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 4

Nuclear localization of CWC22 in inflamed murine BMM is limited by the physical association between CAPN6 and CWC22.

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Nuclear localization of CWC22 in inflamed murine BMM is limited by the p...
(A) CAPN6 associates with CWC22. Protein immunoprecipitates were detected by immunoblotting against CWC22 or GFP. One representative result of 3 independent experiments is shown. (B) Capn6 deficiency potentiates nuclear localization of CWC22 in BMMs. After 20 minutes of a second administration of TNF-α/M-CSF, nuclear and cytoplasmic fractions were separated, and CWC22 was detected by immunoblotting. One representative result of 3 independent experiments is shown. N, nucleus; C, cytosol. (C) Nuclear localization of CWC22 in BMMs was induced by a variety of inflammatory cytokines. TNF-α–primed Capn6–/yLdlr–/– BMMs were stimulated with a variety of cytokines for 20 minutes, and nuclear localization in individual cells was quantified. (D) Subcellular distribution of CWC22 in BMMs. BM cells were stimulated with M-CSF at 50 ng/ml in the presence or absence of TNF-α for 4 days; then CWC22 and CAPN6 were detected by immunocytochemistry. (E) Capn6 deficiency potentiates nuclear localization of CWC22 in BMMs. BM cells were cultured in the presence of TNF-α at 10 ng/ml and M-CSF at 50 ng/ml for 3 days; then the culture medium was replaced with freshly prepared TNF-α/M-CSF–supplemented medium. Following 30-minute incubation, cells were fixed and CWC22 was detected by immunocytochemistry. Arrows represent nuclear localization of CWC22. **P < 0.01, 1-way ANOVA followed by Bonferroni’s test (C); error bars represent mean ± SEM. Scale bars: 10 μm (D); 10 μm (E).