Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).
Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19).
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DOI：
10.1016/j.imbio.2014.03.020
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发表时间：
2014-09
期刊：
影响因子：
2.8
通讯作者：
Palaga, Tanapat
中科院分区：
文献类型：
--
作者：
Sangphech, Naunpun;Osborne, Barbara A.;Palaga, Tanapat
巨噬细胞通过模式识别受体感知微生物，在先天免疫防御中发挥关键作用。脂多糖（LPS）通过TLR刺激巨噬细胞，导致下游信号级联反应的激活。在本研究中，我们研究了一个保守的信号通路Notch在巨噬细胞LPS/TLR4通路下游信号级联中的调节作用。利用磷酸化蛋白质组学方法和γ -分泌酶抑制剂（GSI）抑制Notch信号的加工和激活，我们发现G蛋白信号19的调节因子（RGS19）是一个磷酸化受到GSI处理影响的靶蛋白。RGS19是一种鸟苷三磷酸酶（GTPase）激活蛋白，通过Gαi/Gαq-linked信号通路负调控G蛋白偶联受体。LPS刺激RAW264.7细胞可增加RGS19磷酸化形式的水平，而GSI存在时LPS刺激可降低其水平。GSI处理未改变rgs19 mRNA水平。在LPS刺激下，GSI处理或沉默巨噬细胞中的rgs19可破坏Akt Thr308的磷酸化。此外，巨噬细胞中dna结合蛋白和Notch受体的结合伙伴RBP-Jκ/CSL的靶向缺失导致Akt磷酸化延迟和降低。由于PI3K/Akt通路调节各种细胞类型的细胞存活，因此在GSI处理、磷脂酰肌醇3激酶（PI3K）抑制剂处理或rgs19沉默的情况下，研究了细胞周期和细胞死亡。GSI处理导致G1期和S期细胞数量减少，而死亡细胞数量增加。同样，rgs19的沉默导致G1期细胞数量减少，而subG1期细胞数量增加。在lps刺激的巨噬细胞中，PI3K抑制剂抑制Akt磷酸化可增加G1期的细胞数量，提示可能的细胞周期阻滞。综上所述，这些结果表明Notch信号可能通过磷酸化RGS19而正向调节Akt的磷酸化，抑制这两个分子会影响LPS刺激下巨噬细胞的细胞存活和细胞周期。
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 phosphoproteomic 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 Gαi/Gαq-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 Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/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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