Nuclear Factor-κB/p65 Responds to Changes in the Notch Signaling Pathway in Murine BV-2 Cells and in Amoeboid Microglia in Postnatal Rats Treated With the γ-Secretase Complex Blocker DAPT

Microglial cells constitutively express Notch-1 and nuclear factor-kappa B/p65 (NF-kappa B/p65), and both pathways modulate production of inflammatory mediators. This study sought to determine whether a functional relationship exists between them and, if so, to investigate whether they synergistically regulate common microglial cell functions. By immunofluorescence labeling, real-time polymerase chain reaction (RT-PCR), flow cytometry, and Western blot, BV-2 cells exhibited Notch-1 and NF-kappa B/p65 expression, which was significantly up-regulated in cells challenged with lipopolysaccharide (LPS). This was coupled with an increase in expression of Hes-1, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta). In BV-2 cells pretreated with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenyglycine t-butyl ester (DAFT), a gamma-secretase inhibitor, followed by LPS stimulation, Notch-1 expression level was enhanced but that of all other markers was suppressed. Additionally, Hes-1 expression and NE-kappa B nuclear translocation decreased as shown by flow cytometry. Notch-1's modulation of NF-kappa B/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5-day-old rats given intraperitoneal injections of LPS, Notch-1, NF-kappa B/p65, TNF-alpha, and IL-1 beta immunofluorescence in AMC was markedly enhanced. However, in rats given an intraperitoneal injection of DAFT prior to LPS, Notch-1 labeling was augmented, but that of TNF-alpha and IL-1 beta was reduced. The results suggest that blocking of Notch-1 activation with DAFT would reduce the level of its downstream end product Hes-1 along with suppression of NF-kappa B/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch-1 signaling can trans-activate NF-kappa B/p65 by amplifying NF-kappa B/p65-dependent proinflammatory functions in activated microglia. (C) 2010 Wiley-Liss, Inc.