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Differential control of interleukin-6 mRNA levels by cellular distribution of YB-1.

Kang S, Lee TA, Ra EA, Lee E, Choi Hj, Lee S, Park B - PLoS ONE (2014)

Bottom Line: YB-1 secretion occurs in a cell type-specific manner.Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells.Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems biology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea.

ABSTRACT
Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

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Inflammatory stimuli induce YB-1 secretion in a cell type-specific manner.(A) Immunofluorescence microscopy assay (IFA) of YB-1 in RAW macrophages following exposure to LPS (80 ng/ml) or CpG-DNA (1 µM). LPS or CPG-DNA-stimulated macrophages exhibited YB-1-enriched exporting vesicles. 6.5× digital enlargement of main image, Scale bars, 10 µm. (B) Bone marrow-derived Dendritic cells (BMDC) were stimulated with LPS (80 ng/ml) or CpG-DNA (1 µM). Anti-YB-1 antibody with Alexa 488-conjugated secondary antibody and DAPI were used. YB-1-enriched vesicles were not detected in BMDC. 6.5× digital enlargement of main image, Scale bars, 10 µm. (C) Western blot analyses of YB-1 in macrophages and dendritic cells with anti-YB-1antibody. TCA-precipitated extracellular supernatant from macrophages contained secreted YB-1, which was confirmed by depletion of YB-1. Lineage markers were used to characterize macrophages or dendritic cells (Fig. S1). Asterisk indicates the molecular weight species of extracellular YB-1. Data are representative of three (A–B), or two (C) experiments.
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pone-0112754-g001: Inflammatory stimuli induce YB-1 secretion in a cell type-specific manner.(A) Immunofluorescence microscopy assay (IFA) of YB-1 in RAW macrophages following exposure to LPS (80 ng/ml) or CpG-DNA (1 µM). LPS or CPG-DNA-stimulated macrophages exhibited YB-1-enriched exporting vesicles. 6.5× digital enlargement of main image, Scale bars, 10 µm. (B) Bone marrow-derived Dendritic cells (BMDC) were stimulated with LPS (80 ng/ml) or CpG-DNA (1 µM). Anti-YB-1 antibody with Alexa 488-conjugated secondary antibody and DAPI were used. YB-1-enriched vesicles were not detected in BMDC. 6.5× digital enlargement of main image, Scale bars, 10 µm. (C) Western blot analyses of YB-1 in macrophages and dendritic cells with anti-YB-1antibody. TCA-precipitated extracellular supernatant from macrophages contained secreted YB-1, which was confirmed by depletion of YB-1. Lineage markers were used to characterize macrophages or dendritic cells (Fig. S1). Asterisk indicates the molecular weight species of extracellular YB-1. Data are representative of three (A–B), or two (C) experiments.

Mentions: Studies have shown that YB-1 exhibits various subcellular localization patterns depending on the stimulus [7], [11]. In particular, human monocytes stimulated with LPS secrete YB-1 from micro-vesicles [7]. We began examining the role(s) of YB-1 in the immune response by reproducing documented observations of YB-1 secretion in response to LPS, which promotes robust cytokine production to induce innate and adaptive immunity [7]. First, we assessed YB-1 subcellular localization in LPS-stimulated macrophages and dendritic cells. As shown previously, we observed intracellular secretory vesicle formation with enriched levels of endogenous YB-1 in LPS-treated macrophages, but not in unstimulated cells (Figure 1A). To explore TLR-agonist specificity in YB-1 secretion, we measured the pattern of intracellular vesicles in macrophages exposed to CpG-DNA, which activates the TLR9 signaling cascade. In CpG-DNA-stimulated macrophages, YB-1 secretory vesicles were clearly detected to an extent similar to LPS (Figure 1A). Interestingly, unlike macrophages, YB-1 was distributed throughout the cytoplasm, with one or two speckles close to the nuclear membrane in LPS- or CpG-DNA-stimulated bone marrow-derived dendritic cells (BMDCs) (Figure 1B). Therefore, YB-1 exhibits differential subcellular distribution that varies according to cell type, resulting in the regulation of distinct biological functions in macrophages and dendritic cells.


Differential control of interleukin-6 mRNA levels by cellular distribution of YB-1.

Kang S, Lee TA, Ra EA, Lee E, Choi Hj, Lee S, Park B - PLoS ONE (2014)

Inflammatory stimuli induce YB-1 secretion in a cell type-specific manner.(A) Immunofluorescence microscopy assay (IFA) of YB-1 in RAW macrophages following exposure to LPS (80 ng/ml) or CpG-DNA (1 µM). LPS or CPG-DNA-stimulated macrophages exhibited YB-1-enriched exporting vesicles. 6.5× digital enlargement of main image, Scale bars, 10 µm. (B) Bone marrow-derived Dendritic cells (BMDC) were stimulated with LPS (80 ng/ml) or CpG-DNA (1 µM). Anti-YB-1 antibody with Alexa 488-conjugated secondary antibody and DAPI were used. YB-1-enriched vesicles were not detected in BMDC. 6.5× digital enlargement of main image, Scale bars, 10 µm. (C) Western blot analyses of YB-1 in macrophages and dendritic cells with anti-YB-1antibody. TCA-precipitated extracellular supernatant from macrophages contained secreted YB-1, which was confirmed by depletion of YB-1. Lineage markers were used to characterize macrophages or dendritic cells (Fig. S1). Asterisk indicates the molecular weight species of extracellular YB-1. Data are representative of three (A–B), or two (C) experiments.
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pone-0112754-g001: Inflammatory stimuli induce YB-1 secretion in a cell type-specific manner.(A) Immunofluorescence microscopy assay (IFA) of YB-1 in RAW macrophages following exposure to LPS (80 ng/ml) or CpG-DNA (1 µM). LPS or CPG-DNA-stimulated macrophages exhibited YB-1-enriched exporting vesicles. 6.5× digital enlargement of main image, Scale bars, 10 µm. (B) Bone marrow-derived Dendritic cells (BMDC) were stimulated with LPS (80 ng/ml) or CpG-DNA (1 µM). Anti-YB-1 antibody with Alexa 488-conjugated secondary antibody and DAPI were used. YB-1-enriched vesicles were not detected in BMDC. 6.5× digital enlargement of main image, Scale bars, 10 µm. (C) Western blot analyses of YB-1 in macrophages and dendritic cells with anti-YB-1antibody. TCA-precipitated extracellular supernatant from macrophages contained secreted YB-1, which was confirmed by depletion of YB-1. Lineage markers were used to characterize macrophages or dendritic cells (Fig. S1). Asterisk indicates the molecular weight species of extracellular YB-1. Data are representative of three (A–B), or two (C) experiments.
Mentions: Studies have shown that YB-1 exhibits various subcellular localization patterns depending on the stimulus [7], [11]. In particular, human monocytes stimulated with LPS secrete YB-1 from micro-vesicles [7]. We began examining the role(s) of YB-1 in the immune response by reproducing documented observations of YB-1 secretion in response to LPS, which promotes robust cytokine production to induce innate and adaptive immunity [7]. First, we assessed YB-1 subcellular localization in LPS-stimulated macrophages and dendritic cells. As shown previously, we observed intracellular secretory vesicle formation with enriched levels of endogenous YB-1 in LPS-treated macrophages, but not in unstimulated cells (Figure 1A). To explore TLR-agonist specificity in YB-1 secretion, we measured the pattern of intracellular vesicles in macrophages exposed to CpG-DNA, which activates the TLR9 signaling cascade. In CpG-DNA-stimulated macrophages, YB-1 secretory vesicles were clearly detected to an extent similar to LPS (Figure 1A). Interestingly, unlike macrophages, YB-1 was distributed throughout the cytoplasm, with one or two speckles close to the nuclear membrane in LPS- or CpG-DNA-stimulated bone marrow-derived dendritic cells (BMDCs) (Figure 1B). Therefore, YB-1 exhibits differential subcellular distribution that varies according to cell type, resulting in the regulation of distinct biological functions in macrophages and dendritic cells.

Bottom Line: YB-1 secretion occurs in a cell type-specific manner.Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells.Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems biology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea.

ABSTRACT
Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

Show MeSH
Related in: MedlinePlus