Limits...
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.

Show MeSH

Related in: MedlinePlus

Nur77 enhanced the endocytic activity but suppressed the DNA synthesis of RAW264.7 treated by oxLDL. (A) Flow cytometry analysis of endocytic activity using lucifer yellow (LY) uptake by oxLDL-treated RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Flow cytometry analysis of BrdU incorporation by co-cultures of allogeneic T cells and RAW264.7 cells stably expressing either GFP, GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Nur77 enhanced the endocytic activity but suppressed the DNA synthesis of RAW264.7 treated by oxLDL. (A) Flow cytometry analysis of endocytic activity using lucifer yellow (LY) uptake by oxLDL-treated RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Flow cytometry analysis of BrdU incorporation by co-cultures of allogeneic T cells and RAW264.7 cells stably expressing either GFP, GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control.

Mentions: We have previously shown that oxLDL-treated RAW264.7 cells gain several of the functional characteristics of mature DC [5]. Therefore, we next assayed the role of Nur77 in these oxLDL-induced functional changes. Using LY uptake as a marker of endocytosis (Figure 4A), we found that a large proportion of the four stable RAW264.7 cell lines were LY + following 12 h exposure to oxLDL. Consistent with published results, oxLDL had a dynamic effect on endocytosis. GFP-expressing controlcells exhibited decreased LY uptake by approximately 33% at 24 h and 57% at 48 h after oxLDL treatment. Similar results were obtained from GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD-expressing cells, except that at 48 h LY uptake by GFP-Nur77-ΔDBD-expressing cells was 15% lower than GFP-expressing cells. In contrast, oxLDL had little effect on LY uptake in GFP-Nur77-expressing cells at either 24 h or 48 h. These results indicate that overexpression of Nur77 protein in RAW264.7 cells inhibited the endocytic changes typically displayed by mature DCs, and that this inhibition was dependent upon the DNA binding and transactivation domains of Nur77.Figure 4


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 enhanced the endocytic activity but suppressed the DNA synthesis of RAW264.7 treated by oxLDL. (A) Flow cytometry analysis of endocytic activity using lucifer yellow (LY) uptake by oxLDL-treated RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Flow cytometry analysis of BrdU incorporation by co-cultures of allogeneic T cells and RAW264.7 cells stably expressing either GFP, GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Nur77 enhanced the endocytic activity but suppressed the DNA synthesis of RAW264.7 treated by oxLDL. (A) Flow cytometry analysis of endocytic activity using lucifer yellow (LY) uptake by oxLDL-treated RAW264.7 cells stably expressing GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control. (B) Flow cytometry analysis of BrdU incorporation by co-cultures of allogeneic T cells and RAW264.7 cells stably expressing either GFP, GFP-Nur77, GFP-Nur77-ΔTAD, or GFP-Nur77-ΔDBD. Mean ± SD of three independent experiments is shown. *p <0.05 compared with GFP-expressing control.
Mentions: We have previously shown that oxLDL-treated RAW264.7 cells gain several of the functional characteristics of mature DC [5]. Therefore, we next assayed the role of Nur77 in these oxLDL-induced functional changes. Using LY uptake as a marker of endocytosis (Figure 4A), we found that a large proportion of the four stable RAW264.7 cell lines were LY + following 12 h exposure to oxLDL. Consistent with published results, oxLDL had a dynamic effect on endocytosis. GFP-expressing controlcells exhibited decreased LY uptake by approximately 33% at 24 h and 57% at 48 h after oxLDL treatment. Similar results were obtained from GFP-Nur77-ΔTAD and GFP-Nur77-ΔDBD-expressing cells, except that at 48 h LY uptake by GFP-Nur77-ΔDBD-expressing cells was 15% lower than GFP-expressing cells. In contrast, oxLDL had little effect on LY uptake in GFP-Nur77-expressing cells at either 24 h or 48 h. These results indicate that overexpression of Nur77 protein in RAW264.7 cells inhibited the endocytic changes typically displayed by mature DCs, and that this inhibition was dependent upon the DNA binding and transactivation domains of Nur77.Figure 4

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