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Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).

ABSTRACT: 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 protein that functions to negatively regulate G protein-coupled receptors via G?i/G?q-linked 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 Thr(308) 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.

SUBMITTER: Sangphech N

PROVIDER: S-EPMC4101182 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).

Sangphech Naunpun N Osborne Barbara A BA Palaga Tanapat T

Immunobiology 20140406 9

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 ...[more]

PMID: 24775271

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Project description:Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligand Delta-like protein-1, transcriptional repressor hairy and enhancer of split-1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation-induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta-like protein-1, Notch1, and hairy and enhancer of split-1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation-induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80(+) macrophage polarization was skewed in the uterus of inflammation-induced preterm labor toward M1-positive (CD11c(+)) and double-positive [CD11c(+) (M1) and CD206(+) (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage-associated cytokines in decidual macrophages in response to ?-secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. ?-Secretase inhibitor treatment also diminished the LPS-induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta-like protein-1 ligand enhanced the LPS-induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta-like protein-4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation-induced preterm labor. These results suggest that up-regulation of Notch-related inflammation and down-regulation of angiogenesis factors may be associated with inflammation-induced preterm labor but not with hormonally induced preterm labor.

Dataset Information

Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).

Publications

Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).

Similar Datasets

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