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The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6.

Seto E, Yoshida-Sugitani R, Kobayashi T, Toyama-Sorimachi N - PLoS ONE (2015)

Bottom Line: In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs.These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs.Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Immunology and Inflammation, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, Gunma, Japan.

ABSTRACT
Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.

No MeSH data available.


Related in: MedlinePlus

EDC4 or Dcp1a knockdown affects P-body assembly.THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.
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pone.0123223.g005: EDC4 or Dcp1a knockdown affects P-body assembly.THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.

Mentions: To determine whether the preferential localization of Dcp1a and EDC4 to P-bodies in M1 macrophages has a functional role during the cell's response to LPS stimulation, we examined the effect of EDC4 or Dcp1a knockdown on LPS-triggered cytokine production. Knockdown efficiency was confirmed by western blotting, as shown in Fig 5A. EDC4 knockdown caused most of the EDC4-positive and Dcp1a-positive cytoplasmic foci to disappear, indicating that the EDC4 loss disrupted P-body assembly (Fig 5B). Interestingly, Dcp1a knockdown resulted in an obvious loss of Dcp1a-positive but not EDC4-positive foci; this suggested that EDC4 facilitates the assembly of a different type of P-body in the absence of Dcp1a, although such EDC4-positive foci might differ functionally from P-bodies containing both EDC4 and Dcp1a.


The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6.

Seto E, Yoshida-Sugitani R, Kobayashi T, Toyama-Sorimachi N - PLoS ONE (2015)

EDC4 or Dcp1a knockdown affects P-body assembly.THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4430274&req=5

pone.0123223.g005: EDC4 or Dcp1a knockdown affects P-body assembly.THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.
Mentions: To determine whether the preferential localization of Dcp1a and EDC4 to P-bodies in M1 macrophages has a functional role during the cell's response to LPS stimulation, we examined the effect of EDC4 or Dcp1a knockdown on LPS-triggered cytokine production. Knockdown efficiency was confirmed by western blotting, as shown in Fig 5A. EDC4 knockdown caused most of the EDC4-positive and Dcp1a-positive cytoplasmic foci to disappear, indicating that the EDC4 loss disrupted P-body assembly (Fig 5B). Interestingly, Dcp1a knockdown resulted in an obvious loss of Dcp1a-positive but not EDC4-positive foci; this suggested that EDC4 facilitates the assembly of a different type of P-body in the absence of Dcp1a, although such EDC4-positive foci might differ functionally from P-bodies containing both EDC4 and Dcp1a.

Bottom Line: In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs.These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs.Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Immunology and Inflammation, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, Gunma, Japan.

ABSTRACT
Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.

No MeSH data available.


Related in: MedlinePlus