Exposure to 12-O-Tetradecanoylphorbol-13-acetate (TPA) Induces the Synthesis of Histidine-Rich Protein (Filaggrin) in Monolayer Cultures of Rat Keratinocytes

Abstract

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, on epidermal differentiation has been studied by investigating the appearance of the epidermal differentiative marker protein, histidine-rich protein (HRP), in monolayer cultures of rat keratinocytes grown in low-calcium (0.1 mM) medium. Monoclonal antibody, 3F6-6, raised against the 60K HRP (HRPII) derived from the epidermis of the newborn rat was utilized to detect HRP in cultured keratinocytes. As a major component of keratohyalin in the cells of the granular layer in epidermis, HRP is not normally found in a monolayer culture of rat keratinocytes containing a high population of proliferating cells. However, when these cultures were exposed to 10 ng/ml of TPA, DNA and protein synthesis were decreased by up to 95% and 60%, respectively, and the cells in the monolayer culture became HRP-positive within 18-24 h. TPA has been shown to cause a withdrawal of the basal cells from the cell cycle and a commitment of these cells to an accelerated rate of terminal differentiation. Exposure to TPA at the same time that the calcium level of the monolayer cultures was raised to 2.0 mM in order to allow stratification resulted in positive immunostaining for HRP much earlier and much more intensively than in the absence of TPA. TPA also enhanced stratification of these cultures; however, the positive expression of HRP preceded the stratification. These observations support the view that the induction of terminal differentiation occurs independently of stratification and that the process of terminal differentiation (maturation) of basal cells precedes and triggers the movement of these cells to a suprabasal position (stratification).