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Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

ABSTRACT: Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-J? binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

SUBMITTER: Palaga T

PROVIDER: S-EPMC4003195 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

Palaga Tanapat T Ratanabunyong Siriluk S Pattarakankul Thitiporn T Sangphech Naunpun N Wongchana Wipawee W Hadae Yukihiro Y Kueanjinda Patipark P

Cellular & molecular immunology 20130722 5

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived mac ...[more]

PMID: 23872918

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Project description:Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the 'regulatory sensitivity' of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn't significant changes of this 'regulatory sensitivity' of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the 'regulatory sensitivity' of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.

Project description: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 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 Thr(308) 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.

Project description:BackgroundOur previous studies have reported the down-regulation of EGFL8 correlates to the development and prognosis of colorectal and gastric cancer. The present study is carried out to explore the expression pattern and role of EGFL8 in hepatocellular carcinoma (HCC).Methods and materialsEGFL8 expression in 102 cases of HCC tissues matched with adjacent non-tumorous liver tissues, a normal liver cell line and three liver cancer cell lines with different metastatic capacity was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Moreover, the clinicopathological features and prognosis of HCC patients were correlated with expression of EGFL8. Subsequently, the gain-and loss-of-function experiments were carried out to investigate the biological function of EGFL8 in HCC. We also used N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)- phenylglycine t-butyl ester (DAPT), an inhibitor for Notch signaling pathway, in these experiments to verify the involvement of Notch signaling pathway in the effects of EGFL8. Additionally, a mouse model was established to investigate the effect of EGFL8 on metastasis of HCC cells. The expression of Notch signaling pathway in HCC cells and xenograft mouse tumors were detected by Western blot and immunohistochemistory.ResultsThe expression of EGFL8 was significantly decreased in HCC tissues and cell lines and EGFL8 down-regulation correlated to multiple nodules, vein invasion, high TNM stage and poor prognosis of HCC. Interestingly, the expression levels of EGFL8 in three liver cancer cell lines were negatively associated with their metastatic capacity. In vitro and in vivo experiments indicated that EGFL8 obviously suppressed metastasis and invasion of HCC cells but slightly promoted apoptosis. Meanwhile, the expression of Notch signaling pathway was obviously suppressed in EGFL8 overexpressed HCCLM3 cells and xenograft mouse tumors generated from these cells but markedly elevated in EGFL8 depleted Hep3B cells. Furthermore, the up-regulated expression of Notch signaling pathway and effects induced by EGFL8 knockdown in Hep3B cells could be counteracted by DAPT treatment.ConclusionThe down-regulation of EGFL8 was correlated to progression and poor prognosis of HCC and regulates HCC cell migration, invasion and apoptosis through activating the Notch signaling pathway, suggesting EGFL8 as a novel therapeutic target and a potential prognostic marker for HCC.

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Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

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Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

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