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A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Kueanjinda P, Roytrakul S, Palaga T - Sci Rep (2015)

Bottom Line: Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK.In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb.A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

View Article: PubMed Central - PubMed

Affiliation: 1] Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand [2] Center of Excellence in Immunology and Immune-mediated Diseases, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

No MeSH data available.


Related in: MedlinePlus

Proteomic data analysis of proteins from Numb-silenced macrophages.(A) Expression level of Numb in GFP+ macrophages following LPS stimulation was detected by immunoblotting. (B) Heat map representing expression levels of proteins as identified by GO enrichment in biological process. (C–E) Expression level of Akt1 (C), Map3k10 (D), and Ticam1 (E) in control (open bars) or shNumb (closed bars) macrophages were detected by qPCR. (F) A network of protein-protein interactions generated from proteins in (B) using the software STRING v9.1. (G) Phosphorylation of Akt (Thr308) and total Akt from macrophages containing control or shNumb vector was detected by immunoblotting. (H) Schematic diagram showing how Numb interacts with different proteins and regulates pro-inflammatory cytokines (see text for details). Solid lines depict the confirmed links while the dotted lines depict the potential links.
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f6: Proteomic data analysis of proteins from Numb-silenced macrophages.(A) Expression level of Numb in GFP+ macrophages following LPS stimulation was detected by immunoblotting. (B) Heat map representing expression levels of proteins as identified by GO enrichment in biological process. (C–E) Expression level of Akt1 (C), Map3k10 (D), and Ticam1 (E) in control (open bars) or shNumb (closed bars) macrophages were detected by qPCR. (F) A network of protein-protein interactions generated from proteins in (B) using the software STRING v9.1. (G) Phosphorylation of Akt (Thr308) and total Akt from macrophages containing control or shNumb vector was detected by immunoblotting. (H) Schematic diagram showing how Numb interacts with different proteins and regulates pro-inflammatory cytokines (see text for details). Solid lines depict the confirmed links while the dotted lines depict the potential links.

Mentions: To investigate further the role of Numb in macrophages, we employed a quantitative proteomics approach using Numb-silenced macrophages, or control cells, stimulated with LPS. The expression of Numb was separately confirmed (Fig. 6A) prior to gel loading for SDS-PAGE followed by trypsin digestion. Based on the LC/MS/MS data, we identified 758 proteins that were differentially expressed in the unstimulated or LPS-activated Numb-intact and Numb-deficient macrophages (Supplementary Figure 3). Among these proteins, 551 had previously described functions and so were subjected to analysis for protein-protein interaction using the software STRING v9.147. Gene ontology (GO) enrichments in biological processes of these proteins using the software identified categories of proteins involved in regulating MAPKs, NF-κB signaling pathways and TNF production as represented by a heat map (Fig. 6B). To validate our proteomics results, we performed qPCR to measure Akt1, Map3k10, and Ticam1 in Numb-silenced macrophages and found that their expression pattern correlated well with our proteomics data (Fig. 6C–E).


A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Kueanjinda P, Roytrakul S, Palaga T - Sci Rep (2015)

Proteomic data analysis of proteins from Numb-silenced macrophages.(A) Expression level of Numb in GFP+ macrophages following LPS stimulation was detected by immunoblotting. (B) Heat map representing expression levels of proteins as identified by GO enrichment in biological process. (C–E) Expression level of Akt1 (C), Map3k10 (D), and Ticam1 (E) in control (open bars) or shNumb (closed bars) macrophages were detected by qPCR. (F) A network of protein-protein interactions generated from proteins in (B) using the software STRING v9.1. (G) Phosphorylation of Akt (Thr308) and total Akt from macrophages containing control or shNumb vector was detected by immunoblotting. (H) Schematic diagram showing how Numb interacts with different proteins and regulates pro-inflammatory cytokines (see text for details). Solid lines depict the confirmed links while the dotted lines depict the potential links.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4542673&req=5

f6: Proteomic data analysis of proteins from Numb-silenced macrophages.(A) Expression level of Numb in GFP+ macrophages following LPS stimulation was detected by immunoblotting. (B) Heat map representing expression levels of proteins as identified by GO enrichment in biological process. (C–E) Expression level of Akt1 (C), Map3k10 (D), and Ticam1 (E) in control (open bars) or shNumb (closed bars) macrophages were detected by qPCR. (F) A network of protein-protein interactions generated from proteins in (B) using the software STRING v9.1. (G) Phosphorylation of Akt (Thr308) and total Akt from macrophages containing control or shNumb vector was detected by immunoblotting. (H) Schematic diagram showing how Numb interacts with different proteins and regulates pro-inflammatory cytokines (see text for details). Solid lines depict the confirmed links while the dotted lines depict the potential links.
Mentions: To investigate further the role of Numb in macrophages, we employed a quantitative proteomics approach using Numb-silenced macrophages, or control cells, stimulated with LPS. The expression of Numb was separately confirmed (Fig. 6A) prior to gel loading for SDS-PAGE followed by trypsin digestion. Based on the LC/MS/MS data, we identified 758 proteins that were differentially expressed in the unstimulated or LPS-activated Numb-intact and Numb-deficient macrophages (Supplementary Figure 3). Among these proteins, 551 had previously described functions and so were subjected to analysis for protein-protein interaction using the software STRING v9.147. Gene ontology (GO) enrichments in biological processes of these proteins using the software identified categories of proteins involved in regulating MAPKs, NF-κB signaling pathways and TNF production as represented by a heat map (Fig. 6B). To validate our proteomics results, we performed qPCR to measure Akt1, Map3k10, and Ticam1 in Numb-silenced macrophages and found that their expression pattern correlated well with our proteomics data (Fig. 6C–E).

Bottom Line: Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK.In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb.A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

View Article: PubMed Central - PubMed

Affiliation: 1] Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand [2] Center of Excellence in Immunology and Immune-mediated Diseases, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

No MeSH data available.


Related in: MedlinePlus