Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing
Takuro Miyazaki, … , Hiroki Kurihara, Akira Miyazaki
Takuro Miyazaki, … , Hiroki Kurihara, Akira Miyazaki
Published August 15, 2016
Citation Information: J Clin Invest. 2016;126(9):3417-3432. https://doi.org/10.1172/JCI85880.
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Research Article Vascular biology

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

Abstract

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL–derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex–driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor–deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.

Authors

Takuro Miyazaki, Kazuo Tonami, Shoji Hata, Toshihiro Aiuchi, Koji Ohnishi, Xiao-Feng Lei, Joo-ri Kim-Kaneyama, Motohiro Takeya, Hiroyuki Itabe, Hiroyuki Sorimachi, Hiroki Kurihara, Akira Miyazaki

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Figure 1

Loss of CAPN6 reduces the pinocytotic uptake of native LDL in macrophages.

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Loss of CAPN6 reduces the pinocytotic uptake of native LDL in macrophage...
BMMs at day 4 of differentiation were used in these experiments. (A) Expression of calpain family members in BMMs in response to TNF-α stimulus. (B) Expression of Capn6 mRNA in BMMs in response to variety of inflammatory stimuli. BM cells were stimulated with a variety of cytokines (IL-1β, IL-4, IL-12, IL-17A, IFN-γ, TNF-α, 10 ng/ml; or CCL2, 50 ng/ml) together with M-CSF at 50 ng/ml for 4 days. (C) CAPN6 protein expression in TNF-α/M-CSF–primed BMMs. One representative result of 3 independent experiments is shown. (D) Subcellular distribution of CAPN6 in TNF-α/M-CSF–primed Capn6+/yLdlr–/– BMMs. Capn6–/yLdlr–/– BMMs served as a negative control. Scale bars: 10 μm (D); 3 μm (insets).(E) Cellular movements in BMMs. TNF-α/M-CSF–primed BMMs were tracked for 10 hours in the presence of CCL2 at 50 ng/ml. (F) Uptake of native LDL in TNF-α/M-CSF–primed Capn6+/yLdlr–/– or Capn6–/yLdlr–/– BMMs. Cells were treated with native LDL for 24 hours at the concentrations indicated, either in the presence of DMSO or nocodazole (Noco) at 1 μmol/l. (G) Temporal changes in the uptake of Dil-labeled LDL in TNF-α/M-CSF–primed BMMs. **P < 0.01; *P < 0.05, 1-way ANOVA followed by Bonferroni’s test (A and B), Mann–Whitney U test (E), 2-way ANOVA (F and G). Error bars represent the mean ± SEM.