Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing
Takuro Miyazaki, … , Hiroki Kurihara, Akira Miyazaki
Takuro Miyazaki, … , Hiroki Kurihara, Akira Miyazaki
Published September 1, 2016; First 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 2

Recovery of Rac1 by CAPN6 deficiency antagonizes pinocytotic activity in macrophages.

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Recovery of Rac1 by CAPN6 deficiency antagonizes pinocytotic activity in...
BMMs differentiated with M-CSF/TNF-α for 4 days were utilized in these experiments. (A) Expression of Rho GTPases and their regulatory molecules in Capn6+/yLdlr–/– or Capn6–/yLdlr–/– BMMs. Cells were stimulated with 10 ng/ml TNF-α for the indicated time periods. One representative result of 3 independent experiments is shown. (B) Rac1 activity in Capn6+/yLdlr–/– or Capn6–/yLdlr–/– BMMs. TNF-α–primed BMMs were stimulated with CCL2 at 50 ng/ml or native LDL at 400 μg/ml for 20 minutes. (C) Pinocytotic activity in Capn6-deficient BMMs. Cells were pretreated with y-27632 at 10 μmol/l or NSC23766 at 50 μmol/l for 1 hour. (D) Silencing of endogenous Rac1 protein by siRNA. One representative result of 3 independent experiments is shown. (E) Effects of Rac1 silencing on pinocytotic activity in Capn6-deficient BMMs. **P < 0.01; *P < 0.05, 1-way ANOVA followed by Bonferroni’s test (B, C and E); error bars represent mean ± SEM.