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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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Flow cytometry analysis of the cell surface phenotype of oxLDL-treated RAW264.7 cell lines. (A) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells lines treated with oxLDL for 48 h. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells transfected with either scrambled siRNA or Nur77 siRNA and stimulated with oxLDL for 48 h. *p <0.05 compared with scrambled control.
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Fig3: Flow cytometry analysis of the cell surface phenotype of oxLDL-treated RAW264.7 cell lines. (A) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells lines treated with oxLDL for 48 h. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells transfected with either scrambled siRNA or Nur77 siRNA and stimulated with oxLDL for 48 h. *p <0.05 compared with scrambled control.

Mentions: The changes in cell morphology described above suggest that Nur77 inhibits oxLDL-induced RAW264.7 cell differentiation into DCs through its DNA binding and transactivation domains. To investigate this possibility, we evaluated phenotypic changes in oxLDL-treated RAW264.7 cells stably expressing Nur77 and Nur77 mutant proteins by flow cytometry using specific antibodies against co-stimulatory molecules, antigen-presenting molecules, and markers of DC activation. Following oxLDL treatment, the levels of CD40, CD86, CD83, MHC class II, and CD1d were reduced by 62.4%, 44.69%, 51.7%, 55.2%, and 53.29%, respectively, in RAW264.7 cells stably expressing GFP-Nur77 protein compared with those in GFP-expressing cells. However, there was little difference in the levels of these proteins when comparing cells expressing either GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD with GFP-expressing cells, with the exception that GFP-Nur77-ΔDBD cells had a small but significant increase in the expression of CD83, MHC class II molecules, and CD1d (p <0.05 compared to control cells; Figure 3A). Moreover, all of the aforementioned proteins were significantly up-regulated in Nur77-depleted RAW264.7 cells when compared to control cells transfected with scrambled siRNA (p <0.05; Figure 3B).Figure 3


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)

Flow cytometry analysis of the cell surface phenotype of oxLDL-treated RAW264.7 cell lines. (A) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells lines treated with oxLDL for 48 h. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells transfected with either scrambled siRNA or Nur77 siRNA and stimulated with oxLDL for 48 h. *p <0.05 compared with scrambled control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4274730&req=5

Fig3: Flow cytometry analysis of the cell surface phenotype of oxLDL-treated RAW264.7 cell lines. (A) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells lines treated with oxLDL for 48 h. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Expression of CD40, CD86, CD83, MHC Class II, and CD1d in RAW264.7 cells transfected with either scrambled siRNA or Nur77 siRNA and stimulated with oxLDL for 48 h. *p <0.05 compared with scrambled control.
Mentions: The changes in cell morphology described above suggest that Nur77 inhibits oxLDL-induced RAW264.7 cell differentiation into DCs through its DNA binding and transactivation domains. To investigate this possibility, we evaluated phenotypic changes in oxLDL-treated RAW264.7 cells stably expressing Nur77 and Nur77 mutant proteins by flow cytometry using specific antibodies against co-stimulatory molecules, antigen-presenting molecules, and markers of DC activation. Following oxLDL treatment, the levels of CD40, CD86, CD83, MHC class II, and CD1d were reduced by 62.4%, 44.69%, 51.7%, 55.2%, and 53.29%, respectively, in RAW264.7 cells stably expressing GFP-Nur77 protein compared with those in GFP-expressing cells. However, there was little difference in the levels of these proteins when comparing cells expressing either GFP-Nur77-ΔTAD or GFP-Nur77-ΔDBD with GFP-expressing cells, with the exception that GFP-Nur77-ΔDBD cells had a small but significant increase in the expression of CD83, MHC class II molecules, and CD1d (p <0.05 compared to control cells; Figure 3A). Moreover, all of the aforementioned proteins were significantly up-regulated in Nur77-depleted RAW264.7 cells when compared to control cells transfected with scrambled siRNA (p <0.05; Figure 3B).Figure 3

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