Stratification of the epidermis commences during embryonic development and is a process that continues to occur throughout the life of the organism. The process entails the outward movement of the proliferative basal cells that are adjacent to the basement membrane toward the surface of the skin. The stratification is concurrent with a Ca++-dependent differentiation process and the layer-specific expression of structural and enzymatic markers, with the basal proliferative cell differentiating first to a spinous cell, then to a granular cell, to ultimately terminally differentiate as a cornified, anucleated cell. To elucidate the functional role of the Dlx3 homeodomain protein during early development and during epidermal differentiation, we are currently in the process of determining the target genes and the Dlx3 interacting factors necessary to exert the transcription regulatory function. Furthermore, biochemical characterization of a mutant form of Dlx3 that in humans is associated with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome is also being performed. TDO syndrome is characterized by defects in ectodermal derivatives such as hair and teeth and craniofacial bone abnormalities. Recent findings show that Dlx3 is also expressed in the developing bone, and the expression is retained in trabecular bone of the adult skeleton. Identification and characterization of a novel epidermal- and differentiation-specific gene. Performing a Suppressive Subtractive Hybridization (SSH) screen we obtained a cDNA clone we termed Scarf, which has homology to Calmodulin-like proteins. The ORF encodes a protein that contains four conserved EF-hand motifs, putative Ca++-binding domains. The Ca++ signaling dependent systems, such as keratinocyte differentiation process, must be finely tuned for rapid and effective response to transient variations in Ca++ concentration. A central role in the transduction of Ca++ signals is played by members of the Ca++-binding proteins. The functionality of the EF-hand motifs may contribute to specificity in the interaction with the target molecules. Thus, it is through the binding of Ca++ by the EF-motifs that these proteins are able to bind or liberate target interacting proteins and in this way modulate their functions. Northern blot and in situ hybridization analysis showed restricted differential expression of the Scarf gene in the suprabasal layers of the epidermis. Furthermore, induction of Scarf expression was observed upon in vitro differentiation by Ca++ of primary cultured keratinocytes. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca++-binding method, determining that Scarf EF-hand motifs are functional binding domains. To identify Scarf target proteins we are performing immunoprecipitation assays followed by mass spectrometry. Identification of target proteins will aid in understanding the role of Scarf during keratinocyte differentiation.
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