Limits...
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.

Show MeSH

Related in: MedlinePlus

YB-1 is essential for maintaining intracellular IL-6 mRNAs levels by secreting mRNA to the extracellular space in macrophages.(A) After LPS stimulation for 24 h, total RNAs were purified separately from medium or cell lysates and the presence of IL-6 or TNF-α mRNAs was examined by RT-PCR. (B) The ratio of IL-6 mRNA between intracellular and extracellular fluid was correlated to YB-1 expression level. *P<0.005 (student's t-test). (C) YB-1 does not act as an exoribonuclease enzyme. Cell lysates from macrophages stably expressing YB-1-GFP were immunoprecipitated with anti-GFP antibody. YB-1-GFP proteins were then purified and incubated with the P32-labeled single-stranded 21mer oligonucleotides or 12mer Poly-A tail RNAs. Exonuclease or exoribonuclease assay was performed at 37°C for 2 h and terminated by adding 2× sample buffer and reaction products were separated on a 15% polyacrylamide 7 M urea gel. YB-1-GFP proteins were probed by anti-GFP-antibody. Data are representative of three (A, B), or two (C) experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232504&req=5

pone-0112754-g004: YB-1 is essential for maintaining intracellular IL-6 mRNAs levels by secreting mRNA to the extracellular space in macrophages.(A) After LPS stimulation for 24 h, total RNAs were purified separately from medium or cell lysates and the presence of IL-6 or TNF-α mRNAs was examined by RT-PCR. (B) The ratio of IL-6 mRNA between intracellular and extracellular fluid was correlated to YB-1 expression level. *P<0.005 (student's t-test). (C) YB-1 does not act as an exoribonuclease enzyme. Cell lysates from macrophages stably expressing YB-1-GFP were immunoprecipitated with anti-GFP antibody. YB-1-GFP proteins were then purified and incubated with the P32-labeled single-stranded 21mer oligonucleotides or 12mer Poly-A tail RNAs. Exonuclease or exoribonuclease assay was performed at 37°C for 2 h and terminated by adding 2× sample buffer and reaction products were separated on a 15% polyacrylamide 7 M urea gel. YB-1-GFP proteins were probed by anti-GFP-antibody. Data are representative of three (A, B), or two (C) experiments.

Mentions: Previous studies have shown that YB-1 associates with GM-CSF mRNA and thereby protects it from degradation, resulting in the development of allergic asthma by accumulating eosinophils in the lung parenchyma and airways [13], [16]. However, our results showed that YB-1 depletion clearly increases IL-6 mRNA levels by a mechanism other than enhancing its stability in macrophages (Figure 2). Therefore, we hypothesized that in macrophages, YB-1 may facilitate IL-6 mRNA export to the extracellular space by YB-1 secretory micro-vesicles, resulting in a reduction of total cytosolic IL-6 mRNA levels. It is known that extracellular RNA (exRNA) species present outside the cells from which were transcribed, but their biological function is not fully understood [17], [18]. To explore this possibility, we determined whether IL-6 mRNA exists extracellularly and whether or not secretory YB-1 functions in exporting IL-6 mRNA from cytosol to the extracellular space. Macrophages expressing either control or YB-1 RNAi were incubated in serum-free medium in the absence or presence of LPS for 24 h. Culture medium was collected from the macrophages and then assayed for IL-6 mRNA by RT-PCR. Surprisingly, we found that IL-6 mRNA was secreted to the extracellular space from macrophages (Figure 4A, lane 7 of left panel); in contrast, we did not observe any secreted TNF-α mRNA (Figure 4A, lane 5 of right panel). Furthermore, intracellular and extracellular IL-6 mRNA levels correlated with YB-1 expression levels in macrophages. YB-1 depletion increased the amount of intracellular IL-6 mRNA and decreased levels of extracellular IL-6 mRNA (Figure 4A, comparing lane 3 with 4 and lane 7 with 8 of left panel). TNF-α mRNA levels were unaffected, as shown previously (Figure 4A, right panel). We quantified the IL-6 mRNA band intensities from RT-PCR and showed that IL-6 mRNA increased in the intracellular space by 51% in YB-1-depleted macrophages and extracellular IL-6 mRNA levels decreased by 58% (Figure 4B).


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)

YB-1 is essential for maintaining intracellular IL-6 mRNAs levels by secreting mRNA to the extracellular space in macrophages.(A) After LPS stimulation for 24 h, total RNAs were purified separately from medium or cell lysates and the presence of IL-6 or TNF-α mRNAs was examined by RT-PCR. (B) The ratio of IL-6 mRNA between intracellular and extracellular fluid was correlated to YB-1 expression level. *P<0.005 (student's t-test). (C) YB-1 does not act as an exoribonuclease enzyme. Cell lysates from macrophages stably expressing YB-1-GFP were immunoprecipitated with anti-GFP antibody. YB-1-GFP proteins were then purified and incubated with the P32-labeled single-stranded 21mer oligonucleotides or 12mer Poly-A tail RNAs. Exonuclease or exoribonuclease assay was performed at 37°C for 2 h and terminated by adding 2× sample buffer and reaction products were separated on a 15% polyacrylamide 7 M urea gel. YB-1-GFP proteins were probed by anti-GFP-antibody. Data are representative of three (A, B), or two (C) experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232504&req=5

pone-0112754-g004: YB-1 is essential for maintaining intracellular IL-6 mRNAs levels by secreting mRNA to the extracellular space in macrophages.(A) After LPS stimulation for 24 h, total RNAs were purified separately from medium or cell lysates and the presence of IL-6 or TNF-α mRNAs was examined by RT-PCR. (B) The ratio of IL-6 mRNA between intracellular and extracellular fluid was correlated to YB-1 expression level. *P<0.005 (student's t-test). (C) YB-1 does not act as an exoribonuclease enzyme. Cell lysates from macrophages stably expressing YB-1-GFP were immunoprecipitated with anti-GFP antibody. YB-1-GFP proteins were then purified and incubated with the P32-labeled single-stranded 21mer oligonucleotides or 12mer Poly-A tail RNAs. Exonuclease or exoribonuclease assay was performed at 37°C for 2 h and terminated by adding 2× sample buffer and reaction products were separated on a 15% polyacrylamide 7 M urea gel. YB-1-GFP proteins were probed by anti-GFP-antibody. Data are representative of three (A, B), or two (C) experiments.
Mentions: Previous studies have shown that YB-1 associates with GM-CSF mRNA and thereby protects it from degradation, resulting in the development of allergic asthma by accumulating eosinophils in the lung parenchyma and airways [13], [16]. However, our results showed that YB-1 depletion clearly increases IL-6 mRNA levels by a mechanism other than enhancing its stability in macrophages (Figure 2). Therefore, we hypothesized that in macrophages, YB-1 may facilitate IL-6 mRNA export to the extracellular space by YB-1 secretory micro-vesicles, resulting in a reduction of total cytosolic IL-6 mRNA levels. It is known that extracellular RNA (exRNA) species present outside the cells from which were transcribed, but their biological function is not fully understood [17], [18]. To explore this possibility, we determined whether IL-6 mRNA exists extracellularly and whether or not secretory YB-1 functions in exporting IL-6 mRNA from cytosol to the extracellular space. Macrophages expressing either control or YB-1 RNAi were incubated in serum-free medium in the absence or presence of LPS for 24 h. Culture medium was collected from the macrophages and then assayed for IL-6 mRNA by RT-PCR. Surprisingly, we found that IL-6 mRNA was secreted to the extracellular space from macrophages (Figure 4A, lane 7 of left panel); in contrast, we did not observe any secreted TNF-α mRNA (Figure 4A, lane 5 of right panel). Furthermore, intracellular and extracellular IL-6 mRNA levels correlated with YB-1 expression levels in macrophages. YB-1 depletion increased the amount of intracellular IL-6 mRNA and decreased levels of extracellular IL-6 mRNA (Figure 4A, comparing lane 3 with 4 and lane 7 with 8 of left panel). TNF-α mRNA levels were unaffected, as shown previously (Figure 4A, right panel). We quantified the IL-6 mRNA band intensities from RT-PCR and showed that IL-6 mRNA increased in the intracellular space by 51% in YB-1-depleted macrophages and extracellular IL-6 mRNA levels decreased by 58% (Figure 4B).

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