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.

Tetrahydroxystilbene Glucoside Attenuates Neuroinflammation through the Inhibition of Microglia Activation

“…Increasing evidence has demonstrated that microglial activation and the consequent release of proinflammatory and cytotoxic factors such as TNF α , IL-1 β , NO, and prostaglandin E synthase 2 (PGE 2 ) are thought to contribute to the neurodegenerative diseases [13–15]. Analysis of postmortem brains showed that the increased levels of proinflammatory mediators were investigated in patients with neurological disorders [16].…”

Inflammation-Mediated Notch Signaling Skews Fanconi Anemia Hematopoietic Stem Cell Differentiation

“…For in vivo treatment, mice were subcutaneously injected with γ-secretase inhibitor, DAPT in a range of doses (0, 10, 50, 100 and 200 mg/kg, Tocris, R&D, Minneapolis, MN) daily for 2 days followed by bone marrow cell isolation on day 3 (25–27). For TNF-α treatment, a single injection (100 µg/kg) of mouse recombinant TNF-α(Peprotech, Rocky Hill, NJ) was given by intraperitoneally (i.p.)…”

Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells

“…Although activated microglia scavenge dead cells from the CNS and secrete different neurotrophic factors for neuronal survival [2], [3], it is believed that severe activation causes various autoimmune responses leading to neuronal death and brain injury [4], [5]. Activation of microglia and consequent release of proinflammatory and/or cytotoxic factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), prostaglandin E2 (PGE2), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) are believed to contribute to neuronal damage, particularly in neurodegenerative diseases [6], [7], [8], [9]. Subsequently, the damaged neurons release toxic soluble factors, which in turn induce microglial activation termed as reactive microgliosis [10].…”