The assembly of a stratified, squamous, epidermal epithelium is complex and is accompanied by a sequential series of changes in keratinocyte gene expression as cells migrate from the basal epidermal compartment towards the differentiating suprabasal layers. The AP-2 transcription factor family is presumed to play an important role in the regulation of this squamous differentiation program however, little functional data is available to support this. In the present study, the expression, activity and regulation of AP-2 was examined both in vitro and in vivo in differentiating keratinocytes. In addition the expression of AP-2 isoforms was also examined in skin appendages such as the sebaceous glands, melanocytes and hair follicles. The data presented indicate that 1) AP-2α, β and y mRNA and protein expression occurs in proliferating and differentiated keratinocytes in vitro and in vivo, 2) AP-2 transcriptional activity decreases in differentiated keratinocytes, 3) AP-2 isoforms are expressed in the keratinocyte-derived squamous cell carcinoma however, AP-2 activity is not reduced in squamous cell carcinoma cells, 4) in normal keratinocytes AP-2 transcriptional activity does not appear to be regulated by recruitment of activating histone acetyltransferases or repressive histone deacetylases, 5) diminished AP-2 transcriptional activity is associated with a loss of specific DNA-bound AP-2 complexes, 6) AP-2β and y, but not α, are associated with the AP-2 DNA-binding complexes in proliferative keratinocytes and 7) each AP-2 isoform is phosphorylated by nuclear extracts from proliferative keratinocytes but this phosphorylation is reduced as keratinocytes differentiate. In skin appendages, the data presented indicate that 8) AP-2α, β and y proteins exhibit distinct and different patterns of expression within the anagen hair follicle in vivo and 9) AP-2 factors are expressed in isoform-specific patterns in a) sebaceous glands and b) melanocytes.
Combined, these data indicate that despite expression of AP-2α, β and y mRNA and protein species throughout proliferating and differentiating human epidermis, AP-2 rran5-activation and DNA-binding activity decrease as keratinocytes differentiate. This decrease in AP-2 activity is associated with a reduction in the phosphorylation status of the AP-2 isoforms that is unlikely to be a result of phosphatase activity. In contrast, expression of all AP-2 isoforms was observed in squamous cell carcinoma in vivo however, activity was not decreased in squamous cell carcinoma cells that had been treated with the differentiation inducer TPA. Together these data indicate that, AP-2 expression patterns may not be indicative of activity and that regulation of AP-2 factors (e.g. posttranslational modification such as phosphorylation) may be a better determinant of AP-2 activity. Furthermore, the present study indicates that AP-2 isoforms are likely to have potentially important and specific functions in the anagen hair follicle and that these functions may differ to their role in the interfollicular epidermis. Moreover, the finding that AP-2 factors have isoform-specific staining within melanocytes and sebocytes strongly suggests that they may be implicated in the regulation of specific functions within these cells.