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Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor.

Devries-Seimon T, Li Y, Yao PM, Stone E, Wang Y, Davis RJ, Flavell R, Tabas I - J. Cell Biol. (2005)

Bottom Line: Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis.One involves the type A scavenger receptor (SRA).Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Columbia University, New York, NY 10032, USA.

ABSTRACT
Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.

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SRA ligands induce apoptosis in macrophages that are FC loaded by non-SRA sources of cholesterol, or treated with low-dose thapsigargin. (A) Macrophages were left untreated or incubated with 25 μg/ml fucoidan alone (Fuc), CD-cholesterol plus 58035 alone (CD-Chol), or CD-cholesterol/58035 plus fucoidan for 24 h. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent and bright field images are shown. Quantitative apoptosis data for each condition are shown as described in the legend for Fig. 4. Data are expressed as mean ± SEM (n = 8). Bar, 25 μm. (B) The experiment was conducted as in (A) except the macrophages were incubated for 24 h with 100 μg/ml of CML-BSA, CD-cholesterol/58035, or CD-cholesterol/58035 plus CML-BSA. Bar, 25 μm. As a control, FC-loaded macrophages also were treated with nonmodified BSA, which did not induce apoptosis (not depicted). (C) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with 50 μg/ml β-VLDL plus 58035, β-VLDL plus 58035 plus fucoidan (25 μg/ml), or 100 μg/ml ac-LDL plus 58035. Data are expressed as mean ± SEM (n = 8). (D) Quantitative apoptosis data for WT or Mkk3−/− macrophages left untreated or incubated for 24 h with CD-cholesterol/58035 or CD-cholesterol/58035 plus fucoidan. Cells were stained with Alexa 488 Annexin V and propidium iodide. Representative quantitative apoptosis data are from four fields of cells under each condition. Data are expressed as mean ± SEM (n = 4). (E) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with fucoidan (25 μg/ml), 0.5 μM thapsigargin (Tg), or fucoidan plus thapsigargin. Data are expressed as mean ± SEM (n = 8). Representative fluorescent and bright field images are shown. Bar, 25 μm.
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fig6: SRA ligands induce apoptosis in macrophages that are FC loaded by non-SRA sources of cholesterol, or treated with low-dose thapsigargin. (A) Macrophages were left untreated or incubated with 25 μg/ml fucoidan alone (Fuc), CD-cholesterol plus 58035 alone (CD-Chol), or CD-cholesterol/58035 plus fucoidan for 24 h. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent and bright field images are shown. Quantitative apoptosis data for each condition are shown as described in the legend for Fig. 4. Data are expressed as mean ± SEM (n = 8). Bar, 25 μm. (B) The experiment was conducted as in (A) except the macrophages were incubated for 24 h with 100 μg/ml of CML-BSA, CD-cholesterol/58035, or CD-cholesterol/58035 plus CML-BSA. Bar, 25 μm. As a control, FC-loaded macrophages also were treated with nonmodified BSA, which did not induce apoptosis (not depicted). (C) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with 50 μg/ml β-VLDL plus 58035, β-VLDL plus 58035 plus fucoidan (25 μg/ml), or 100 μg/ml ac-LDL plus 58035. Data are expressed as mean ± SEM (n = 8). (D) Quantitative apoptosis data for WT or Mkk3−/− macrophages left untreated or incubated for 24 h with CD-cholesterol/58035 or CD-cholesterol/58035 plus fucoidan. Cells were stained with Alexa 488 Annexin V and propidium iodide. Representative quantitative apoptosis data are from four fields of cells under each condition. Data are expressed as mean ± SEM (n = 4). (E) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with fucoidan (25 μg/ml), 0.5 μM thapsigargin (Tg), or fucoidan plus thapsigargin. Data are expressed as mean ± SEM (n = 8). Representative fluorescent and bright field images are shown. Bar, 25 μm.

Mentions: One of the differences between ac-LDL and CD-cholesterol or β-VLDL is that only ac-LDL is a ligand for the SRA (Goldstein et al., 1979). Thus, ac-LDL might trigger apoptosis because it delivers cholesterol and engages the SRA, whereas CD-cholesterol and β-VLDL only affect the cholesterol loading step. To test this idea, we determined whether apoptosis could be “reconstituted” by incubating macrophages with a CD-cholesterol (or β-VLDL) plus a noncholesterol-containing ligand for the SRA. As shown in Fig. 6 A, neither fucoidan, a ligand for the SRA, nor CD-cholesterol, induced a substantial degree of apoptosis when the reagents were added individually. However, the combination of reagents caused a marked apoptotic response. The data in Fig. 6 B show similar results with another SRA ligand, carboxymethyllysine-BSA (CML-BSA), that has possible relevance to diabetic macrovascular disease. CML-BSA is an advance glycation end-product (AGE) that may be formed during the oxidation of lipoproteins in the vascular wall (Fu et al., 1996; Miyazaki et al., 2002). In addition, the same results were obtained when the source of non-SRA cholesterol was β-VLDL, instead of CD-cholesterol (Fig. 6 C). To assess the relationship of CD-cholesterol/fucoidan-induced apoptosis with ac-LDL–induced apoptosis further, we tested dependency on the MKK3/p38 MAPK pathway. As shown in Fig. 6 D, CD-cholesterol/fucoidan-induced apoptosis, like ac-LDL–induced apoptosis, was decreased markedly in Mkk3−/− macrophages; similar results were found in Mk2−/− macrophages (not depicted).


Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor.

Devries-Seimon T, Li Y, Yao PM, Stone E, Wang Y, Davis RJ, Flavell R, Tabas I - J. Cell Biol. (2005)

SRA ligands induce apoptosis in macrophages that are FC loaded by non-SRA sources of cholesterol, or treated with low-dose thapsigargin. (A) Macrophages were left untreated or incubated with 25 μg/ml fucoidan alone (Fuc), CD-cholesterol plus 58035 alone (CD-Chol), or CD-cholesterol/58035 plus fucoidan for 24 h. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent and bright field images are shown. Quantitative apoptosis data for each condition are shown as described in the legend for Fig. 4. Data are expressed as mean ± SEM (n = 8). Bar, 25 μm. (B) The experiment was conducted as in (A) except the macrophages were incubated for 24 h with 100 μg/ml of CML-BSA, CD-cholesterol/58035, or CD-cholesterol/58035 plus CML-BSA. Bar, 25 μm. As a control, FC-loaded macrophages also were treated with nonmodified BSA, which did not induce apoptosis (not depicted). (C) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with 50 μg/ml β-VLDL plus 58035, β-VLDL plus 58035 plus fucoidan (25 μg/ml), or 100 μg/ml ac-LDL plus 58035. Data are expressed as mean ± SEM (n = 8). (D) Quantitative apoptosis data for WT or Mkk3−/− macrophages left untreated or incubated for 24 h with CD-cholesterol/58035 or CD-cholesterol/58035 plus fucoidan. Cells were stained with Alexa 488 Annexin V and propidium iodide. Representative quantitative apoptosis data are from four fields of cells under each condition. Data are expressed as mean ± SEM (n = 4). (E) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with fucoidan (25 μg/ml), 0.5 μM thapsigargin (Tg), or fucoidan plus thapsigargin. Data are expressed as mean ± SEM (n = 8). Representative fluorescent and bright field images are shown. Bar, 25 μm.
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fig6: SRA ligands induce apoptosis in macrophages that are FC loaded by non-SRA sources of cholesterol, or treated with low-dose thapsigargin. (A) Macrophages were left untreated or incubated with 25 μg/ml fucoidan alone (Fuc), CD-cholesterol plus 58035 alone (CD-Chol), or CD-cholesterol/58035 plus fucoidan for 24 h. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent and bright field images are shown. Quantitative apoptosis data for each condition are shown as described in the legend for Fig. 4. Data are expressed as mean ± SEM (n = 8). Bar, 25 μm. (B) The experiment was conducted as in (A) except the macrophages were incubated for 24 h with 100 μg/ml of CML-BSA, CD-cholesterol/58035, or CD-cholesterol/58035 plus CML-BSA. Bar, 25 μm. As a control, FC-loaded macrophages also were treated with nonmodified BSA, which did not induce apoptosis (not depicted). (C) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with 50 μg/ml β-VLDL plus 58035, β-VLDL plus 58035 plus fucoidan (25 μg/ml), or 100 μg/ml ac-LDL plus 58035. Data are expressed as mean ± SEM (n = 8). (D) Quantitative apoptosis data for WT or Mkk3−/− macrophages left untreated or incubated for 24 h with CD-cholesterol/58035 or CD-cholesterol/58035 plus fucoidan. Cells were stained with Alexa 488 Annexin V and propidium iodide. Representative quantitative apoptosis data are from four fields of cells under each condition. Data are expressed as mean ± SEM (n = 4). (E) Quantitative apoptosis data for macrophages left untreated or incubated for 24 h with fucoidan (25 μg/ml), 0.5 μM thapsigargin (Tg), or fucoidan plus thapsigargin. Data are expressed as mean ± SEM (n = 8). Representative fluorescent and bright field images are shown. Bar, 25 μm.
Mentions: One of the differences between ac-LDL and CD-cholesterol or β-VLDL is that only ac-LDL is a ligand for the SRA (Goldstein et al., 1979). Thus, ac-LDL might trigger apoptosis because it delivers cholesterol and engages the SRA, whereas CD-cholesterol and β-VLDL only affect the cholesterol loading step. To test this idea, we determined whether apoptosis could be “reconstituted” by incubating macrophages with a CD-cholesterol (or β-VLDL) plus a noncholesterol-containing ligand for the SRA. As shown in Fig. 6 A, neither fucoidan, a ligand for the SRA, nor CD-cholesterol, induced a substantial degree of apoptosis when the reagents were added individually. However, the combination of reagents caused a marked apoptotic response. The data in Fig. 6 B show similar results with another SRA ligand, carboxymethyllysine-BSA (CML-BSA), that has possible relevance to diabetic macrovascular disease. CML-BSA is an advance glycation end-product (AGE) that may be formed during the oxidation of lipoproteins in the vascular wall (Fu et al., 1996; Miyazaki et al., 2002). In addition, the same results were obtained when the source of non-SRA cholesterol was β-VLDL, instead of CD-cholesterol (Fig. 6 C). To assess the relationship of CD-cholesterol/fucoidan-induced apoptosis with ac-LDL–induced apoptosis further, we tested dependency on the MKK3/p38 MAPK pathway. As shown in Fig. 6 D, CD-cholesterol/fucoidan-induced apoptosis, like ac-LDL–induced apoptosis, was decreased markedly in Mkk3−/− macrophages; similar results were found in Mk2−/− macrophages (not depicted).

Bottom Line: Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis.One involves the type A scavenger receptor (SRA).Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Columbia University, New York, NY 10032, USA.

ABSTRACT
Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.

Show MeSH
Related in: MedlinePlus