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

Chaturvedi, V., Qin, J., Miele, L. and Nickoloff, B.J. Pathology, Loyola University Chicago, Maywood, IL, USA.
Abstract

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.

Keywords

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

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