Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis
Biqing Wang, … , Hongfeng Jiang, Ding Ai
Biqing Wang, … , Hongfeng Jiang, Ding Ai
Published April 7, 2022
Citation Information: J Clin Invest. 2022;132(10):e154217. https://doi.org/10.1172/JCI154217.
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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 7

K63-linked ubiquitination of SR-A1 promotes its internalization.

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K63-linked ubiquitination of SR-A1 promotes its internalization. (A) Wes...
(A) Western blot analysis of internalized biotinylated proteins in BMDMs from WT mice treated with or without WP1130 for 24 hours before surface receptor biotinylation and internalization (see Supplemental Methods). (B) Quantification of SR-A1 internalization in A. Two-way ANOVA with Bonferroni’s post hoc test (n = 5). (C) Flow cytometric analysis of surface expression of SR-A1 protein by cells after ox-LDL stimulation for the indicated times. Two-way ANOVA with Bonferroni’s post hoc test (n = 5). (D) Immunofluorescence analysis of RAW264.7 cells stably overexpressing SR-A1-WT-EGFP or SR-A1-27R-EGFP and transfected with control or Usp9x siRNA for 48 hours before incubation with ox-LDL for 30 minutes to induce internalization. Colocalization of SR-A1 (EGFP, green) and EEA1 (red) is shown (left) and quantified (right). Two-way ANOVA with Bonferroni’s post hoc test (n = 5). Scale bar: 10 μm. (E) Western blot analysis of the indicated proteins in RAW264.7 cells stably overexpressing SR-A1-WT-EGFP and treated with or without WP1130 for 24 hours before incubation with ox-LDL for 30 minutes to induce internalization and IP of whole cell lysates with MYC magnetic beads (n = 5). (F) Western blot analysis of the indicated proteins in RAW264.7 cells stably overexpressing SR-A1-WT-EGFP or SR-A1-K27R-EGFP and transfected with siCtrl or siUsp9x for 48 hours before incubation with ox-LDL for 30 minutes to induce internalization and IP of whole cell lysates with MYC magnetic beads (n = 5).