Deimination is a posttranslational modification catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). In a protein substrate, PADs transform arginyl into citrullyl residues. Although deimination has been associated with numerous physiological and pathological processes, its cellular function is still misunderstood. Three PADs are expressed in the epidermis and skin appendages. To study the importance of PADs in the epidermis homeostasis, three-dimensional reconstructed human epidermis (RHEs) were treated for two days with increased concentrations (from 0 to 800 µM) of Cl-amidine, a specific pan-PAD inhibitor. The efficacy of Cl-amidine treatment was analyzed by western blotting with antibodies specific for deiminated proteins and its effect on the morphology of the epidermis by transmission electron microscopy. We showed that PAD1 and PAD3 were expressed in RHEs, deiminated proteins being detected in the cornified layer. Cl-amidine treatments inhibited deimination in a dose-dependent manner and were not cytotoxic. At 800 µM, Cl-amidine was shown to decrease deimination by half, to reduce the number of corneocyte layers, to increase the number of transitional cells, to induce an accumulation of mitochondria and of heterogeneous cytoplasmic vesicles in the granular keratinocytes, and to up-regulate LC3B-II, a marker of autophagic vesicles. These results demonstrated that Cl-amidine treatments slow down the transition of granular keratinocytes to corneocytes and disturb the autophagic flux in the granular layer. This suggests that PAD1 and/or PAD3 play a key role in the constitutive epidermal autophagy process that appears as an important step in cornification.

In reconstructed human epidermis, peptidylarginine deiminase inhibitor Cl-amidine modulates cornification and keratinocyte autophagic flux