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Retinoic acid and GM-CSF coordinately induce retinal dehydrogenase 2 (RALDH2) expression through cooperation between the RAR/RXR complex and Sp1 in dendritic cells.

Ohoka Y, Yokota-Nakatsuma A, Maeda N, Takeuchi H, Iwata M - PLoS ONE (2014)

Bottom Line: The DNA sequences around these sites were highly conserved among different species.GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated.These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki-shi, Kagawa, Japan; Japan Science and Technology Agency, CREST, Chiyoda-ku, Tokyo, Japan.

ABSTRACT
Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.

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Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity.(A) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3. (B) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. (C) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*p<0.05, **p<0.01, ***p<0.001). Data are representative of three independent experiments.
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pone-0096512-g005: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity.(A) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3. (B) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. (C) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*p<0.05, **p<0.01, ***p<0.001). Data are representative of three independent experiments.

Mentions: Sp1 can directly interact with RARα and RXRα [34], [35]. We examined whether Sp1 and RARα/RXRα mutually enhanced their binding to Probe C. As shown in Figure 5A, when Sp1 or the combination of RARα and RXRα was expressed in COS-7 cells, each of these could bind to this probe. However, when Sp1, RARα, and RXRα were expressed together, the binding of each component was significantly enhanced. These results suggest that Sp1 and RARα/RXRα mutually enhance their binding to the Aldh1a2 promoter region. Accordingly, Sp1 and RARα/RXRα cooperatively enhanced Aldh1a2 promoter-reporter activity in the presence of RA (Figure 5B). A truncated form of Sp1 that consisted only of the DNA-binding factor could not enhance the reporter activity in the presence or absence of RARα/RXRα (Figure 5B), and it suppressed Sp1-induced promoter activity in a dose-dependent manner (Figure S3). Mithramycin A and the MAPK pathway inhibitors PD98059 and SB204580 inhibited Aldh1a2 expression that was induced by the combination of RA and GM-CSF (Figure S4A). These results suggest that Sp1 and RARα/RXRα cooperatively contribute to GM-CSF/RA-induced Aldh1a2 expression, depending on MAPK activation.


Retinoic acid and GM-CSF coordinately induce retinal dehydrogenase 2 (RALDH2) expression through cooperation between the RAR/RXR complex and Sp1 in dendritic cells.

Ohoka Y, Yokota-Nakatsuma A, Maeda N, Takeuchi H, Iwata M - PLoS ONE (2014)

Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity.(A) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3. (B) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. (C) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*p<0.05, **p<0.01, ***p<0.001). Data are representative of three independent experiments.
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pone-0096512-g005: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity.(A) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3. (B) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. (C) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*p<0.05, **p<0.01, ***p<0.001). Data are representative of three independent experiments.
Mentions: Sp1 can directly interact with RARα and RXRα [34], [35]. We examined whether Sp1 and RARα/RXRα mutually enhanced their binding to Probe C. As shown in Figure 5A, when Sp1 or the combination of RARα and RXRα was expressed in COS-7 cells, each of these could bind to this probe. However, when Sp1, RARα, and RXRα were expressed together, the binding of each component was significantly enhanced. These results suggest that Sp1 and RARα/RXRα mutually enhance their binding to the Aldh1a2 promoter region. Accordingly, Sp1 and RARα/RXRα cooperatively enhanced Aldh1a2 promoter-reporter activity in the presence of RA (Figure 5B). A truncated form of Sp1 that consisted only of the DNA-binding factor could not enhance the reporter activity in the presence or absence of RARα/RXRα (Figure 5B), and it suppressed Sp1-induced promoter activity in a dose-dependent manner (Figure S3). Mithramycin A and the MAPK pathway inhibitors PD98059 and SB204580 inhibited Aldh1a2 expression that was induced by the combination of RA and GM-CSF (Figure S4A). These results suggest that Sp1 and RARα/RXRα cooperatively contribute to GM-CSF/RA-induced Aldh1a2 expression, depending on MAPK activation.

Bottom Line: The DNA sequences around these sites were highly conserved among different species.GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated.These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki-shi, Kagawa, Japan; Japan Science and Technology Agency, CREST, Chiyoda-ku, Tokyo, Japan.

ABSTRACT
Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.

Show MeSH
Related in: MedlinePlus