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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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JNK2 is necessary for apoptosis, but activation is not dependent on SRA engagement. (A) Macrophages were preincubated for 30 min with 10 μM SP600125 or with vehicle and then incubated for 18 h in medium alone or medium containing ac-LDL plus 58035 ± SP600125. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent images and quantitative apoptosis data from four fields of cells for each condition are shown. The data are expressed as the percent of total cells that stained with Annexin V and propidium iodide. Data are expressed as mean ± SEM (n = 4). Bar, 25 μm. (B) Macrophages were preincubated for 30 min with 15 μM SP600125 (SP) or with vehicle and then incubated for 18 h in medium containing 25 μg/ml fucoidan alone (Fuc), 0.5 μM thapsigargin (Tg) alone, or both compounds. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (C) WT or Jnk2−/− macrophages were incubated for 18 h in medium alone or medium containing ac-LDL plus 58035, 0.5 μM thapsigargin, or 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (D) Macrophages were incubated with 0.5 μM thapsigargin or 0.5 μM thapsigargin plus 25 μg/ml fucoidan for 0, 1, 2, and 3 h. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (phospho-JNK, top panel) and total JNK (bottom panel). (E) Macrophages were preincubated for 30 min with medium alone or medium containing the anti-SRA antibody 2f8 or isotype control IgG2b (30 μg/ml). The cells were incubated for 3 h in medium alone or medium containing 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (top panel), and total JNK (bottom panel). (F) WT and Jnk2−/− macrophages (Mφs) were FC loaded for 0 or 12, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for CHOP (top panel), total JNK (middle panel), and actin (bottom panel).
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fig9: JNK2 is necessary for apoptosis, but activation is not dependent on SRA engagement. (A) Macrophages were preincubated for 30 min with 10 μM SP600125 or with vehicle and then incubated for 18 h in medium alone or medium containing ac-LDL plus 58035 ± SP600125. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent images and quantitative apoptosis data from four fields of cells for each condition are shown. The data are expressed as the percent of total cells that stained with Annexin V and propidium iodide. Data are expressed as mean ± SEM (n = 4). Bar, 25 μm. (B) Macrophages were preincubated for 30 min with 15 μM SP600125 (SP) or with vehicle and then incubated for 18 h in medium containing 25 μg/ml fucoidan alone (Fuc), 0.5 μM thapsigargin (Tg) alone, or both compounds. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (C) WT or Jnk2−/− macrophages were incubated for 18 h in medium alone or medium containing ac-LDL plus 58035, 0.5 μM thapsigargin, or 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (D) Macrophages were incubated with 0.5 μM thapsigargin or 0.5 μM thapsigargin plus 25 μg/ml fucoidan for 0, 1, 2, and 3 h. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (phospho-JNK, top panel) and total JNK (bottom panel). (E) Macrophages were preincubated for 30 min with medium alone or medium containing the anti-SRA antibody 2f8 or isotype control IgG2b (30 μg/ml). The cells were incubated for 3 h in medium alone or medium containing 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (top panel), and total JNK (bottom panel). (F) WT and Jnk2−/− macrophages (Mφs) were FC loaded for 0 or 12, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for CHOP (top panel), total JNK (middle panel), and actin (bottom panel).

Mentions: Previous studies suggested roles for JNK in SRA-induced cytokine production and SRA internalization (Hsu et al., 2001; Ricci et al., 2004). Therefore, we considered the possibility that JNK signaling played a role in SRA-dependent apoptosis in UPR-activated macrophages. In this regard, we showed recently that JNK is activated when macrophages are FC-loaded with ac-LDL plus 58035 (Li et al., 2005). To test a possible role for JNK in UPR-SRA–induced apoptosis, we first used the JNK inhibitor SP600125 (Bennett et al., 2001). As shown in Fig. 9, A and B, pretreatment of macrophages with SP600125 blocked apoptosis that was induced by ac-LDL plus 58035 and by thapsigargin plus fucoidan. To assess the specific role of JNK2, we used Jnk2−/− macrophages instead of the JNK inhibitor, and found a similar inhibition of apoptosis (Fig. 9 C). Thus, JNK activation, in general, and JNK2 activation, in particular, is necessary for UPR-SRA–dependent apoptosis in macrophages.


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)

JNK2 is necessary for apoptosis, but activation is not dependent on SRA engagement. (A) Macrophages were preincubated for 30 min with 10 μM SP600125 or with vehicle and then incubated for 18 h in medium alone or medium containing ac-LDL plus 58035 ± SP600125. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent images and quantitative apoptosis data from four fields of cells for each condition are shown. The data are expressed as the percent of total cells that stained with Annexin V and propidium iodide. Data are expressed as mean ± SEM (n = 4). Bar, 25 μm. (B) Macrophages were preincubated for 30 min with 15 μM SP600125 (SP) or with vehicle and then incubated for 18 h in medium containing 25 μg/ml fucoidan alone (Fuc), 0.5 μM thapsigargin (Tg) alone, or both compounds. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (C) WT or Jnk2−/− macrophages were incubated for 18 h in medium alone or medium containing ac-LDL plus 58035, 0.5 μM thapsigargin, or 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (D) Macrophages were incubated with 0.5 μM thapsigargin or 0.5 μM thapsigargin plus 25 μg/ml fucoidan for 0, 1, 2, and 3 h. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (phospho-JNK, top panel) and total JNK (bottom panel). (E) Macrophages were preincubated for 30 min with medium alone or medium containing the anti-SRA antibody 2f8 or isotype control IgG2b (30 μg/ml). The cells were incubated for 3 h in medium alone or medium containing 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (top panel), and total JNK (bottom panel). (F) WT and Jnk2−/− macrophages (Mφs) were FC loaded for 0 or 12, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for CHOP (top panel), total JNK (middle panel), and actin (bottom panel).
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fig9: JNK2 is necessary for apoptosis, but activation is not dependent on SRA engagement. (A) Macrophages were preincubated for 30 min with 10 μM SP600125 or with vehicle and then incubated for 18 h in medium alone or medium containing ac-LDL plus 58035 ± SP600125. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent images and quantitative apoptosis data from four fields of cells for each condition are shown. The data are expressed as the percent of total cells that stained with Annexin V and propidium iodide. Data are expressed as mean ± SEM (n = 4). Bar, 25 μm. (B) Macrophages were preincubated for 30 min with 15 μM SP600125 (SP) or with vehicle and then incubated for 18 h in medium containing 25 μg/ml fucoidan alone (Fuc), 0.5 μM thapsigargin (Tg) alone, or both compounds. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (C) WT or Jnk2−/− macrophages were incubated for 18 h in medium alone or medium containing ac-LDL plus 58035, 0.5 μM thapsigargin, or 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (D) Macrophages were incubated with 0.5 μM thapsigargin or 0.5 μM thapsigargin plus 25 μg/ml fucoidan for 0, 1, 2, and 3 h. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (phospho-JNK, top panel) and total JNK (bottom panel). (E) Macrophages were preincubated for 30 min with medium alone or medium containing the anti-SRA antibody 2f8 or isotype control IgG2b (30 μg/ml). The cells were incubated for 3 h in medium alone or medium containing 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr 183/Tyr185 JNK (top panel), and total JNK (bottom panel). (F) WT and Jnk2−/− macrophages (Mφs) were FC loaded for 0 or 12, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for CHOP (top panel), total JNK (middle panel), and actin (bottom panel).
Mentions: Previous studies suggested roles for JNK in SRA-induced cytokine production and SRA internalization (Hsu et al., 2001; Ricci et al., 2004). Therefore, we considered the possibility that JNK signaling played a role in SRA-dependent apoptosis in UPR-activated macrophages. In this regard, we showed recently that JNK is activated when macrophages are FC-loaded with ac-LDL plus 58035 (Li et al., 2005). To test a possible role for JNK in UPR-SRA–induced apoptosis, we first used the JNK inhibitor SP600125 (Bennett et al., 2001). As shown in Fig. 9, A and B, pretreatment of macrophages with SP600125 blocked apoptosis that was induced by ac-LDL plus 58035 and by thapsigargin plus fucoidan. To assess the specific role of JNK2, we used Jnk2−/− macrophages instead of the JNK inhibitor, and found a similar inhibition of apoptosis (Fig. 9 C). Thus, JNK activation, in general, and JNK2 activation, in particular, is necessary for UPR-SRA–dependent apoptosis in macrophages.

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