphA2 to increase the strength of epithelial cell–cell adhesion. Notably, we did not find any evidence for marked changes in E-cadherin incorporation into junctions (data not shown) or changes in expression levels. Alternatively, a marked increase in the expression of desmosomal cadherins was observed. In particular, desmosomal cadherins restricted to the more differentiated layers of the epidermis, namely desmoglein 1 and desmocollin 1 a/b, were up-regulated by ligand targeting of EphA2, uncovering a novel link between EphA2 and desmosomes. EphA2 not only enhanced desmosomal adhesion but also increased terminal differentiation in a population of stratified keratinocytes while allowing cells in contact with the substrate to continue proliferating. Finally, we demonstrated that the increase in desmoglein 1 expression contributed to these differentiation-promoting effects of soluble ephrin ligands.
Although ligand targeting of EphA2 did not inhibit human keratinocyte proliferation, we believe that our results remain consistent with previous reports in mouse keratinocytes where colony growth was inhibited and proliferation was not directly measured (Guo et al., 2006
). Instead of suppressing proliferation, the primary event in limiting the expansion of mouse and human keratinocyte colonies may be increased differentiation and stratification. The ability of ephrin ligands to enhance keratinocyte differentiation may also help explain how EphA2 serves as a tumor suppressor in the skin. Recent studies in mouse models of skin carcinogenesis have demonstrated a critical role for Raf-1 in suppressing differentiation; this differentiation phenotype was unrelated to the mitogenic effects of Raf-1 mediated through activation of Mek-Erk1/2 signaling cascade (Ehrenreiter et al., 2009
). Importantly, tumors were resolved by Raf-1 ablation. Because EphA2 is abundantly expressed in skin cancer, it may serve as a good candidate to be targeted for the purpose of promoting tumor cell differentiation and regression. Systemic administration of ephrin peptidomimetics may not be the best approach to trigger EphA2 activation in the epidermis as this leads to an increase in keratinocyte proliferation (Genander et al., 2010
). Because our studies show that EphA2 is not required for the negative regulation of keratinocyte proliferation, the growth-promoting effects observed in peptide-treated mice may reflect differences in cell culture or species, agonistic or antagonistic effects of these peptides on other EphA receptor subtypes, changes in epidermal differentiation and barrier function that indirectly lead to epidermal hyperplasia, or actions on other targeted cell types, such as immunoregulatory cells, that drive inflammatory-associated epidermal hyperproliferation (Segre, 2006
; Hanifin, 2009
). The differentiation-promoting effects of ephrins may be restricted to stratified epithelia and corresponds well with the spatiotemporal distribution of ephrin-A ligands in the basal layer and the more broad distribution of EphA receptors (EphA1 and EphA2) that extend into the upper layers of the epidermis (de Saint-Vis et al., 2003
; Guo et al., 2006
; Hafner et al., 2006
Finally, it is important to consider that EphA2 expression in keratinocytes is increased in response to hypoxia and UV irradiation, where it plays an important role in promoting apoptosis (Vihanto et al., 2005
; Zhang et al., 2008
). Ligand targeting of EphA2 did not alter keratinocyte growth but effects on survival pathways may contribute to the differentiated phenotype because these distinct cellular processes share many of the same effector molecules and both ultimately lead to cell death (Candi et al., 2005
). Taken together, these studies demonstrate that ligand targeting of EphA2 increases the adhesive strength and terminal differentiation of keratinocytes via up-regulation of desmoglein 1 and provide a novel mechanism beyond the inhibition in proliferation to explain ephrin-mediated restriction in the lateral expansion of epithelial colonies. Future studies will be aimed at delineating the pathways downstream of EphA receptors that help maintain epidermal homeostasis.