Protein Deimination and Peptidylarginine Deiminase Expression during Cornification of Rat Epidermal Keratinocytes

Abstract

We have studied protein deimination, which is catalyzed by peptidylarginine deiminases (PADs), in a line of epidermal keratinocytes from a newborn rat. Such cells, when inoculated into cultures at a density of 1.3 X 10 4 cells/cm 2 , became confluent at 6 days, began to construct stratified colonies at 8 days, and formed multiple cell layers at 15 days. Deiminated proteins were initially detected on the 11 t h day, then gradually increased as the cell layers multiplied. However, PAD activity was detected 5 days earlier than the appearance of deiminated proteins when cell homogenates were incubated in the presence of 1 mM Ca 2 + . To elucidate the precise stage of growth and differentiation when keratinocytes express PAD, we tested for filaggrin, which is a terminal differentiation marker synthesized in granular layers of keratinocytes. Filaggrin content was represented by its high-molecular-weight precursor protein, profilaggrin. Profilaggrin was initially detected on the 6 t h day of culture and reached maximum production at 8 days. Processed intermediate filaggrins and filaggrin monomers were also detected at 8 days. Since profilaggrin and PAD were expressed after the same interval of cultivation, these proteins must arise at the same point of keratinocytes' terminal differentiation. The foregoing results indicate that protein deimination is involved in the cornification of epidermal keratinocytes and that PAD is expressed during their terminal differentiation process.