Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis
Biqing Wang, Xuening Tang, Liu Yao, Yuxin Wang, Zhipeng Chen, Mengqi Li, Naishi Wu, Dawei Wu, Xiangchen Dai, Hongfeng Jiang, Ding Ai
Biqing Wang, Xuening Tang, Liu Yao, Yuxin Wang, Zhipeng Chen, Mengqi Li, Naishi Wu, Dawei Wu, Xiangchen Dai, Hongfeng Jiang, Ding Ai
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Research Article Cardiology Immunology

Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis

Abstract

Subendothelial macrophage internalization of modified lipids and foam cell formation are hallmarks of atherosclerosis. Deubiquitinating enzymes (DUBs) are involved in various cellular activities; however, their role in foam cell formation is not fully understood. Here, using a loss-of-function lipid accumulation screening, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a factor that suppressed lipid uptake in macrophages. We found that USP9X expression in lesional macrophages was reduced during atherosclerosis development in both humans and rodents. Atherosclerotic lesions from macrophage USP9X-deficient mice showed increased macrophage infiltration, lipid deposition, and necrotic core content than control apolipoprotein E–KO (Apoe–/–) mice. Additionally, loss-of-function USP9X exacerbated lipid uptake, foam cell formation, and inflammatory responses in macrophages. Mechanistically, the class A1 scavenger receptor (SR-A1) was identified as a USP9X substrate that removed the K63 polyubiquitin chain at the K27 site. Genetic or pharmacological inhibition of USP9X increased SR-A1 cell surface internalization after binding of oxidized LDL (ox-LDL). The K27R mutation of SR-A1 dramatically attenuated basal and USP9X knockdown–induced ox-LDL uptake. Moreover, blocking binding of USP9X to SR-A1 with a cell-penetrating peptide exacerbated foam cell formation and atherosclerosis. In this study, we identified macrophage USP9X as a beneficial regulator of atherosclerosis and revealed the specific mechanisms for the development of potential therapeutic strategies for atherosclerosis.

Authors

Biqing Wang, Xuening Tang, Liu Yao, Yuxin Wang, Zhipeng Chen, Mengqi Li, Naishi Wu, Dawei Wu, Xiangchen Dai, Hongfeng Jiang, Ding Ai

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Figure 6

Mutation of the ubiquitylation site K27 of SR-A1 reduces foam cell formation and pro-inflammatory gene expression.

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Mutation of the ubiquitylation site K27 of SR-A1 reduces foam cell forma...
(A) Oil Red O staining of RAW264.7 cells stably overexpressing SR-A1-WT-EGFP or SR-A1-K27R-EGFP and transfected with siCtrl or siUsp9x for 48 hours followed by treatment with ox-LDL for 24 hours. Scale bar: 20 μm. (B) Quantification of intensity of staining in A. Two-way ANOVA with Bonferroni’s post hoc test (n = 5). (C) Representative images of Dil-ox-LDL uptake in RAW264.7 cells stably overexpressing SR-A1-WT or SR-A1-K27R and transfected with siCtrl or siUsp9x for 48 hours followed by treatment with Dil-ox-LDL (30 μg/mL) for 4 hours. Scale bar: 10 μm. (D) Quantification of Dil-ox-LDL fluorescence intensity. Two-way ANOVA with Bonferroni’s post hoc test (n = 5). (E) Quantitative PCR analysis of indicated gene expression in RAW264.7 cells stably overexpressing SR-A1-WT or SR-A1-K27R and treated with PBS or TNF-α (30 ng/mL) for 24 hours. Target gene expression was normalized to Actb mRNA levels. Two-way ANOVA with Bonferroni’s post hoc test (n = 5).