title: 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.
abstract: Microglial cells constitutively express Notch-1 and nuclear factor-kappaB/p65 (NF-kappaB/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-kappaB/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-1beta (IL-1beta). In BV-2 cells pretreated with N-[N-(3,5-difluorophenacetyl)-1-alany1]-S-phenyglycine t-butyl ester (DAPT), 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 NF-kappaB nuclear translocation decreased as shown by flow cytometry. Notch-1's modulation of NF-kappaB/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5-day-old rats given intraperitoneal injections of LPS, Notch-1, NF-kappaB/p65, TNF-alpha, and IL-1beta 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-alpha and IL-1beta 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-kappaB/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch-1 signaling can trans-activate NF-kappaB/p65 by amplifying NF-kappaB/p65-dependent proinflammatory functions in activated microglia.
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
authors.forename: Q
authors.initials: Q
authors.lastname: Cao
authors.forename: P
authors.initials: P
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Lu
authors.forename: S T
authors.initials: ST
authors.lastname: Dheen
authors.forename: C
authors.initials: C
authors.lastname: Kaur
authors.forename: E-A
authors.initials: EA
authors.lastname: Ling
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D004151:Dipeptides / Q000494:pharmacology*; D005911:Gliosis / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism / Q000502:physiology; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D018836:Inflammation Mediators; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C497763:Notch1 protein, rat; D011480:Protease Inhibitors; D051881:Receptor, Notch1; D051996:Transcription Factor RelA; D053829:Amyloid Precursor Protein Secretases