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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 inhibits DC morphological changes in oxLDL-treated RAW264.7 cells. (A) RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD were treated with oxLDL (10 μg/ml) for 48 h and visualized by phase contrast microscopy (400×). Results are representative of three separate experiments. (B) Cells with DC morphology were calculated as the percentage of all cells observed in 10 different fields at 400× magnification. The bars represent mean ± SD from three experiments. *p <0.05 compared with GFP-expressing control. (C) Western blots showing endogenous Nur77 in RAW264.7 cells 48 h after transfection with either scrambled siRNA or Nur77 siRNA. Similar results were obtained in three separate experiments. (D) Phase contrast images showing the morphology of oxLDL-treated RAW264.7 cells transfected with either scramble siRNA or Nur77 siRNA. Cultured cells were visualized by phase contrast microscopy as described in (A) and cells with DC morphology were calculated as described in (B). (E) *p <0.05 compared with scrambled control.
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Fig2: Nur77 inhibits DC morphological changes in oxLDL-treated RAW264.7 cells. (A) RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD were treated with oxLDL (10 μg/ml) for 48 h and visualized by phase contrast microscopy (400×). Results are representative of three separate experiments. (B) Cells with DC morphology were calculated as the percentage of all cells observed in 10 different fields at 400× magnification. The bars represent mean ± SD from three experiments. *p <0.05 compared with GFP-expressing control. (C) Western blots showing endogenous Nur77 in RAW264.7 cells 48 h after transfection with either scrambled siRNA or Nur77 siRNA. Similar results were obtained in three separate experiments. (D) Phase contrast images showing the morphology of oxLDL-treated RAW264.7 cells transfected with either scramble siRNA or Nur77 siRNA. Cultured cells were visualized by phase contrast microscopy as described in (A) and cells with DC morphology were calculated as described in (B). (E) *p <0.05 compared with scrambled control.

Mentions: We tested the effects of oxLDL on the morphology, DC surface marker expression, endocytic activity, allostimulatory activity, and cytokine secretion of the RAW264.7 stable cell lines. Consistent with previous results, 72.50% of GFP control cells had DC morphology after oxLDL treatment as determined by increased cell size, the presence of multiple prominent cytoplasmic processes, and prominent nucleoli (Figure 2A and B). In contrast, although most GFP-Nur77-expressing cells increased in size, only 28.94% had DC morphology following oxLDL treatment. In contrast, 72.30% oxLDL-treated GFP-Nur77-ΔTAD or 82.8% of oxLDL-treated GFP-Nur77-ΔDBD cell lines were of DC morphology, which was similar to control GFP-expressing cells (p >0.05). There was a small but statistically significant increase in the proportion of DCs in GFP-Nur77-ΔDBD cells compared to GFP-expressing cells (p <0.05; Figure 2A,B). To determine whether endogenous Nur77 played a role in macrophage–DC differentiation, we used siRNAs to deplete Nur77 and assayed the effect on oxLDL-induced morphological changes. Transfection of Nur77 siRNA successfully depleted endogenous Nur77 in RAW264.7 cells compared to the scrambled siRNA (Figure 2C) and led to a 17% increase in the proportion of cells with DC morphology following oxLDL treatment compared to that in the scrambled siRNA group ( 66.5 ± 12.4% vs. 83.8 ± 12.1%, p <0.05; Figure 2D,E).Figure 2


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 inhibits DC morphological changes in oxLDL-treated RAW264.7 cells. (A) RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD were treated with oxLDL (10 μg/ml) for 48 h and visualized by phase contrast microscopy (400×). Results are representative of three separate experiments. (B) Cells with DC morphology were calculated as the percentage of all cells observed in 10 different fields at 400× magnification. The bars represent mean ± SD from three experiments. *p <0.05 compared with GFP-expressing control. (C) Western blots showing endogenous Nur77 in RAW264.7 cells 48 h after transfection with either scrambled siRNA or Nur77 siRNA. Similar results were obtained in three separate experiments. (D) Phase contrast images showing the morphology of oxLDL-treated RAW264.7 cells transfected with either scramble siRNA or Nur77 siRNA. Cultured cells were visualized by phase contrast microscopy as described in (A) and cells with DC morphology were calculated as described in (B). (E) *p <0.05 compared with scrambled control.
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Fig2: Nur77 inhibits DC morphological changes in oxLDL-treated RAW264.7 cells. (A) RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD were treated with oxLDL (10 μg/ml) for 48 h and visualized by phase contrast microscopy (400×). Results are representative of three separate experiments. (B) Cells with DC morphology were calculated as the percentage of all cells observed in 10 different fields at 400× magnification. The bars represent mean ± SD from three experiments. *p <0.05 compared with GFP-expressing control. (C) Western blots showing endogenous Nur77 in RAW264.7 cells 48 h after transfection with either scrambled siRNA or Nur77 siRNA. Similar results were obtained in three separate experiments. (D) Phase contrast images showing the morphology of oxLDL-treated RAW264.7 cells transfected with either scramble siRNA or Nur77 siRNA. Cultured cells were visualized by phase contrast microscopy as described in (A) and cells with DC morphology were calculated as described in (B). (E) *p <0.05 compared with scrambled control.
Mentions: We tested the effects of oxLDL on the morphology, DC surface marker expression, endocytic activity, allostimulatory activity, and cytokine secretion of the RAW264.7 stable cell lines. Consistent with previous results, 72.50% of GFP control cells had DC morphology after oxLDL treatment as determined by increased cell size, the presence of multiple prominent cytoplasmic processes, and prominent nucleoli (Figure 2A and B). In contrast, although most GFP-Nur77-expressing cells increased in size, only 28.94% had DC morphology following oxLDL treatment. In contrast, 72.30% oxLDL-treated GFP-Nur77-ΔTAD or 82.8% of oxLDL-treated GFP-Nur77-ΔDBD cell lines were of DC morphology, which was similar to control GFP-expressing cells (p >0.05). There was a small but statistically significant increase in the proportion of DCs in GFP-Nur77-ΔDBD cells compared to GFP-expressing cells (p <0.05; Figure 2A,B). To determine whether endogenous Nur77 played a role in macrophage–DC differentiation, we used siRNAs to deplete Nur77 and assayed the effect on oxLDL-induced morphological changes. Transfection of Nur77 siRNA successfully depleted endogenous Nur77 in RAW264.7 cells compared to the scrambled siRNA (Figure 2C) and led to a 17% increase in the proportion of cells with DC morphology following oxLDL treatment compared to that in the scrambled siRNA group ( 66.5 ± 12.4% vs. 83.8 ± 12.1%, p <0.05; Figure 2D,E).Figure 2

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