CAMKIIγ suppresses an efferocytosis pathway in macrophages and promotes atherosclerotic plaque necrosis
Amanda C. Doran, Lale Ozcan, Bishuang Cai, Ze Zheng, Gabrielle Fredman, Christina C. Rymond, Bernhard Dorweiler, Judith C. Sluimer, Joanne Hsieh, George Kuriakose, Alan R. Tall, Ira Tabas
Amanda C. Doran, Lale Ozcan, Bishuang Cai, Ze Zheng, Gabrielle Fredman, Christina C. Rymond, Bernhard Dorweiler, Judith C. Sluimer, Joanne Hsieh, George Kuriakose, Alan R. Tall, Ira Tabas
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Research Article Cardiology Cell biology

CAMKIIγ suppresses an efferocytosis pathway in macrophages and promotes atherosclerotic plaque necrosis

Abstract

Atherosclerosis is the underlying etiology of cardiovascular disease, the leading cause of death worldwide. Atherosclerosis is a heterogeneous disease in which only a small fraction of lesions lead to heart attack, stroke, or sudden cardiac death. A distinct type of plaque containing large necrotic cores with thin fibrous caps often precipitates these acute events. Here, we show that Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) in macrophages plays a major role in the development of necrotic, thin-capped plaques. Macrophages in necrotic and symptomatic atherosclerotic plaques in humans as well as advanced atherosclerotic lesions in mice demonstrated activation of CaMKII. Western diet–fed LDL receptor–deficient (Ldlr–/–) mice with myeloid-specific deletion of CaMKII had smaller necrotic cores with concomitantly thicker collagen caps. These lesions demonstrated evidence of enhanced efferocytosis, which was associated with increased expression of the macrophage efferocytosis receptor MerTK. Mechanistic studies revealed that CaMKIIγ-deficient macrophages and atherosclerotic lesions lacking myeloid CaMKIIγ had increased expression of the transcription factor ATF6. We determined that ATF6 induces liver X receptor-α (LXRα), an Mertk-inducing transcription factor, and that increased MerTK expression and efferocytosis in CaMKIIγ-deficient macrophages is dependent on LXRα. These findings identify a macrophage CaMKIIγ/ATF6/LXRα/MerTK pathway as a key factor in the development of necrotic atherosclerotic plaques.

Authors

Amanda C. Doran, Lale Ozcan, Bishuang Cai, Ze Zheng, Gabrielle Fredman, Christina C. Rymond, Bernhard Dorweiler, Judith C. Sluimer, Joanne Hsieh, George Kuriakose, Alan R. Tall, Ira Tabas

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Figure 6

CaMKII deficiency in macrophages disrupts an HDAC4/DACH1/ATF6 pathway that regulates LXRα and MerTK.

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CaMKII deficiency in macrophages disrupts an HDAC4/DACH1/ATF6 pathway th...
(A) Whole cell or nuclear extracts from control and CaMKII-KO macrophages were immunoblotted using antibodies against p-HDAC4 (pHDAC4), HDAC4, DACH1, β-actin, ATF6, and nucleophosmin (NPM). Left: representative immunoblot. Right: densitometry. *P < 0.05; **P < 0.01, unpaired t test. (B) Macrophage mRNA was quantified by RT-qPCR for Atf6. Data are presented as relative to the value for control macrophages. ***P < 0.001, unpaired t test. (C) Left: conserved ATF6 consensus sequence in intron 1 of the Nr1h3 (LXRα) gene. Right: macrophages from control and CaMKII-KO mice were subjected to ChIP analysis using anti-ATF6 or IgG control. The intronic region containing the ATF6-binding site and a nonconsensus sequence were amplified by RT-qPCR and normalized to input DNA. n = 4 biologic replicates. *P < 0.05, unpaired t test. (D) Macrophages were transfected with siRNA targeting ATF6 (siATF6) and analyzed for the indicated mRNAs as in Figure 5C. **P < 0.01; ***P < 0.001, 2-way ANOVA with post hoc Tukey’s analysis. (E–G) Macrophages were transfected with siATF6 as above and analyzed for Mertk mRNA, cell-surface MerTK by flow cytometry, and efferocytosis. ***P < 0.001, 2-way ANOVA with post hoc Tukey’s analysis. (H) RNA from aortic root sections was quantified for Atf6 mRNA as in Figure 5F. n = 5 mice per group. **P < 0.01, Mann-Whitney U test. (I) Human monocyte-derived macrophages were transfected with scrambled RNA or siRNA targeting CAMK2G and harvested 72 hours later for analysis of CAMK2G, ATF6, NR1H3 (LXRα), and MERTK. Dotted line represents the average value obtained with scramble RNA for each gene of interest. **P < 0.01; ***P < 0.001, unpaired t test. For all experiments, results are shown as mean + SEM. n = 3 experiments unless noted otherwise.