204 Is the proteasome activity involved in filaggrin breakdown?

Abstract

Epidermal hydration is maintained mainly by the retention of water in the stratum corneum through the natural moisturizing factor (NMF). NMF is a complex mixture of components, including pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA), two amino acid derivatives. In the stratum corneum, breakdown of filaggrin (FLG) is the main source of PCA and UCA. The proteases involved in this degradation are still incompletely known. In the keratosis linearis with ichthyosis congenital and sclerosing keratoderma (KLICK) syndrome, a defect in the proteasome maturation protein POMP causes an altered epidermal distribution of this multi-protein complex, associated with enlarge and rounded shaped keratohyalin granules and a disturbed detection of FLG. We hypothesized that the proteasome is involved in FLG degradation. 3D reconstructed human epidermis, produced at the air-liquid interface in a low relative humidity condition (30-50%), were treated for 24 hours with various concentrations of the irreversible proteasome inhibitor clasto-lactacystin b-lactone, and analyzed using histochemistry, indirect immuno-fluorescence, transmission electron microscopy, western blotting and UHPLC linked to mass spectrometry. After clasto-lactacystin b-lactone treatment, as expected, a significant accumulation of ubiquitinated proteins was observed, without any major cytotoxic effects compared to controls. The morphological integrity of the epidermis was maintain, even if many small vesicles and spherical keratohyalin granules were observed in the cytoplasm of differentiated keratinocytes. The treatment induced a significant decrease of FLG immunodetection. However, the amount of PCA and UCA did not show any drastic differences between treated and untreated conditions. Therefore, proteasome activity seems not directly involved in FLG breakdown but may act during keratinocyte differentiation and epidermal homeostasis.