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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

Numb expression in macrophages after LPS stimulation and effects of Numb silencing on Notch signaling.(A) Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL) and Numb mRNA was measured. (B) Numb protein expression was shown and its intensity was measured as fold-increase compared to unstimulated cells. (C–J) Immunofluorescent staining of macrophages retrovirally infected with control vector (C–F) or shNumb vector (G–J), and stained for Numb (red). White arrows indicate macrophages containing shRNA vector (green). (K–L) Expression level of Numb (K) and Numb-like (L) mRNA from GFP+ macrophages as determined by qPCR. (M) Cleaved Notch1 (Val1744) was detected in LPS-stimulated macrophages by immunoblot. (N) Hes1 mRNA was measured in LPS-stimulated macrophages by qPCR. N.D. = not detectable. Data are the mean ± SEM from three independent experiments, and a representative blot from at least two independent replicates.
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f1: Numb expression in macrophages after LPS stimulation and effects of Numb silencing on Notch signaling.(A) Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL) and Numb mRNA was measured. (B) Numb protein expression was shown and its intensity was measured as fold-increase compared to unstimulated cells. (C–J) Immunofluorescent staining of macrophages retrovirally infected with control vector (C–F) or shNumb vector (G–J), and stained for Numb (red). White arrows indicate macrophages containing shRNA vector (green). (K–L) Expression level of Numb (K) and Numb-like (L) mRNA from GFP+ macrophages as determined by qPCR. (M) Cleaved Notch1 (Val1744) was detected in LPS-stimulated macrophages by immunoblot. (N) Hes1 mRNA was measured in LPS-stimulated macrophages by qPCR. N.D. = not detectable. Data are the mean ± SEM from three independent experiments, and a representative blot from at least two independent replicates.

Mentions: We began our investigation by exploring the expression of Numb in LPS-stimulated macrophages. We found that the expression level of Numb mRNA significantly decreased to approximately one-half and remained at this level after LPS stimulation for 3 hrs and up to 24 hrs (Fig. 1A). For protein expression, we observed a slight increase of Numb at 1 hr post stimulation and a gradual decrease over the next 24 hrs (Fig. 1B and Supplementary Figure 1A). To further investigate the effect of Numb in macrophages, we constructed a retroviral shRNA vector, pMKO.1-shNumb-GFP, introduced it into BMMs and confirmed its target specificity. In BMMs retrovirally transduced with pMKO.1-shNumb-GFP vectors, the expression level of Numb decreased significantly compared to that of the control-transduced macrophages (Fig. 1C–J). To further assess the target specificity of the construct, macrophages containing pMKO.1-shNumb-GFP, or control vector, were sorted to obtain homogenous GFP+ populations. The mRNA level of Numb and its homolog, Numb-like, that has been reported to have redundant functions with Numb in other cell types were measured303839. The expression level of Numb mRNA was reduced to one-half, whereas the level of Numb-like mRNA was not affected (Fig. 1K,L). These results confirmed that the shRNA vector specifically targeted only Numb. Numb has been reported to be a negative regulator of Notch signaling. When we assessed cleaved Notch1 expression (Val1744), and the level of Hes1 mRNA, we observed that both were increased in LPS-activated Numb-silenced macrophages, compared to control cells (Fig. 1M,N). These results confirmed the role of Numb as a negative regulator of Notch signaling, as previously described.


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)

Numb expression in macrophages after LPS stimulation and effects of Numb silencing on Notch signaling.(A) Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL) and Numb mRNA was measured. (B) Numb protein expression was shown and its intensity was measured as fold-increase compared to unstimulated cells. (C–J) Immunofluorescent staining of macrophages retrovirally infected with control vector (C–F) or shNumb vector (G–J), and stained for Numb (red). White arrows indicate macrophages containing shRNA vector (green). (K–L) Expression level of Numb (K) and Numb-like (L) mRNA from GFP+ macrophages as determined by qPCR. (M) Cleaved Notch1 (Val1744) was detected in LPS-stimulated macrophages by immunoblot. (N) Hes1 mRNA was measured in LPS-stimulated macrophages by qPCR. N.D. = not detectable. Data are the mean ± SEM from three independent experiments, and a representative blot from at least two independent replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4542673&req=5

f1: Numb expression in macrophages after LPS stimulation and effects of Numb silencing on Notch signaling.(A) Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL) and Numb mRNA was measured. (B) Numb protein expression was shown and its intensity was measured as fold-increase compared to unstimulated cells. (C–J) Immunofluorescent staining of macrophages retrovirally infected with control vector (C–F) or shNumb vector (G–J), and stained for Numb (red). White arrows indicate macrophages containing shRNA vector (green). (K–L) Expression level of Numb (K) and Numb-like (L) mRNA from GFP+ macrophages as determined by qPCR. (M) Cleaved Notch1 (Val1744) was detected in LPS-stimulated macrophages by immunoblot. (N) Hes1 mRNA was measured in LPS-stimulated macrophages by qPCR. N.D. = not detectable. Data are the mean ± SEM from three independent experiments, and a representative blot from at least two independent replicates.
Mentions: We began our investigation by exploring the expression of Numb in LPS-stimulated macrophages. We found that the expression level of Numb mRNA significantly decreased to approximately one-half and remained at this level after LPS stimulation for 3 hrs and up to 24 hrs (Fig. 1A). For protein expression, we observed a slight increase of Numb at 1 hr post stimulation and a gradual decrease over the next 24 hrs (Fig. 1B and Supplementary Figure 1A). To further investigate the effect of Numb in macrophages, we constructed a retroviral shRNA vector, pMKO.1-shNumb-GFP, introduced it into BMMs and confirmed its target specificity. In BMMs retrovirally transduced with pMKO.1-shNumb-GFP vectors, the expression level of Numb decreased significantly compared to that of the control-transduced macrophages (Fig. 1C–J). To further assess the target specificity of the construct, macrophages containing pMKO.1-shNumb-GFP, or control vector, were sorted to obtain homogenous GFP+ populations. The mRNA level of Numb and its homolog, Numb-like, that has been reported to have redundant functions with Numb in other cell types were measured303839. The expression level of Numb mRNA was reduced to one-half, whereas the level of Numb-like mRNA was not affected (Fig. 1K,L). These results confirmed that the shRNA vector specifically targeted only Numb. Numb has been reported to be a negative regulator of Notch signaling. When we assessed cleaved Notch1 expression (Val1744), and the level of Hes1 mRNA, we observed that both were increased in LPS-activated Numb-silenced macrophages, compared to control cells (Fig. 1M,N). These results confirmed the role of Numb as a negative regulator of Notch signaling, as previously described.

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