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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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Activation of Mkk3/p38 MAPK is necessary for CHOP induction in FC-loaded macrophages. (A) WT and Mkk3−/− macrophages (Mφs) were FC-loaded for 0, 2, 5, 7, 10, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described under “Materials and methods” and immunoblotted for CHOP (top panel), activated phospho-Thr180/Tyr182 p38 MAPK (phospho-p38, middle panel), and total p38 (bottom panel). Lines indicate areas of the same gel that were spliced together. (B) WT and Chop−/− macrophages were left untreated (−) or were FC-loaded (+) for 5 h, and lysates were immunoblotted for phospho-p38 and total p38. (C) WT and Mkk3−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). In some experiments, WT cells were treated with ac-LDL alone, which showed only minimal MK2 phosphorylation (not depicted). Whole cell lysates were immunoblotted for activated phospho-Thr334 MK2 (phospho-MK2, top panel) and total MK2 (bottom panel). (D) WT and Mk2−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). Whole cell lysates were immunoblotted for CHOP (top panel), total MK2 (second panel), total p38 (third panel), or tubulin as a loading control (bottom panel). Lines indicate areas of the same gel that were spliced together.
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fig1: Activation of Mkk3/p38 MAPK is necessary for CHOP induction in FC-loaded macrophages. (A) WT and Mkk3−/− macrophages (Mφs) were FC-loaded for 0, 2, 5, 7, 10, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described under “Materials and methods” and immunoblotted for CHOP (top panel), activated phospho-Thr180/Tyr182 p38 MAPK (phospho-p38, middle panel), and total p38 (bottom panel). Lines indicate areas of the same gel that were spliced together. (B) WT and Chop−/− macrophages were left untreated (−) or were FC-loaded (+) for 5 h, and lysates were immunoblotted for phospho-p38 and total p38. (C) WT and Mkk3−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). In some experiments, WT cells were treated with ac-LDL alone, which showed only minimal MK2 phosphorylation (not depicted). Whole cell lysates were immunoblotted for activated phospho-Thr334 MK2 (phospho-MK2, top panel) and total MK2 (bottom panel). (D) WT and Mk2−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). Whole cell lysates were immunoblotted for CHOP (top panel), total MK2 (second panel), total p38 (third panel), or tubulin as a loading control (bottom panel). Lines indicate areas of the same gel that were spliced together.

Mentions: Ongoing work in our laboratory has revealed that p38 MAPK is phosphorylated and activated in the early, preapoptotic stages of FC loading. p38 activation is downstream of MKK3 as indicated by the absence of p38 phosphorylation in FC-loaded macrophages from Mkk3−/− mice (Fig. 1 A and Li et al., 2005). Because the commonly used p38 pathway inhibitors (e.g., SB203580) are inhibitors of cholesterol trafficking (unpublished data), Mkk3-deficient macrophages are an essential tool to determine the consequences of FC-induced p38 activation.


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)

Activation of Mkk3/p38 MAPK is necessary for CHOP induction in FC-loaded macrophages. (A) WT and Mkk3−/− macrophages (Mφs) were FC-loaded for 0, 2, 5, 7, 10, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described under “Materials and methods” and immunoblotted for CHOP (top panel), activated phospho-Thr180/Tyr182 p38 MAPK (phospho-p38, middle panel), and total p38 (bottom panel). Lines indicate areas of the same gel that were spliced together. (B) WT and Chop−/− macrophages were left untreated (−) or were FC-loaded (+) for 5 h, and lysates were immunoblotted for phospho-p38 and total p38. (C) WT and Mkk3−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). In some experiments, WT cells were treated with ac-LDL alone, which showed only minimal MK2 phosphorylation (not depicted). Whole cell lysates were immunoblotted for activated phospho-Thr334 MK2 (phospho-MK2, top panel) and total MK2 (bottom panel). (D) WT and Mk2−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). Whole cell lysates were immunoblotted for CHOP (top panel), total MK2 (second panel), total p38 (third panel), or tubulin as a loading control (bottom panel). Lines indicate areas of the same gel that were spliced together.
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Related In: Results  -  Collection

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fig1: Activation of Mkk3/p38 MAPK is necessary for CHOP induction in FC-loaded macrophages. (A) WT and Mkk3−/− macrophages (Mφs) were FC-loaded for 0, 2, 5, 7, 10, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described under “Materials and methods” and immunoblotted for CHOP (top panel), activated phospho-Thr180/Tyr182 p38 MAPK (phospho-p38, middle panel), and total p38 (bottom panel). Lines indicate areas of the same gel that were spliced together. (B) WT and Chop−/− macrophages were left untreated (−) or were FC-loaded (+) for 5 h, and lysates were immunoblotted for phospho-p38 and total p38. (C) WT and Mkk3−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). In some experiments, WT cells were treated with ac-LDL alone, which showed only minimal MK2 phosphorylation (not depicted). Whole cell lysates were immunoblotted for activated phospho-Thr334 MK2 (phospho-MK2, top panel) and total MK2 (bottom panel). (D) WT and Mk2−/− macrophages were left untreated (−) or were FC-loaded for 7 h (+). Whole cell lysates were immunoblotted for CHOP (top panel), total MK2 (second panel), total p38 (third panel), or tubulin as a loading control (bottom panel). Lines indicate areas of the same gel that were spliced together.
Mentions: Ongoing work in our laboratory has revealed that p38 MAPK is phosphorylated and activated in the early, preapoptotic stages of FC loading. p38 activation is downstream of MKK3 as indicated by the absence of p38 phosphorylation in FC-loaded macrophages from Mkk3−/− mice (Fig. 1 A and Li et al., 2005). Because the commonly used p38 pathway inhibitors (e.g., SB203580) are inhibitors of cholesterol trafficking (unpublished data), Mkk3-deficient macrophages are an essential tool to determine the consequences of FC-induced p38 activation.

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