NOTCH-1 MEDIATED SIGNALING INCREASES IKKα LEVELS – A CRITICAL DETERMINANT CONTROLLING KERATINOCYTE TERMINAL DIFFERENTIATION AND CELL SURVIVAL.

Epidermal formation depends on a balance between cell proliferation, differentiation, and death. While deletion of IKKα gene blocks terminal differentiation in mice, biochemical pathways regulating IKKα in epidermis are unknown. Notch receptors regulate cell fate in response to ligands such as Jagged-1. In normal skin, Jagged-1 is expressed by keratinocytes (KCs) in basal cells and stratum spinosum, but not in granular cell layer or by corneocytes. To explore a role for Notch signaling in epidermopoiesis, human KCs were infected with retroviral constructs containing truncated (constitutively activated) forms of Notch-1 receptor (NIC-1) and Notch-4 receptor (NIC-4). Compared to empty linker control infection, which had no effect on KCs, infection with either NIC-1 or NIC-4, triggered growth arrest accompanied by stratification. Biochemical analysis confirmed induction of Notch-target genes such as Hes-1, and cell cycle regulator p21, as well as early/late differentiation markers (involucrin-loricrin). Importantly, only NIC-1 triggered accumulation of IKKα accompanied by decrease in pro-apoptotic PKCδ, whereas neither NIC-1 nor NIC-4 induced IKKß levels. By contrast, cytokines such as IFN-γ plus TNF-α induced IKKß but not IKKα. When Notch signaling was interrupted by addition of a γ -secretase inhibitor (GSI), activated Notch receptor levels were reduced, and KCs underwent apoptosis in a concentration and time dependent fashion blocked by addition of caspase inhibitors. Apoptotic machinery involved in GSI-induced cell death included activation of caspase 3 and release of cytochrome C from mitochondria. Given the critical role for IKKα in terminal differentiation, we suggest that Notch-1 mediated signaling is responsible for endogenous IKKα accumulation. Furthermore, abrupt loss of Jagged-1 in upper epidermal layers may trigger cell death necessary for cornification following terminal differentiation. Thus, we propose Notch signaling regulates KC proliferation, differentiation, and cell death.

Apoptosis, Apoptotic machinery, Basal cells, Caspase 3, Caspase inhibitors, Cell cycle regulator p21, Cell death, Cell differentiation, Cell proliferation, Constitutively activated, Corneocytes, Cytochrome C, Cytokines, Early/late differentiation markers, EpiDerm, Epidermal formation, Epidermopoiesis, GSI-induced cell death, Gamma secretase inhibitor (GSI), Granular cell layer, Hes-1, IFN-gamma, IKK-alpha gene, Involucrin-loricrin, Jagged-1, KC, Keratinocytes, Ligands, Mitochondria, Notch receptors, Notch signaling, Notch-1 receptor (NIC-1), Notch-4 receptor (NIC-4), Notch-target genes, Pro-apoptotic PKC-delta, Stratum spinosum, TNF-a, Terminal differentiation