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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
Author(s) -
Cao Q.,
Li P.,
Lu J.,
Dheen S.T.,
Kaur C.,
Ling E.A.
Publication year - 2010
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.22429
Subject(s) - notch signaling pathway , notch 1 , microglia , tumor necrosis factor alpha , lipopolysaccharide , flow cytometry , nf κb , microbiology and biotechnology , western blot , chemistry , immunofluorescence , signal transduction , biology , inflammation , immunology , biochemistry , gene , antibody
Microglial cells constitutively express Notch‐1 and nuclear factor‐κB/p65 (NF‐κB/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‐κB/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‐α (TNF‐α), and interleukin‐1β (IL‐1β). In BV‐2 cells pretreated with N‐[N‐(3,5‐difluorophenacetyl)‐1‐alany1]‐S‐phenyglycine t ‐butyl ester (DAPT), a γ‐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 NF‐κB nuclear translocation decreased as shown by flow cytometry. Notch‐1's modulation of NF‐κB/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5‐day‐old rats given intraperitoneal injections of LPS, Notch‐1, NF‐κB/p65, TNF‐α, and IL‐1β immunofluorescence in AMC was markedly enhanced. However, in rats given an intraperitoneal injection of DAPT prior to LPS, Notch‐1 labeling was augmented, but that of TNF‐α and IL‐1β was reduced. The results suggest that blocking of Notch‐1 activation with DAPT would reduce the level of its downstream end product Hes‐1 along with suppression of NF‐κB/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch‐1 signaling can trans‐activate NF‐κB/p65 by amplifying NF‐κB/p65‐dependent proinflammatory functions in activated microglia. © 2010 Wiley‐Liss, Inc.