Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFR<strong>β</strong>

doi:10.1007/s11684-024-1056-8

Front. Med.

2024, Vol. 18

Issue (3) : 465-483 https://doi.org/10.1007/s11684-024-1056-8

Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ

Fei Xu^1,^2,^3,⁴, Han Chen^2,^3,⁴, Changyi Zhou^2,^3,⁴, Tongtong Zang^2,^3,⁴, Rui Wang^2,^3,⁴, Shutong Shen^2,^3,⁴, Chaofu Li^2,^3,⁴, Yue Yu^2,^3,⁴, Zhiqiang Pei^2,^3,⁴, Li Shen^2,^3,⁴(

), Juying Qian^2,^3,⁴(

), Junbo Ge^2,^3,⁴(

)

¹. Department of Cardiology and Laboratory of Heart Valve Disease, West China Hospital, Sichuan University, Chengdu 610041, China
². Department of Cardiology, Zhongshan Hospital, Fudan University, Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Shanghai 200032, China
³. Shanghai Institute of Cardiovascular Diseases, Shanghai 200032, China
⁴. National Clinical Research Center for Interventional Medicine & Shanghai Clinical Research Center for Interventional Medicine (19MC1910300), Shanghai 200032, China

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Abstract

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe^−/− mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβ with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRβ. Inhibiting OTUB1 in VSMCs could promote PDGFRβ degradation via the ubiquitin–proteasome pathway, so it was beneficial in preventing VSMCs’ phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs’ phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Keywords atherosclerosis vascular smooth muscle cell ubiquitylation deubiquitinase OTUB1 PDGFRβ

Corresponding Author(s): Li Shen,Juying Qian,Junbo Ge

Just Accepted Date: 12 March 2024 Online First Date: 22 April 2024 Issue Date: 17 June 2024

Cite this article:

Fei Xu,Han Chen,Changyi Zhou, et al. Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ[J]. Front. Med., 2024, 18(3): 465-483.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-024-1056-8
https://academic.hep.com.cn/fmd/EN/Y2024/V18/I3/465

Fig.1 OTUB1 facilitates the phenotype switch of vascular smooth muscle cell (VSMC). (A) Schematic illustration of in vitro RNAi screening of the effects of OTU family deubiquitinases on VSMC phenotype switch induced by PDGF-BB. PDGF-BB: platelet-derived growth factor BB. (B) Quantitative RT-PCR (qRT-PCR) results confirmed that the knockdown efficiency of siRNAs targeting OTU deubiquitinases was over 70%. Data were normalized to GAPDH and compared with the control group. (C–F) HASMCs were transfected with siRNAs targeting OTUs and treated with PDGF-BB or not for 48 h. Results of qRT-PCR analysis for the relative mRNA expression of TAGLN (C), ACTA2 (D), OPN (E), and BMP2 (F) after 48 h of PDGF-BB treatment are shown. Data were normalized to GAPDH and shown as mean values ± SEM of three independent experiments. NC: normal control, NS: not significant. For comparisons with the si-NC and PDGF-BB (-) groups: **, P < 0.01; ***, P < 0.001. For comparisons with the si-NC and PDGF-BB (+) groups: #, P < 0.05; ##, P < 0.01; ###, P < 0.001.

Fig.2 Mass spectrometry analysis on the impact of OTUB1 deficiency in protein ubiquitylation in HASMCs. (A) Schematic illustration of HASMC sample collection and mass spectrometry (MS) analysis on ubiquitylated protein. (B) Volcano map showing the differentially ubiquitylated proteins. (C) Modification motif (MoMo) analysis of the ubiquitylated sites on the differentially ubiquitylated proteins. (D) Heatmap of the 40 proteins with the most significantly increased and decreased ubiquitylation levels. Genes implicated in atherosclerosis from previous research were highlighted. (E, F) GO analyses of genes encoding the proteins with significantly decreased (E) or increased (F) ubiquitylation levels from MS results.

Fig.3 OTUB1 is required for the PDGF-BB-induced proliferation, phenotypic switch, and migration of HASMC. (A–C) Representative EdU staining images (A), Ki-67 immunofluorescence images (B), and quantitative analyses of EdU- or Ki-67-positive cells (C) in HASMCs transfected with control siRNA (si-NC) and siRNA targeting OTUB1 (si-OTUB1) and treated with PDGF-BB (n = 3 independent experiments). (D) CCK-8 results of HASMCs showing the proliferation of cells decreased after knocking down OTUB1. Data are shown as relative to the control/si-NC group (n = 3 independent experiments). (E) qRT-PCR analysis for the relative mRNA expression of phenotype markers (ACTA2, TAGLN, and OPN) in HASMCs. The qRT-PCR data were normalized to GAPDH and shown as mean values ± SEM of three independent experiments. (F, G) Representative images (F) and quantification (G) of migrated cells transfected with si-NC or si-OTUB1 per microscopic field from Boyden chamber transwell migration assay. (H–J) RNA sequencing results of OTUB1 knockdown HASMCs compared with control cells. (H) Volcano map of significantly differentially expressed genes (DEGs). GO (I) and KEGG (J) analyses were performed based on the differentially expressed genes. NC: normal control, NS: not significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig.4 OTUB1 restrains proteasomal degradation of PDGFRβ. (A–C) qRT-PCR results of PDGFRβ (A), SLC3A2 (B), and SLC7A5 (C) mRNA expression in HASMCs transfected with si-OTUB1 or normal control siRNA (si-NC) (n = 3 independent experiments). Data were normalized to GAPDH and shown as mean values ± SEM. (D, E) Representative images and quantification of Western blot analyses of PDGFRβ, SLC3A2, and SLC7A5 protein levels in HASMCs transfected with si-OTUB1 or si-NC (D) and HASMCs infected with OTUB1-overexpression or normal control lentivirus (E) (n = 4 independent experiments). Data were normalized to GAPDH, and all were compared with their own group’s si-NC/control. (F) Representative Western blot images on protein from HASMCs transfected with si-OTUB1 or normal control (si-NC), which were later immunoprecipitated with PDGFRβ, showing that OTUB1 influenced total and K48-linked ubiquitylation. (G) Quantification of ubiquitin expression levels in samples immunoprecipitated by PDGFRβ. Immunoprecipitated PDGFRβ was used as an internal control. Data were compared with the corresponding si-NC group (n = 3 independent experiments). (H) Quantification of PDGFRβ and OTUB1 expression in input samples from Fig. 4F (n = 3 independent experiments). GAPDH was used as an internal control. Data were normalized to the si-NC group. (I, J) Western blot analyses of PDGFRβ and OTUB1 in HASMCs treated with CHX for 0–8 h in HASMCs transfected with si-OTUB1 or normal control siRNA (I) and HASMCs infected with OTUB1-OE or NC lentivirus (J). PDGFRβ expression levels were normalized to GAPDH, and all were compared with the 0 h level of their own group. Data were shown as mean values ± SEM of three independent experiments. (K, L) Western blot analyses and quantification of PDGFRβ in HASMCs treated with DMSO (K) or MG132 (L) at 15–36 h after transfection with si-OTUB1 or normal control in HASMCs. PDGFRβ expression was normalized to GAPDH, and all were compared with the 0 h level of their own group. Data were shown as mean values ± SEM of three independent experiments. NC: normal control; OE: overexpression; NS: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig.5 OTUB1 specifically interacts with and regulates PDGFRβ through K48-linked ubiquitylation. (A, B) Co-IP assays showed that OTUB1 and PDGFRβ could be coimmunoprecipitated in vitro. (C) Co-IP assays showed that depleting c-CBL prevented the interaction between OTUB1 and PDGFRβ. (D) Western blot analyses of PDGFRβ expression in HASMCs treated with CHX for 0–8 h. HASMCs were transfected with WT- or K707R mutated-PDGFRβ plasmids after depleting endogenous PDGFRβ (n = 3 independent experiments). PDGFRβ expression levels were normalized to GAPDH, and all were compared with the 0 h level of their own group. (E) Representative IP results showed that K707 mutation decreased the ubiquitylation of PDGFRβ and abrogated OTUB1’s deubiquitinating effects on PDGFRβ. (F) Quantification of ubiquitin levels in PDGFRβ-immunoprecipitated samples, showing that the K707 site in PDGFRβ was necessary for OTUB1’s deubiquitylation on PDGFRβ. Data were normalized to immunoprecipitated PDGFRβ and compared with the WT/control group (n = 3 independent experiments). (G) Schematic of mutated OTUB1 constructs. (H) Representative Western blot images of protein immunoprecipitated with PDGFRβ showed that the catalytic triad was necessary for the deubiquitinating effects of OTUB1 on PDGFRβ. (I) Quantification of relative ubiquitin levels in PDGFRβ-immunoprecipitated samples, showing that OTUB1 deubiquitinated PDGFRβ with its catalytic triad. Data were normalized to immunoprecipitated PDGFRβ and compared with the WT group (n = 3 independent experiments). WT: wild type; NC: normal control; KD: knockdown.

Fig.6 PDGFRβ is necessary for OTUB1-mediated HASMC phenotype switching. (A, B) Representative EdU staining images (A) and quantitative analyses of EdU-positive cells (B) in PDGF-BB-treated HASMC. (C, D) Representative images (C) and quantification (D) of Western blot on PDGF-BB-treated HASMCs showed that overexpressing PDGFRβ ameliorated the expression changes of contractile markers (TAGLN and ACTA2) and a synthetic marker (OPN). Data were normalized to GAPDH and compared with the si-NC/control level of its own group. (E) qRT-PCR results on PDGF-BB-treated HASMCs confirmed the changes in the expression levels of TAGLN, ACTA2, and OPN. GAPDH was used as the internal control (n = 3 independent experiments). (F, G) Representative images (F) and quantification (G) of migrated cells per microscopic field in Boyden chamber transwell migration assay (n = 3 independent experiments). (H, I) Representative images (H) and quantification (I) of migrated cells per microscopic field in cell scratch assay (n = 3 independent experiments). NC: normal control; OE: overexpression; NS: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig.7 OTUB1 knockdown prevents advanced atherogenesis and stabilizes atherosclerotic plaque in vivo. (A) Schematic illustration of animal experiments on Apoe^−/− mice. (B, C) Representative Western blot images (B) and quantification (C) of PDGFRβ and OTUB1 in control-AAV9 or Otub1-shRNA-AAV9 treated early- and late-stage atherosclerotic mice aortas. GAPDH was used as the internal control (n = 6 per group). (D, E) Representative images (D) and quantification (E) of Oil-Red O staining in atherosclerotic aortic roots (n = 6–9 per group). (F, G) Representative images of Masson’s trichrome staining (F) and collagen quantification (G) in atherosclerotic aortic roots (n = 7–10 per group). (H) Quantification of fibrous cap area proportion and average cap thickness in aortic roots in Masson’s trichrome staining (n = 7–8 per group). (I, J) Representative images of H&E staining (I) and quantification of necrotic core area percentages (J) in atherosclerotic aortic roots (n = 7–10 per group). NC: necrotic core. (K, L) Representative images (K) and quantification (L) of F4/80 immunofluorescence staining in early- and late-stage atherosclerotic aortic roots, indicating macrophage infiltration (n = 6 per group). Plaque was indicated by dotted lines. (M) Plaque vulnerability index in early- and late-stage atherosclerotic aortic roots (n = 6 per group). KD: knock down; NS: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. ORO: Oil-Red O.

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