Differentiation of stratified squamous epithelial tissues like the corneal epithelium and the epidermis of the skin involve multiple alterations in gene expression in order to enable the basal cells to leave the basement membrane and migrate to the surface where they are shed. Central to the changes required for terminal differentiation of the basal cells are a cessation of cell division and alterations in adhesive properties. The underlying cue(s) for terminal differentiation of these cells is not well understood.
AP-2 transcription factors have been implicated in regulating genes involved in epithelial differentiation, and defects in null mutant mice have revealed a requirement for AP-2α in early development of the ocular epithelium (West-Mays et al., 1999
; Hilger-Eversheim et al., 2000
). In the current study, we have shown that the two AP-2 (α, β) proteins are differentially expressed in the developing ocular surface. AP-2β protein was found to be broadly expressed throughout all of the stratified layers of the corneal epithelium, whereas AP-2α expression was more restricted to the basal cell layers including the stratum basale and the stratum spinosum. AP-2α protein expression was also highly expressed in the developing eyelid epidermis with particularly strong staining in the more basal cells of the epidermal surface. In contrast, AP-2β expression was not detected in these tissues. Stable transfection of a corneal epithelial cell line with the AP-2α gene resulted in an alteration in cell phenotype and an increase in levels of a 135–140 kD cadherin protein identified as N-cadherin. Examination of the ocular surface of AP-2α chimeric mice further revealed a positive correlation between the knockout cell location and regions of absent E-cadherin expression. Together, these findings suggest that AP-2α participates in differentiation of the ocular surface through the regulation of cadherin gene expression.
The more restricted expression pattern of AP-2α protein in the more basal cell compartments of the ocular surface epithelium as compared to AP-2β suggests that it is positioned to contribute to the mechanisms which control differentiation. A number of genes involved in differentiation exhibit a differential expression pattern within the stratified layers of epithelial tissues, such as members of the integrins, keratins, and importantly, the cadherins (Schermer et al., 1989
; Maytin et al., 1999
). For example, E-cadherin expression in the epidermis is lost in the upper keratinized layers, and as a result, the decreased cell-cell adhesion is thought to contribute to terminal cell differentiation. In the cornea, the keratin 3 (K3) gene is expressed in the upper stratified layers, whereas its expression is repressed in the undifferentiated basal epithelial cell compartment. In a culture model for corneal epithelial cell differentiation, AP-2 was shown to act as a repressor of K3 expression (Chen et al., 1997
). A down-regulation in AP-2 binding activity in the more suprabasal cells was found to coincide with differentiation and activation of K3. Paradoxically, the decrease in binding activity for AP-2 in the suprabasal cells was reported despite findings of a steady-state level of AP-2 mRNA throughout all layers of the cultures. Based on our in vivo
findings that the AP-2 proteins are differentially expressed in the corneal epithelium, with AP-2α expression restricted to the more basal cell layers, it is reasonable to propose that the repression of the K3 gene in the cultures was due to specific binding of AP-2α.
Additional studies support a role for AP-2α in regulating genes involved in differentiation. A subtractive hybridization screen of AP-2α-/- mutant mice revealed premature expression of a target gene involved in the induction of terminal differentiation of embryonic fibroblasts, the Kruppel-box transcription factor, KLF-4 (Pfisterer et al., 2002
). Similarly, our group has recently demonstrated that ectopic expression of AP-2α in the developing lens resulted in repression of Membrane Instrinsic Protein (MIP) expression and inhibition in fiber cell differentiation (West-Mays et al., 2002
). AP-2 has also been implicated in regulating genes involved in the cell cycle and promoting proliferation (Bosher et al., 1996
; West-Mays et al., 1999
; Pfisterer et al., 2002
). Thus, the findings of the current study, including the localization of AP-2α to the more undifferentiated cell populations of the ocular surface, support a growing body of evidence that AP-2α may act in the maintenance of cells in the cell cycle and in preventing them from undergoing premature differentiation.
Overexpression of AP-2α in SIRC cells resulted in a significant alteration in cell phenotype and gene expression profile. SIRC cells are generally fusiform in shape and form a confluent monolayer. In the stable clones, the cells clustered and did not plate well. A number of genes may have been altered in these clones and responsible for the observed phenotype. While Western blots indicated similar levels of β1 family integrins, changes in integrin activation cannot be ruled out. We did, however, observe an alteration in the levels of as N-cadherin, a 135–140 kDa protein belonging to the family of cell adhesion molecules, the cadherins. N-cadherin is a Ca + + dependent cell adhesion molecule originally identified in mouse and chicken nervous tissues (Hatta et al., 1985
) and is classically described as being involved in homophillic cell-to-cell adhesion (Hatta et al., 1987
). Thus, induced expression of N-cadherin in the clones may have been directly responsible for the clustering phenotype observed in the SIRC clones. Such an increase in cell-to-cell contact may have further resulted in less cell surface area available for adhesion to matrix, thereby resulting in reduced ability of the SIRC clones to adhere to ECM. Alternatively, since cadherins have also been shown to mediate cell-to-matrix adhesion through cross talk with integrin molecules (Hodivala and Watt, 1994
: Higgins et al., 1998
), a change in the levels of N-cadherin may have altered the ability of the integrins expressed in the SIRC clones to interact with particular ECM molecules.
Decreased staining for E-cadherin was detected in the ocular surface tissue regions of AP-2α chimeric mice that contained a high proportion of AP-2α-/- cells. Regulation of E-cadherin promoter activity by AP-2α has been shown previously in cultured keratinocytes (Behrens et al., 1991
; Faraldo et al., 1997
), and a positive correlation between localization patterns of AP-2 and E-cadherin expression have also been reported in vivo
(Baldi et al., 2001
). Recent findings by our group have further shown that ectopic expression of AP-2α in the developing ocular lens resulted in expanded expression of E-cadherin protein in the lens transitional zone, the region in which epithelial cells terminally differentiate into fiber cells (West-Mays et al., 2002
). Thus, E-cadherin is likely to be an important downstream target for AP-2α during differentiation. We also found that overexpression of AP-2α in a corneal epithelial cell line resulted in an upregulation of N-cadherin protein expression. Earlier characterization of the promoter region of the chicken N-cadherin gene had revealed several consensus AP-2 binding sequences which were required for promoter activity (Li et al., 1997
). This suggests the possibility that AP-2α may control N-cadherin expression through direct transactivation of the promoter, although this remains to be determined. Together, these findings suggest that AP-2α may regulate the expression of multiple members of the cadherin family.
Corneal epithelial cells have been shown to express the classical cadherins, E-cadherin and P-cadherin (Takahashi et al., 1992
; Mohan et al., 1995
). Reports of N-cadherin expression in the cornea typically include that found in the neural crest cells which contribute to the corneal stroma and endothelium (Takeichi, 1988
). Although transient N-cadherin expression has been reported in the corneal epithelium during embryonic development (Hatta et al., 1987
), N-cadherin is not usually detected in the adult corneal epithelium. Thus, our observation of a low level of N-cadherin expression in the SIRC cells and no E-cadherin expression was unexpected and may suggest that this cell line does not express all corneal epithelial cell specific characteristics. In fact, SIRC cells have been known to lack other corneal epithelial specific genes (Niederkorn et al., 1990
). However, like other ocular epithelial cells, SIRC cells express Pax-6 protein (Sivak et al., 2000
) and form adhesive colonies. The SIRC clones (transfected with AP–2α) also formed circular clusters that closely resembled lentoid bodies, differentiated fiber cells derived from cultured lens epithelial cells, another ocular epithelial cell type. Thus, one possibility is that SIRC cells more closely resemble lens epithelial cells. Since AP-2α acts in a combinatorial fashion with multiple transcription factors in governing epidermal gene expression (Sinha et al., 2000
), the final outcome as to which of the cadherin subclasses is ultimately expressed in an ocular epithelial tissue is likely dependent on the milieu of additional factors expressed in each cell type. Further studies examining the factors that AP-2 interacts with and how this influences cadherin gene expression should help shed light on how the cadherins are differentially expressed in the developing and adult ocular epithelia.
In summary, we have shown that the AP-2α and AP-2β proteins are uniquely expressed in the differentiating ocular surface, with AP-2α expression correlated with the more undifferentiated cell compartments. Altered expression of the AP-2α gene also resulted in defects in differentiation of the corneal epithelium and stratified epithelium of the eyelids, and this was positively correlated with altered cadherin expression. Together, these findings suggest that AP-2α acts as an important switch for regulating differentiation of the stratified epithelium of the ocular surface.