ACTIVATION OF NOTCH SIGNALING IN KERATINOCYTES (KC’S) IS NECESSARY AND SUFFICIENT TO CREATE MATURE HUMAN EPIDERMIS.

The signaling system regulating the highly coordinated and programmed series of molecular events that generate a mature, fully differentiated and cornified epidermis is unknown. To determine if Notch ligands and receptors that regulate cell fate decisions in other organ systems may be operative in epidermis, normal human skin was initially examined by immunostaining and Western blot analysis. Fractionating KCs by state of maturation using discontinuous Percoll gradients, followed by immunoblotting confirmed immunohistology results in which KCs in suprabasal and mid epidermal levels prominently expressed a Notch ligand – Jagged 1 on the plasma membrane; which were the same KC layers coexpressing several Notch receptors; including Notch1, Notch3, and Notch4. Based on these localization studies, Jagged-1 appeared to be strategically located in epidermis to influence both early and late stages of KC differentiation. To establish a cause:effect relationship between notch signaling and KC differentiation, submerged living epidermal equivalents (EEs) consisting of multilayered but relatively undifferentiated KCs grown on a permeable membrane were raised to an air/liquid interface (A/L-I), which triggers KC terminal differentiation/ corneogenesis. Western blot analysis of proteins extracted from submerged EEs vs. EEs raised to A/L-I for 2 days revealed an ~4-fold increase in levels of these notch ligands/receptors. This result prompted use of soluble notch ligands-peptides derived from the most conspicuously expressed ligand in human skin, Jagged-1. Exposing human KC monolayers to peptides synthesized to correspond to the most conserved DSL domain of hJaggedl (designated: JAG-1 or R-JAG), but not a scrambled control (SC) peptide, produced stratification and loricrin/involucrin expression. Moreover, addition of JAG-1 or R-JAG, but not SC, peptides to submerged EEs was sufficient to induce epidermal maturation resembling submerged EEs raised to A/L-I. Finally, a soluble decoy notch inhibitor (rh11-12) prevented KC differentiation and corneogenesis when submerged EEs were pretreated with inhibitor prior to being lifted to A/L-I. Overall, these results point to a key role for notch ligand/receptor-dependent signaling in governing the complex process of epidermal maturation.

Apoptosis, Differentiation, EpiDerm, Involucrin, Jag-1, Jagged 1, Jagged-1, Loricrin, Notch signaling, Notch1, Notch3, Notch4, R-Jag