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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

Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch.(A) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP+ macrophages were analyzed. Data are representative of two independent experiments. (B,C) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in (C). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. (D) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. (E) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. (F) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. (G) Expression of Itch in macrophages containing control or shNumb vector was detected by immunoblotting following LPS stimulation. (H) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.
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f5: Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch.(A) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP+ macrophages were analyzed. Data are representative of two independent experiments. (B,C) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in (C). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. (D) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. (E) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. (F) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. (G) Expression of Itch in macrophages containing control or shNumb vector was detected by immunoblotting following LPS stimulation. (H) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.

Mentions: Regulatory mechanisms of TNFα, IL-6, and IL-12p40 expression have been extensively studied and shown to be regulated through different MAPK and NF-κB pathways1242. Therefore, we tested the effect of Numb silencing on activation of MAPK and NF-κB pathways in macrophages. We found that the phosphorylation levels of both NF-κB p65 (RelA) and p38 MAPK were compromised in Numb-deficient macrophages after LPS stimulation, while the phosphorylation levels of ERK1/2 and SAP/JNK MAPKs remained similar between control and Numb-deficient macrophages (Fig. 5A). This selective effect silencing Numb has on p38 MAPK and NF-κB p65 activation following TLR4 stimulation may act to decrease TNFα, IL-6 and IL-12p40. Our previous results showed that the mRNA levels of Tnfα did not differ between Numb-deficient and control macrophages, consistent with several studies that demonstrated Tnfα mRNA is regulated by p38 MAPK, followed by post-transcriptional regulation4344. Therefore, to address whether Numb exerts an effect on Tnfα mRNA stability, we performed a Tnfα mRNA decay assay, using actinomycin D to inhibit mRNA synthesis. We found that Tnfα mRNA was degraded at a faster rate in activated macrophages wherein Numb was silenced (Fig. 5B,C). On the other hand, we found that the mRNA stability of Il6 was similar in the presence or absence of Numb (Supplementary Figure 2). Taken together, these results suggested that Numb positively regulates phosphorylation of p65 in the NF-κB pathway, possibly facilitating Il6 and Il12p40 mRNA transcription, as well as phosphorylation of p38 in MAPK, which may further mediate post-transcriptional regulation of Tnfα mRNA.


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)

Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch.(A) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP+ macrophages were analyzed. Data are representative of two independent experiments. (B,C) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in (C). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. (D) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. (E) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. (F) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. (G) Expression of Itch in macrophages containing control or shNumb vector was detected by immunoblotting following LPS stimulation. (H) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.
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Related In: Results  -  Collection

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f5: Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch.(A) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP+ macrophages were analyzed. Data are representative of two independent experiments. (B,C) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in (C). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. (D) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. (E) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. (F) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. (G) Expression of Itch in macrophages containing control or shNumb vector was detected by immunoblotting following LPS stimulation. (H) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.
Mentions: Regulatory mechanisms of TNFα, IL-6, and IL-12p40 expression have been extensively studied and shown to be regulated through different MAPK and NF-κB pathways1242. Therefore, we tested the effect of Numb silencing on activation of MAPK and NF-κB pathways in macrophages. We found that the phosphorylation levels of both NF-κB p65 (RelA) and p38 MAPK were compromised in Numb-deficient macrophages after LPS stimulation, while the phosphorylation levels of ERK1/2 and SAP/JNK MAPKs remained similar between control and Numb-deficient macrophages (Fig. 5A). This selective effect silencing Numb has on p38 MAPK and NF-κB p65 activation following TLR4 stimulation may act to decrease TNFα, IL-6 and IL-12p40. Our previous results showed that the mRNA levels of Tnfα did not differ between Numb-deficient and control macrophages, consistent with several studies that demonstrated Tnfα mRNA is regulated by p38 MAPK, followed by post-transcriptional regulation4344. Therefore, to address whether Numb exerts an effect on Tnfα mRNA stability, we performed a Tnfα mRNA decay assay, using actinomycin D to inhibit mRNA synthesis. We found that Tnfα mRNA was degraded at a faster rate in activated macrophages wherein Numb was silenced (Fig. 5B,C). On the other hand, we found that the mRNA stability of Il6 was similar in the presence or absence of Numb (Supplementary Figure 2). Taken together, these results suggested that Numb positively regulates phosphorylation of p65 in the NF-κB pathway, possibly facilitating Il6 and Il12p40 mRNA transcription, as well as phosphorylation of p38 in MAPK, which may further mediate post-transcriptional regulation of Tnfα mRNA.

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