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 6

Rac1 downregulation in hyperpinocytotic macrophages is due to a disorder of CWC22-mediated mRNA splicing by CAPN6.

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Rac1 downregulation in hyperpinocytotic macrophages is due to a disorder...
BMMs differentiated with M-CSF/TNF-α for 4 days were used in these experiments. (A) Splicing of Rac1, Arhgef2, and Tpi1 pre-mRNA but not of Rhoa and Arhgdia pre-mRNA is upregulated by Capn6 deficiency. Spliced mRNA, pre-mRNA ratio served as a statistical value. (B) siRNA-based silencing of Cwc22 abrogates the upregulated Rac1 splicing caused by Capn6 deficiency. (C) Silencing of Cwc22 cancels the upregulation of Rac1 protein expression caused by Capn6 deficiency. One representative result of 3 independent experiments is shown. Con, control; Si, siRNA. (D) Impaired pinocytotic activity in Capn6–/yLdlr–/– BMMs is rescued by silencing of Cwc22. (E) Accelerated cellular motility in Capn6–/yLdlr–/– BMMs is diminished by silencing of Cwc22. BMMs were stimulated with CCL2 at 50 ng/ml. **P < 0.01; *P < 0.05, 1-way ANOVA followed by Bonferroni’s test (A, B, D and E); error bars represent mean ± SEM.