S100A6 expression in keratinocytes and its impact on epidermal differentiation.

S100A6 is a calcium binding protein expressed in many types of epithelia including epidermis. S100A6 is a binding partner of a number of proteins engaged in cytoskeletal organization, cell cycle control, stress response or apoptosis. So far the effect of its overexpression or knock-down on cell physiology has been studied only at the cellular level. Here, we used an in vitro model of differentiating epidermis to study the role of S100A6 at the tissue level and in the context of tissue differentiation. First of all we have shown that S100A6 mRNA level diminished several fold during primary keratinocyte differentiation and investigated the epigenetic and transcriptional mechanisms involved in this tight expression control. Using bisulfite treatment, luciferase assay and chromatin immunoprecipitation we found that changes in S100A6 expression were DNA methylation independent but could be orchestrated by epidermal specific factors: the ΔNp63 transcription factor and retinoic acid. To investigate if the drop-down in S100A6 expression is indeed critical for keratinocyte differentiation we developed HaCaT cells with stable S100A6 knock-down or overexpression and tested them in 2- and 3-dimensional (organotypic) culture conditions. S100A6 overexpressing cells exhibited accelerated proliferation, enhanced adhesion properties and suppressed loricrin expression - features typical for undifferentiated keratinocytes. In organotypic culture these cells formed thicker epidermis with more Ki67 positive cells, keratin 10 expression spatially limited to the uppermost cell layers and non-detectable loricrin expression. Together, results obtained in both culture models proved that increased S100A6 content in keratinocytes dramatically changed the pace and extent of epidermal differentiation.