Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition
receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream
signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch
signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages.
Using a phospho-proteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing
and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target
protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase
(GTPase)-activating proteinthatfunctions tonegatively regulate G protein-coupled receptors via Gi/Gqlinked
signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form
of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the
mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation
of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and
binding partner of the Notch receptor, RBP-J/CSL, in macrophages resulted in delayed and decreased Akt
phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle
and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment
or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases,
while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased
cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt
phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase,
suggesting a possible cell cycle arrest. Taken together, these results indicate that Notch signaling positively
regulates phosphorylation of Akt, possibly via phosphorylation of RGS19, and inhibition of both
molecules affects the cell survival and cell cycle of macrophages upon LPS stimulation.
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