Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis
Canan Kasikara, … , Muredach P. Reilly, Ira Tabas
Canan Kasikara, … , Muredach P. Reilly, Ira Tabas
Published February 25, 2021
Citation Information: J Clin Invest. 2021;131(8):e145275. https://doi.org/10.1172/JCI145275.
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Research Article Cardiology Cell biology

Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis

Abstract

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte–derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet–fed Ldlr–/– mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps — all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Authors

Canan Kasikara, Maaike Schilperoort, Brennan Gerlach, Chenyi Xue, Xiaobo Wang, Ze Zheng, George Kuriakose, Bernhard Dorweiler, Hanrui Zhang, Gabrielle Fredman, Danish Saleheen, Muredach P. Reilly, Ira Tabas

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Figure 5

PHACTR1 sequesters PP1α in the nucleus and decreases the dephosphorylation of MLC during efferocytosis.

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PHACTR1 sequesters PP1α in the nucleus and decreases the dephosphorylati...
(A) Immunoblot of PHACTR1 (~72 kDa), PP1α (38 kDa), and laminin B (input control; 68 kDa) of anti-PP1α immunoprecipitates from BMDMs incubated in the absence or presence of ACs. (B–D) Phactr1+/+ and Phactr1–/– BMDMs treated with scrambled RNA or PP1α siRNA. One set of cells was immunoblotted for PPA1α (38 kDa) and GAPDH (36 kDa), one set was stained with phospho- and total MLC antibody and quantified as phospho- to total MLC MFI ratio, and one set was assayed for efferocytosis. (E) Quantification of nuclear and cytoplasmic PHACTR1 in BMDMs incubated in the absence or presence of ACs. (F) Quantification of nuclear and cytoplasmic PP1α in Phactr1+/+ and Phactr1–/– BMDMs incubated in the absence or presence of ACs. (G) Immunofluorescence microscopy images of BMDMs incubated in the absence or presence of ACs (green, PHACTR; yellow, PP1α; red, AC; blue, DAPI [nucleus]). (H) Graphic scheme of PHACTR1 WT and mutant protein structure and immunoblots of Myc (PHACTR1 tag) and GAPDH in Phactr1–/– BMDMs transfected with WT and mutant PHACTR1 (Myc-tagged PHACTR1 protein is ~73 kDa in WT and ΔB1, and ~70 kDa in ΔC). (I–K) Quantification of nuclear and cytoplasmic PHACTR1 and PP1α and efferocytosis in the BMDMs depicted in H. In C–F and I–K, mean ± SEM, including individual data points; n = 3 experiments, with n > 5–10 macrophages quantified for each group; **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed Student’s unpaired t test (C–F), 1-way ANOVA with post hoc Dunnet’s analysis compared with WT (I), or 2-way ANOVA with post hoc Tukey’s analysis, compared with mock (J and K).