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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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Characterization of stable RAW264.7 cell lines expressing Nur77 and Nur77 deletion mutants. (A) Schematic structure of the Nur77 gene and deletion mutants lacking either the transactivation domain (TAD) or DNA binding domain (DBD). (B) Expression of Nur77 protein in untransfected RAW264.7 cells and in RAW264.7 cell lines expressing GFP or GFP-Nur77. Expression of GFP-Nur77 is 3–4-fold higher than endogenous Nur77 in untransfected or GFP-transfected cells. (C) Expression of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD fusion proteins in stably transfected RAW264.7 cells. β-actin expression was used to control for protein loading. A representative of three separate experiments is shown. (D) Subcellular localization of GFP-Nur77, GFP-Nur77-ΔTAD, and GFP-Nur77-ΔDBD in RAW264.7 cells.
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Fig1: Characterization of stable RAW264.7 cell lines expressing Nur77 and Nur77 deletion mutants. (A) Schematic structure of the Nur77 gene and deletion mutants lacking either the transactivation domain (TAD) or DNA binding domain (DBD). (B) Expression of Nur77 protein in untransfected RAW264.7 cells and in RAW264.7 cell lines expressing GFP or GFP-Nur77. Expression of GFP-Nur77 is 3–4-fold higher than endogenous Nur77 in untransfected or GFP-transfected cells. (C) Expression of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD fusion proteins in stably transfected RAW264.7 cells. β-actin expression was used to control for protein loading. A representative of three separate experiments is shown. (D) Subcellular localization of GFP-Nur77, GFP-Nur77-ΔTAD, and GFP-Nur77-ΔDBD in RAW264.7 cells.

Mentions: We have shown previously that macrophages exposed to oxLDL in vitro differentiate into mature DC. Here, we have investigated a possible role for the orphan nuclear receptor Nur77 on the differentiation of oxLDL-treated RAW264.7 cells, a murine macrophage cell line. Nur77, a steroid/thyroid hormone nuclear receptor superfamily, contains three characteristic functional domains involved in transactivation, DNA binding, and ligand binding (Figure 1A). We established clonal RAW264.7 cell lines stably expressing either wild-type GFP-Nur77 or GFP fusion proteins with Nur77 lacking either the transactivation or DNA binding domains (GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD, respectively). GFP-Nur77 expression was 3–4 fold the level of endogenous Nur77 (Figure 1B). The two deletion mutants of Nur77 were expressed to similar extents (Figure 1C). Fluorescent microscopy revealed that GFP-Nur77-ΔDBD was cytosolic, whereas GFP-Nur77 and GFP-Nur77-ΔTAD were strictly nuclear (Figure 1D) suggesting that DNA binding is required for nuclear localization.Figure 1


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)

Characterization of stable RAW264.7 cell lines expressing Nur77 and Nur77 deletion mutants. (A) Schematic structure of the Nur77 gene and deletion mutants lacking either the transactivation domain (TAD) or DNA binding domain (DBD). (B) Expression of Nur77 protein in untransfected RAW264.7 cells and in RAW264.7 cell lines expressing GFP or GFP-Nur77. Expression of GFP-Nur77 is 3–4-fold higher than endogenous Nur77 in untransfected or GFP-transfected cells. (C) Expression of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD fusion proteins in stably transfected RAW264.7 cells. β-actin expression was used to control for protein loading. A representative of three separate experiments is shown. (D) Subcellular localization of GFP-Nur77, GFP-Nur77-ΔTAD, and GFP-Nur77-ΔDBD in RAW264.7 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4274730&req=5

Fig1: Characterization of stable RAW264.7 cell lines expressing Nur77 and Nur77 deletion mutants. (A) Schematic structure of the Nur77 gene and deletion mutants lacking either the transactivation domain (TAD) or DNA binding domain (DBD). (B) Expression of Nur77 protein in untransfected RAW264.7 cells and in RAW264.7 cell lines expressing GFP or GFP-Nur77. Expression of GFP-Nur77 is 3–4-fold higher than endogenous Nur77 in untransfected or GFP-transfected cells. (C) Expression of GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD fusion proteins in stably transfected RAW264.7 cells. β-actin expression was used to control for protein loading. A representative of three separate experiments is shown. (D) Subcellular localization of GFP-Nur77, GFP-Nur77-ΔTAD, and GFP-Nur77-ΔDBD in RAW264.7 cells.
Mentions: We have shown previously that macrophages exposed to oxLDL in vitro differentiate into mature DC. Here, we have investigated a possible role for the orphan nuclear receptor Nur77 on the differentiation of oxLDL-treated RAW264.7 cells, a murine macrophage cell line. Nur77, a steroid/thyroid hormone nuclear receptor superfamily, contains three characteristic functional domains involved in transactivation, DNA binding, and ligand binding (Figure 1A). We established clonal RAW264.7 cell lines stably expressing either wild-type GFP-Nur77 or GFP fusion proteins with Nur77 lacking either the transactivation or DNA binding domains (GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD, respectively). GFP-Nur77 expression was 3–4 fold the level of endogenous Nur77 (Figure 1B). The two deletion mutants of Nur77 were expressed to similar extents (Figure 1C). Fluorescent microscopy revealed that GFP-Nur77-ΔDBD was cytosolic, whereas GFP-Nur77 and GFP-Nur77-ΔTAD were strictly nuclear (Figure 1D) suggesting that DNA binding is required for nuclear localization.Figure 1

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