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Orphan nuclear receptor Nur77 Inhibits Oxidized LDL-induced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells.

Hu LH, Yu Y, Jin SX, Nie P, Cai ZH, Cui ML, Sun SQ, Xiao H, Shao Q, Shen LH, He B - BMC Immunol. (2014)

Bottom Line: GFP-Nur77 overexpression significantly suppressed the effect of oxLDL treatment on DC morphologic changes, expression of DC maturation markers, endocytic activity, allogeneic activation of T cell proliferation, and the activity and secretion of pro-inflammatory cytokines.GFP-Nur77-ΔDBD consistently had the opposite effect to GFP-Nur77, increasing DC-type differentiation in all assays.The effects of Nur77 on the macrophage phenotype may involve changes in its subcellular distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200127, People's Republic of China. iam565@126.com.

ABSTRACT

Background: Nur77 is an orphan nuclear receptor expressed in human atheroma. In vascular cells in vitro, Nur77 expression is induced by pro-inflammatory factors, such as oxidized LDL (oxLDL).

Methods: We analyze the role of Nur77 in the oxLDL-induced differentiation of macrophages into dendritic cells (DC). The murine RAW264.7 macrophage cell line was stably transfected with expression plasmids encoding either GFP or GFP fusions with either full-length Nur77 (GFP-Nur77), Nur77 lacking the DNA binding domain (GFP-Nur77-ΔDBD) or Nur77 lacking the transactivation domain (GFP-Nur77-ΔTAD).

Results: GFP-Nur77 overexpression significantly suppressed the effect of oxLDL treatment on DC morphologic changes, expression of DC maturation markers, endocytic activity, allogeneic activation of T cell proliferation, and the activity and secretion of pro-inflammatory cytokines. Analysis of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD indicated that the Nur77 DNA binding and transactivation domains were both required for this effect. GFP-Nur77-ΔDBD consistently had the opposite effect to GFP-Nur77, increasing DC-type differentiation in all assays. Interestingly, GFP-Nur77-ΔDBD protein was cytosolic, whereas GFP-Nur77 and GFP-Nur77-ΔTAD were both nuclear.

Conclusions: These data show that GFP-Nur77 inhibited differentiation of oxLDL-treated macrophages into DC. The effects of Nur77 on the macrophage phenotype may involve changes in its subcellular distribution.

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Nur77 overexpression in RAW264.7 cells reduces oxLDL-induced inflammatory cytokine synthesis. Sandwich ELISA analysis of TNF-α (A) and IL-12 (C) for the supernatants of oxLDL-treated RAW264.7 cells stably expressing GFP, GFP-Nur77, GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD. Data represent mean ± SD of three independent experiments for 1 × 106 cells, *p <0.05 compared with GFP-expressing control cell. (B) and (D) showed the level of TNF-α and IL-12 in GFP-expressing control cells with or without stimulated by oxLDL or LPS, respectively. The bars represent mean ± SD from three experiments. *p <0.05 compared with control cells.
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Fig5: Nur77 overexpression in RAW264.7 cells reduces oxLDL-induced inflammatory cytokine synthesis. Sandwich ELISA analysis of TNF-α (A) and IL-12 (C) for the supernatants of oxLDL-treated RAW264.7 cells stably expressing GFP, GFP-Nur77, GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD. Data represent mean ± SD of three independent experiments for 1 × 106 cells, *p <0.05 compared with GFP-expressing control cell. (B) and (D) showed the level of TNF-α and IL-12 in GFP-expressing control cells with or without stimulated by oxLDL or LPS, respectively. The bars represent mean ± SD from three experiments. *p <0.05 compared with control cells.

Mentions: Finally, we assayed the effect of Nur77 on cytokine secretion by oxLDL-treated RAW264.7 cells. Here we show that, IL-12 and TNF-α from oxLDL-treated GFP-Nur77 cells were decreased to 46.8% and 41.5%, respectively, compared with that from GFP-expressing control cells. In contrast, expression of the Nur77 mutant proteins did not decrease oxLDL-induced IL-12 and TNF-α secretion. GFP-Nur77-ΔDBD-expressing cells demonstrated a 24.9% and 12.7% increase in IL-12 and TNF-α secretion, respectively (Figure 5A,C). These data show that Nur77 had an inhibitory effect on cytokine secretion, which required the Nur77 DNA binding domain and transactivation domains. To test the ability of producing several cytokines by these cells, we also analyzed TNF-α and IL-12 including a stimulated control with LPS in GFP-expressing control cells. Results showed that both of the two cytokines can be stimulated by LPS and the levels were 3-fold higher than by the stimulation with oxLDL (TNF-α: 490.12 ± 14.14 vs. 125.60 ± 7.07 ng/ml; IL-12: 15.47 ± 0.28 vs. 4.15 ± 0.21 pg/ml ) (Figure 5B,D).Figure 5


Orphan nuclear receptor Nur77 Inhibits Oxidized LDL-induced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells.

Hu LH, Yu Y, Jin SX, Nie P, Cai ZH, Cui ML, Sun SQ, Xiao H, Shao Q, Shen LH, He B - BMC Immunol. (2014)

Nur77 overexpression in RAW264.7 cells reduces oxLDL-induced inflammatory cytokine synthesis. Sandwich ELISA analysis of TNF-α (A) and IL-12 (C) for the supernatants of oxLDL-treated RAW264.7 cells stably expressing GFP, GFP-Nur77, GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD. Data represent mean ± SD of three independent experiments for 1 × 106 cells, *p <0.05 compared with GFP-expressing control cell. (B) and (D) showed the level of TNF-α and IL-12 in GFP-expressing control cells with or without stimulated by oxLDL or LPS, respectively. The bars represent mean ± SD from three experiments. *p <0.05 compared with control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274730&req=5

Fig5: Nur77 overexpression in RAW264.7 cells reduces oxLDL-induced inflammatory cytokine synthesis. Sandwich ELISA analysis of TNF-α (A) and IL-12 (C) for the supernatants of oxLDL-treated RAW264.7 cells stably expressing GFP, GFP-Nur77, GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD. Data represent mean ± SD of three independent experiments for 1 × 106 cells, *p <0.05 compared with GFP-expressing control cell. (B) and (D) showed the level of TNF-α and IL-12 in GFP-expressing control cells with or without stimulated by oxLDL or LPS, respectively. The bars represent mean ± SD from three experiments. *p <0.05 compared with control cells.
Mentions: Finally, we assayed the effect of Nur77 on cytokine secretion by oxLDL-treated RAW264.7 cells. Here we show that, IL-12 and TNF-α from oxLDL-treated GFP-Nur77 cells were decreased to 46.8% and 41.5%, respectively, compared with that from GFP-expressing control cells. In contrast, expression of the Nur77 mutant proteins did not decrease oxLDL-induced IL-12 and TNF-α secretion. GFP-Nur77-ΔDBD-expressing cells demonstrated a 24.9% and 12.7% increase in IL-12 and TNF-α secretion, respectively (Figure 5A,C). These data show that Nur77 had an inhibitory effect on cytokine secretion, which required the Nur77 DNA binding domain and transactivation domains. To test the ability of producing several cytokines by these cells, we also analyzed TNF-α and IL-12 including a stimulated control with LPS in GFP-expressing control cells. Results showed that both of the two cytokines can be stimulated by LPS and the levels were 3-fold higher than by the stimulation with oxLDL (TNF-α: 490.12 ± 14.14 vs. 125.60 ± 7.07 ng/ml; IL-12: 15.47 ± 0.28 vs. 4.15 ± 0.21 pg/ml ) (Figure 5B,D).Figure 5

Bottom Line: GFP-Nur77 overexpression significantly suppressed the effect of oxLDL treatment on DC morphologic changes, expression of DC maturation markers, endocytic activity, allogeneic activation of T cell proliferation, and the activity and secretion of pro-inflammatory cytokines.GFP-Nur77-ΔDBD consistently had the opposite effect to GFP-Nur77, increasing DC-type differentiation in all assays.The effects of Nur77 on the macrophage phenotype may involve changes in its subcellular distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200127, People's Republic of China. iam565@126.com.

ABSTRACT

Background: Nur77 is an orphan nuclear receptor expressed in human atheroma. In vascular cells in vitro, Nur77 expression is induced by pro-inflammatory factors, such as oxidized LDL (oxLDL).

Methods: We analyze the role of Nur77 in the oxLDL-induced differentiation of macrophages into dendritic cells (DC). The murine RAW264.7 macrophage cell line was stably transfected with expression plasmids encoding either GFP or GFP fusions with either full-length Nur77 (GFP-Nur77), Nur77 lacking the DNA binding domain (GFP-Nur77-ΔDBD) or Nur77 lacking the transactivation domain (GFP-Nur77-ΔTAD).

Results: GFP-Nur77 overexpression significantly suppressed the effect of oxLDL treatment on DC morphologic changes, expression of DC maturation markers, endocytic activity, allogeneic activation of T cell proliferation, and the activity and secretion of pro-inflammatory cytokines. Analysis of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD indicated that the Nur77 DNA binding and transactivation domains were both required for this effect. GFP-Nur77-ΔDBD consistently had the opposite effect to GFP-Nur77, increasing DC-type differentiation in all assays. Interestingly, GFP-Nur77-ΔDBD protein was cytosolic, whereas GFP-Nur77 and GFP-Nur77-ΔTAD were both nuclear.

Conclusions: These data show that GFP-Nur77 inhibited differentiation of oxLDL-treated macrophages into DC. The effects of Nur77 on the macrophage phenotype may involve changes in its subcellular distribution.

Show MeSH
Related in: MedlinePlus