CROSS-TALK BETWEEN NOTCH SIGNALING AND TWO PATHWAYS REGULATING EPIDERMAL DIFFERENTIATION.

Recently we established that peptides derived from the Notch ligand-Jagged-1 could trigger complete epidermal differentiation using submerged living epidermal equivalents (EEs). Moreover, using a selective inhibitor designed to block Notch receptor activity, epidermal differentiation was also blocked when submerged EEs were raised to an air/liquid interface. To determine the molecular basis by which Notch signaling triggered epidermal differentiation, focus was directed on established pathways that impact epidermal maturation -, i.e. NF-кB and PPAR. Addition of a peptide synthesized to correspond to the most conserved DSL domain of hJaggedl (designated JAG-1 peptide), but not scrambled control peptide, rapidly induced (7-60 mins) p50 and p65 translocation to the nucleus as detected by Western blot analysis, accompanied by increased DNA binding activity of NF-кB (EMSA), and transcriptional activity (luciferase reporter assay). Supershift analysis revealed both p65 and p50 subunits were present in the protein:DNA complex. Moreover, this DNA binding returned to baseline after 2 h, despite continued presence of intranuclear p65 subunits. This suggested a possible inhibitor of p65-mediated DNA binding activity. Since PPARγ has been previously observed by others to inhibit NF-кB transcriptional activity in monocytes, and because we previously observed that JAG-1 peptide could induce PPARγ in adipocytes, the ability of JAG-1 to induce PPARγ was explored. Addition of JAG-1 peptide to KCs also induced increased nuclear levels of PPARγ and PPARα between 30 min and 6 h. Moreover, in separate experiments in which both p65 and PPARγ were elevated, immunoprecipitation followed by Western blot analysis demonstrated physical association between p65 and PPARγ. Furthermore, using a dominant negative retroviral vector for NF-кB (i.e. iкBαDN), there was inhibition of PPARγ- induction indicating a link between NF-кB and PPARγ. Thus, we propose a series of reactions in which JAG-1 peptide initially activates Notch signaling, followed by NF-кB activation, and then PPARγ. A negative feedback loop can be envisioned whereby the PPARγ can bind to p65 and thereby prevent excessive or uncontrolled NF-кB activation. These complex biochemical pathways sug-gest extensive crosstalk amongst several mediators of epidermal differentiation – Notch, NF-кB, and PPAR.

Apoptosis, Differentiation, EpiDerm, Jag-1, Jagged 1, Jagged-1, NF-kappaB, Notch signaling, PPAR gamma