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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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 3

Loss of CAPN6 decelerates pinosome velocity and maturation and facilitates the leakage of pinocytotic particles in macrophages.

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Loss of CAPN6 decelerates pinosome velocity and maturation and facilitat...
(A) Pinosome maturity in Capn6-deficient BMMs. BMMs were fixed following pulsed application of TRITC-labeled dextran at 5 mg/ml for 30 minutes, and fluorescence intensity in individual pinosomes was measured. Scale bars: 5 μm. (B) Leakage of pinocytotic particles in BMMs. Spontaneous leakage of TRITC-labeled dextran for 24 hours was measured in the presence or absence of NSC23766 at 50 μmol/l. EE, early endosome; LE, late endosome; RE, recycling endosome; Lyso, lysosome. (C) Pinosome velocity in Capn6-deficient BMMs. Cells were pretreated with y-27632 at 10 μmol/l or NSC23766 at 50 μmol/l for 1 hour. **P < 0.01, Mann–Whitney U test (A) and 1-way ANOVA followed by Bonferroni’s test (B and C); error bars represent mean ± SEM.