USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101258
ISSN1083-351X
AutoresXiaohong Xia, Xiaolin Liu, Renjie Chai, Qiong Xu, Zhenyu Luo, Jielei Gu, Yangshuo Jin, Tumei Hu, Cuifu Yu, Bijun Du, Hongbiao Huang, Wenchao Ou, Shiming Liu, Ningning Liu,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThe underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression. The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression. Cardiovascular diseases are one of the most common causes of death worldwide and mainly arise due to atherogenesis (1Cannon B. Cardiovascular disease: Biochemistry to behaviour.Nature. 2013; 493: S2-S3Crossref PubMed Scopus (47) Google Scholar, 2Glass C.K. Witztum J.L. Atherosclerosis. The road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2526) Google Scholar). For the treatment of atherogenesis, some surgical procedures, such as stenting, angioplasty, atherectomy, and bypass surgery, are indispensable methods. However, mechanical-injury-induced restenosis is the major threat to the prognosis of these patients (3Weintraub W.S. The pathophysiology and burden of restenosis.Am. J. Cardiol. 2007; 100: 3K-9KAbstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A large number of studies have reported that arterial neointima formation is a complicated pathological process caused by vascular smooth muscle cells (VSMCs) dedifferentiation, proliferation, migration, and secretion of extracellular matrix; these are the hallmarks of restenosis (4O' Brien E.R. Ma X. Simard T. Pourdjabbar A. Hibbert B. Pathogenesis of neointima formation following vascular injury.Cardiovasc. Hematol. Disord. Drug Targets. 2011; 11: 30-39Crossref PubMed Scopus (49) Google Scholar). The proliferation of VSMCs from the media into the intima leads to neointima formation or intimal hyperplasia. This progression is rare in normal adult arteries in response to stimuli. Notably, VSMC proliferation underlies venous graft failure, the development of postangioplasty restenosis, and transplant arteriosclerosis. Hence, developing strategies targeting VSMC proliferation is essential. Several factors are involved in the progression of VSMC proliferation, but the final common pathway is cell cycle (5Dzau V.J. Braun-Dullaeus R.C. Sedding D.G. Vascular proliferation and atherosclerosis: New perspectives and therapeutic strategies.Nat. Med. 2002; 8: 1249-1256Crossref PubMed Scopus (698) Google Scholar). Focus on cell cycle is a promising strategy in patients with surgical procedures. The different phases of the cell cycle are controlled by a series of protein complexes composed of cyclin-dependent kinase inhibitors and catalytic cyclin-dependent kinases (CDKs) (6Tanner F.C. Yang Z.Y. Duckers E. Gordon D. Nabel G.J. Nabel E.G. Expression of cyclin-dependent kinase inhibitors in vascular disease.Circ. Res. 1998; 82: 396-403Crossref PubMed Scopus (235) Google Scholar). In vivo injury or addition of growth factors allows G1/S phase transition via activation of CDK complexes (CDK2, CDK4, and CDK6) and downregulating CDKIs (p21, p27, and p57) (6Tanner F.C. Yang Z.Y. 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Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation.Circulation. 2000; 101: 2022-2025Crossref PubMed Scopus (207) Google Scholar). p27 protein can be polyubiquitinated and degraded by E3 ligases and proteasome (10Nakayama K.I. Nakayama K. Ubiquitin ligases: Cell-cycle control and cancer.Nat. Rev. Cancer. 2006; 6: 369-381Crossref PubMed Scopus (1083) Google Scholar). Skp2, an F-box component of SCFSkp2 ubiquitin ligase, is closely related to and mediates the polyubiquitination of p27 (11Carrano A.C. Eytan E. Hershko A. Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1312) Google Scholar). Reportedly, Skp2 is highly expressed in isolated VSMCs. Skp2 deletion in VSMCs inhibits cell proliferation by increasing p27 levels. The latest evidence showed that Skp2 is a direct player in VSMC proliferation in vivo to affect the final neointima thickening (12Bond M. Sala-Newby G.B. Newby A.C. Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability. A novel mechanism regulating smooth muscle cell proliferation.J. Biol. Chem. 2004; 279: 37304-37310Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Bond M. Wu Y.J. Sala-Newby G.B. Newby A.C. Rho GTPase, Rac1, regulates Skp2 levels, vascular smooth muscle cell proliferation, and intima formation in vitro and in vivo.Cardiovasc. Res. 2008; 80: 290-298Crossref PubMed Scopus (48) Google Scholar, 14Wu Y.J. Bond M. Sala-Newby G.B. Newby A.C. Altered S-phase kinase-associated protein-2 levels are a major mediator of cyclic nucleotide-induced inhibition of vascular smooth muscle cell proliferation.Circ. Res. 2006; 98: 1141-1150Crossref PubMed Google Scholar, 15Wu Y.J. Sala-Newby G.B. Shu K.T. Yeh H.I. Nakayama K.I. Nakayama K. Newby A.C. Bond M. S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo.J. Vasc. Surg. 2009; 50: 1135-1142Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). VSMC plasticity implies rapid change and adaptability through protein turnover. Importantly, the ubiquitin-proteasome system (UPS) plays a critical role in protein turnover, i.e., degradation of proteins related to differentiation, apoptosis, cell-cycle regulation, and signaling (16Demasi M. Laurindo F.R. Physiological and pathological role of the ubiquitin-proteasome system in the vascular smooth muscle cell.Cardiovasc. Res. 2012; 95: 183-193Crossref PubMed Scopus (34) Google Scholar). UPS also interacts with VSMC proliferation. Inhibiting proteasome remarkably induces VSMC apoptosis and inhibits neointima. The proteasome inhibitors also sensitize VSMC to increased ER stress, triggering cell death (17Marciniak S.J. 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CHIP represses myocardin-induced smooth muscle cell differentiation via ubiquitin-mediated proteasomal degradation.Mol. Cell. Biol. 2009; 29: 2398-2408Crossref PubMed Scopus (61) Google Scholar). Thus, UPS is a vital effector in VSMC proliferation. However, it contains approximately 100 deubiquitinases (DUBs); whether DUBs have a role in VSMC proliferation is yet elusive. Therefore, in this study, we aimed to investigate whether DUB regulates VSMC proliferation and found that ubiquitin-specific protease 10 (USP10) inhibition reduced VSMC proliferation by promoting Skp2 degradation in vitro and in vivo. Firstly, we performed complete carotid ligation for 21 days. Hematoxylin-eosin (HE) results showed neointima formation in a vascular injury model of artery ligation (Fig. 1A). Next, we examined the USP10 level in the carotid. Compared with normal arteries, the level of USP10 was upregulated with complete ligation in carotid arteries. Notably, the expression was more in the neointima than the media in the injured carotid artery. DAPI (blue) represents the expression in the nucleus and α-SMA (green) in the cytoplasm of SMCs. Subsequently, we found that USP10 was located more in the cytoplasm than in the nucleus (Fig. 1B). Immunohistochemical analysis indicated that the expression of USP10 was higher in neointima (Fig. 1C). These findings suggested that USP10 expression was increased in SMCs during neointima formation. In addition to the model of carotid ligation, we tested the expression of USP10 in human aortic smooth muscle cell (HASMCs). HASMCs are treated with a potent stimulus of VSMCs (PDGF-BB), and RT-qPCR results showed that USP10 mRNA level was increased after 12 h of stimulation (Fig. 1D). Next, we assessed the level of USP10 protein in HASMCs and A7r5 cells and found that it was expressed in the cytoplasm of the mouse carotid tissue and was increased following PDGF-BB stimulation (Fig. 1E). The expression of USP10 protein started to upregulate understimulation and remained elevated with prolonged treatment (Fig. 1F). VSMCs proliferation is a necessary process for neointima formation (21Lacolley P. Regnault V. Nicoletti A. Li Z. Michel J.B. The vascular smooth muscle cell in arterial pathology: A cell that can take on multiple roles.Cardiovasc. Res. 2012; 95: 194-204Crossref PubMed Scopus (455) Google Scholar). To detect whether USP10 is involved in VSMCs proliferation, cell viability was assessed using MTS assay in HASMCs and A7r5 cells. USP10 inhibitor (Spautin-1) decreased the viability of VSMCs in a time- and dose-dependent manner (Fig. 2A). Moreover, we employed USP10 siRNA to determine cell viability, and the results showed that it was inhibited in HASMCs (Fig. 2, B and C). Since Spautin-1 affects the activity of USP10 and USP13, we determined whether USP13 is involved in the effect of Spautin-1 in cell viability. MTS assay indicated that USP13 knockdown inhibited cell viability (Fig. S1, A and B). To investigate the effect of Spautin-1, we knocked down USP13 and combined with Spautin-1 treatment. The results confirmed that USP13 deletion enhanced Spautin-1 induced-cell growth inhibition and Skp2 downregulation, suggesting that the effect of Spautin-1 did not depend on USP13 (Fig. S1, C and D). Furthermore, PDGF-BB stimuli also promoted cell viability. Under the treatment of PDGF-BB, USP10 inhibition decreased cell viability in VSMCs (Fig. 2D). To evaluate the long-term antiproliferation effect of USP10 deletion, colony formation assays were conducted. We found that the cell number of the colony was decreased after USP10 inhibition (Fig. 2, E and F). Moreover, EdU staining assays showed that USP10 inhibitor-induced DNA replication capacity downregulated with or without PDGF-BB stimuli (Fig. 2, G and H). Moreover, flow cytometry analysis indicated that USP10 deletion inhibited BrdU incorporation into cells, suggesting that USP10 inhibition/knockdown induced cell proliferation inhibition (Fig. 2, I–L). Since USP10 promotes VSMC proliferation, we speculated that it affects cell cycle progression. To investigate whether USP10 mediates cell cycle transition, flow cytometry was employed. As expected, Spautin-1 blocked cell cycle transition from G0/G1 to S phase in HASMCs and A7r5 cells (Figs. 3, A and B and S2A). USP10 knockdown using USP10 siRNA also induced cell cycle arrest (Figs. 3D and S2B). Additionally, the proteins related to the cell cycle were detected. Western blot assay showed that USP10 inhibitor or siRNA decreased the expression of p-Rb, CDK4, and Cyclin D1 and increased the expression of p27 (Fig. 3, C and E). Reportedly, VSMC migration plays a critical role in neointima formation (21Lacolley P. Regnault V. Nicoletti A. Li Z. Michel J.B. The vascular smooth muscle cell in arterial pathology: A cell that can take on multiple roles.Cardiovasc. Res. 2012; 95: 194-204Crossref PubMed Scopus (455) Google Scholar). However, cell apoptosis could not be affected by USP10 (Fig. 3, F and G). To examine the effect of USP10 on VSMC migration, we performed scratch and transwell migration assays. The migration of VSMCs was significantly inhibited by USP10 loss (Fig. 3, H–N). Western blot assay demonstrated that MMP2 was downregulated by Spautin-1, suggesting that USP10 inhibition reduced cell migration (Fig. 3O). These findings indicated that USP10 promoted cell proliferation via regulation of cell cycle progression and migration in both treatments with and without PDGF-BB but did not trigger cell apoptosis. In a previous study, we reported that as a DUB, USP10 stabilizes Skp2 protein expression in cancer (22Liao Y. Liu N. Xia X. Guo Z. Li Y. Jiang L. Zhou R. Tang D. Huang H. Liu J. USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia.Cell Discov. 2019; 5: 24Crossref PubMed Scopus (36) Google Scholar). Considering that Skp2 regulates neointima formation, we investigated whether USP10 mediated-VSMC proliferation is regulated by Skp2 expression. Western blot assay showed that Spautin-1 downregulated the level of Skp2 protein in HASMCs and A7r5 cells. Under the treatment of PDGF-BB, Skp2 protein level was increased. In addition, increased PDGF-BB-induced Skp2 was inhibited by Spautin-1 (Fig. 4A). The Skp2 expression was also reduced after USP10 knockdown in the treatment with and without PDGF-BB, excluding the off-target effects of Spautin-1 (Fig. 4B). Next, we evaluated USP10-induced-Skp2 stability using cycloheximide (CHX). CHX treatment decreased the expression of Skp2 protein in a time-dependent manner. Spautin-1 addition promoted Skp2 protein decrease (Fig. 4, C and D). In addition to USP10 inhibitor, USP10 siRNA rapidly degraded Skp2 protein, suggesting that USP10 is essential for Skp2 stability (Fig. 4, E and F). DUBs regulate their substrate proteins via protein–protein interaction and posttranslation. We found that Skp2 protein was regulated by USP10; however, the specific correlations are unknown. Firstly, we used coimmunoprecipitation (Co-IP) assay to detect the interaction between USP10 and Skp2. The results showed that USP10 interacts with Skp2 protein in HASMCs, and Skp2 also binds with USP10 (Fig. 5A). Moreover, HEK293T transfected with FLAG-USP10 and MYC-Skp2 were subjected to Co-IP and Western blot assays. We found that exogenic MYC-tagged Skp2 interacted with FLAG-tagged USP10, which confirmed a direct interaction between USP10 and Skp2 protein (Fig. 5B). The confocal assay showed that Skp2 was mainly located in the nucleus, while cytoplasm exhibited only a slight expression. Therefore, USP10 was colocalized with Skp2 in the cytoplasm of HASMCs. (Fig. 5C). The localization of Skp2 and USP10 was observed in the carotid artery after ligation. Consistently, the colocalization of USP10 and Skp2 was detected in the cytoplasm of the artery tissue (Fig. 5D). Next, we hypothesized that USP10 mediates the posttranslation of Skp2. In cancer, USP10 decreased Skp2 ubiquitination, and we assessed the level of poly-ubiquitinated Skp2 using Co-IP assay (22Liao Y. Liu N. Xia X. Guo Z. Li Y. Jiang L. Zhou R. Tang D. Huang H. Liu J. USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia.Cell Discov. 2019; 5: 24Crossref PubMed Scopus (36) Google Scholar). The results showed that the inhibition or silencing of USP10 increased the level of ubiquitinated Skp2 remarkably (Fig. 5, E and F). USP10 had a proproliferative effect on VSMCs and stabilized Skp2 protein expression. To study the regulatory mechanism of USP10 on VSMCs, we tested the effects of USP10 and Skp2 on cell growth and found that USP10 overexpression using lentivirus containing human USP10 induced G0/G1 to S phase progression (Figs. 6A and S3A). Western blot assay assessed the proteins associated with cell cycle. Cyclin-dependent kinase inhibitor, p27, was decreased, and Skp2 expression was increased by overexpressing USP10 in HASMCs (Fig. 6B). In addition, lentivirus containing human Skp2 was employed to transfect HASMCs, followed by MTS and colony formation analysis. The results showed that overexpression of Skp2 promoted cell proliferation (Fig. 6, C–E). To determine whether USP10-induced the biological effect depended on Skp2 status, HASMCs were transfected with lentivirus containing human Skp2 and then treated with USP10 inhibitor (Spautin-1). Skp2 overexpression abrogated the cells from USP10 inhibition-triggered G1 arrest. The altered p27 and Cyclin D1 proteins were rescued after overexpressing Skp2 (Figs. 6, F and G and S3B). Meanwhile, Skp2 rescued USP10 siRNA induced-cell cycle arrest. (Figs. 6, H and I and S3C). USP10 was upregulated in VSMCs after carotid ligation, and whether USP10 is a promotor in neointima formation under vascular injury was elucidated. Thus, we established the mice model of carotid ligation. USP10 inhibitor (Spautin-1) was employed to treat the mice. As shown in Figure 7A, USP10 inhibition significantly decreased neointima formation and reduced intima/media rations (Fig. 7B). In addition, we observed that their vitality was influenced by blood pressure (BP) and heart rate, which were associated with vascular remodeling but did not differ markedly. The body weight was also similar between the control and treatment groups after 3-week complete carotid ligation (Fig. 7C). Immunohistochemistry (IHC) results showed that USP10 inhibition inhibited Skp2 expression in the model of carotid ligation. Also, USP10 ablation in SMCs decreased injury-induced upregulation of MMP2 (Fig. 7D). Collectively, these data suggested that USP10 plays a role during restenosis. To further investigate the effect of USP10, we applied EGFP-tagged AAV-USP10 shRNA to knockdown USP10 expression in vivo. Immunofluorescence analysis demonstrated the success of USP10 deletion in mice (Fig. 7, E and F). Intima areas indicated that USP10 deficiency inhibited neointimal formation (Fig. 7, G and H); however, no significant difference was detected between the two groups (Fig. 7I). Neointima formation commonly occurs during vein bypass graft failure, arterial restenosis development, and atherosclerosis (5Dzau V.J. Braun-Dullaeus R.C. Sedding D.G. Vascular proliferation and atherosclerosis: New perspectives and therapeutic strategies.Nat. Med. 2002; 8: 1249-1256Crossref PubMed Scopus (698) Google Scholar). However, the underlying molecular mechanisms remain to be elucidated in this complicated pathophysiological progress. In this study, we identified USP10-Skp2 axis and investigated the role of USP10 in vascular remodeling. USP10 expression was highly expressed in the carotid artery of intimal hyperplasia compared with the normal carotid artery. Skp2 was essential for USP10-induced vascular proliferative pathology. These findings indicated a critical role of USP10 in orchestrating a complex pathological process of vascular restenosis (Fig. 8). Since its discovery, ubiquitin-specific peptidase 10 (USP10) has been explored primarily in the UPS system with respect to its deubiquitinase activity in cancer progression (23Takayama K.I. Suzuki T. Fujimura T. Takahashi S. Inoue S. Association of USP10 with G3BP2 inhibits p53 signaling and contributes to poor outcome in prostate cancer.Mol. 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Over the past decade, the role of protein degradation by UPS gained further support in regulating the transition from contractile to proliferative smooth muscle cell phenotype. Several E3-ligases, Skp2, CHIP, and β-Trcp, regulate VSMC function via promoting protein degradation (11Carrano A.C. Eytan E. Hershko A. Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1312) Google Scholar, 20Xie P. Fan Y. Zhang H. Zhang Y. She M. Gu D. Patterson C. Li H. CHIP represses myocardin-induced smooth muscle cell differentiation via ubiquitin-mediated proteasomal degradation.Mol. Cell. Biol. 2009; 29: 2398-2408Crossref PubMed Scopus (61) Google Scholar, 26Wang X. Adhikari N. Li Q. Guan Z. Hall J.L. The role of [beta]-transducin repeat-containing protein ([beta]-TrCP) in the regulation of NF-[kappa]B in vascular smooth muscle cells.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 85-90Crossref PubMed Scopus (30) Google Scholar). However, DUBs play key roles in the progress of protein degradation. Next, we explored whether there is DUB involved in vascular remodeling. In a recent study, we showed that USP10 triggered the proliferation in chronic myeloid leukemia. Surprisingly, we also identified that USP10 regulated VSMC proliferation. While exploring the effect and expression of USP10 in VSMCs, we used α-SMA as a marker for the identification of SMCs. The gene is expressed in adult SMCs and is considered a classic marker for the recognition of differentiated SMCs (27Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2409) Google Scholar, 28Rensen S.S. Doevendans P.A. van Eys G.J. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.Neth. 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The current results strongly suggested that USP10 is a key promotor in neointima formation. VSMCs proliferation and migration are critical steps in intimal hyperplasia. As a consequence to arterial injury, endothelium-derived growth-inhibiting factors are downregulated, resulting from endothelial damage, accompanied by platelet activation (31Gareri C. De Rosa S. Indolfi C. MicroRNAs for restenosis and thrombosis after vascular injury.Circ. Res. 2016; 118: 1170-1184Crossref PubMed Scopus (87) Google Scholar, 32Shirotani M. Yui Y. Hattori R. Kawai C. U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.Prostaglandins. 1991; 41: 97-110Crossref PubMed Scopus (64) Google Scholar). Subsequently, growth-promoting factors, PDGF products, and VSMCs are elevated (33Zohlnhofer D. Richter T. Neumann F. Nuhrenberg T. Wessely R. Brandl R. Murr A. Klein C.A. Baeuerle P.A. Transcriptome analysis reveals a role of interferon-gamma in human neointima formation.Mol. Cell. 2001; 7: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). PDGF-BB induces VSMC proliferation. In vitro, we discovered that USP10 was increased at mRNA and protein levels under PDGF-BB stimuli. The inhibition or knockdown of USP10 decreased the cell viability by blocking the cell cycle transition from G1 to S phase in VSMCs. The cycle protein regulators, such as CDK4, p-Rb/Rb, and p27, were affected by USP10. Additionally, VSMC migration was inhibited by USP10 deletion with or without PDGF-BB stimuli. Importantly, in the molecular mechanism of USP10-regulated cell proliferation, Skp2 was recognized as a target of USP10. Thus, silencing USP10 promoted the degradation of Skp2 protein by increasing its ubiquitination. The protein rescued the events induced by USP10 inhibition. The findings indicated that Skp2 was involved in the progression of USP10-promoted VSMC proliferation. Taken together, this study demonstrated a major role of USP10 in neointima formation and established it as a deubiquitinating enzyme of Skp2 in VSMCs via interaction with Skp2 and removal of ubiquitin. Spautin-1 (S7888) and cycloheximide (S7418) were obtained from Selleckchem. Antibodies, anti-USP10 (#8501), anti-Skp2 (#2652), anti-p-Rb (#8516), anti-Rb (#9309), anti-CDK4 (#12790), anti-Cyclin D1 (#55506), anti-p27 (#3686), anti-MMP2 (#40994), anti-ubiquitin (#3936), anti-α-SMA (#19245), and anti-GAPDH (#5174) were purchased from Cell Signaling Technology (CST). Human PDGF-BB was obtained from Peprotech. HASMCs were purchased from ScienCell, and A7r5 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37 °C containing 5% CO2. In order to assess cell proliferation, we utilized the following assays: cell viability, colony formation, EDU staining, and cell cycle. These assays were performed as described previously (34Xia X. Liao Y. Huang C. Liu Y. He J. Shao Z. Jiang L. Dou Q.P. Liu J. Huang H. Deubiquitination and stabilization of estrogen receptor alpha by ubiquitin-specific protease 7 promotes breast tumorigenesis.Cancer Lett. 2019; 465: 118-128Crossref PubMed Scopus (35) Google Scholar). Cell viability was tested by MTS (Promega) assay. Briefly, VSMCs were plated into 96-well plates. After 24 h, the indicated reagents were applied to treat the adherent cells. A volume of 20 μl MTS was added to the well and incubated for 3 h. The cell viability was evaluated based on the absorbance of optical density. The cells were seeded into 6 cm dishes and treated at the indicated concentrations. The treated cells were digested and transferred to 6-well plate for culture for 10 to 14 days. Then, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, and stained with 1% crystal violet solution. EdU staining was carried out to recognize replicating DNA. The kit was obtained from Ribobio. VSMCs were plated into chamber slider and treated with Spautin-1 and PDGF-BB for 48 h and then 50 μM EdU for 2 h. Subsequently, glycine and 0.5% Triton X-100, Apollo reaction cocktail, and DAPI were applied successively for 5, 10, 30, and 5 min, respectively. The images were captured by the Olympus microscope. For cell cycle assay, the cells were plated, treated, washed with PBS, resuspended in 2 ml of 70% ethanol and 500 μl PBS at 4 °C for one night. The cells were then stained with propidium iodide (PI), RNase A, and 0.2% Triton X-100 for half an hour. The cell distribution was tested by flow cytometry. Cell apoptosis assay was performed using Annexin V-FITC/PI kit (Cat.# KGA108). The treated cells were washed with PBS and stained for 15 min before flow cytometry was used to analyze cell apoptosis. For measuring the cell migration activity, we used scratch and transwell assays. To perform scratch assay, cells were seeded into 6-well plates and treated with Spautin-1. When the cells were cultured to 90% confluency, a 200 μl pipette tip was used to scrape the cell monolayer to make a wound. The images were acquired digitally at 0 and 72 h using an inverted Olympus microscope. For transwell assay, the treated cells were suspended in a serum-free medium and seeded into the upper chamber. The lower chamber was supplemented with 600 μl of 10% FBS medium. After the indicated time point, the transwell was fixed with 4% paraformaldehyde for 10 min and then stained with 1% crystal violet. This assay was carried out as described previously (35Liao Y. Guo Z. Xia X. Liu Y. Huang C. Jiang L. Wang X. Liu J. Huang H. Inhibition of EGFR signaling with Spautin-1 represents a novel therapeutics for prostate cancer.J. Exp. Clin. Cancer Res. 2019; 38: 157Crossref PubMed Scopus (34) Google Scholar). The cells were treated as indicated, and proteins were extracted with RIPA buffer and protease inhibitors (CST). The BCA kit was utilized to estimate the protein concentration. An equivalent of protein was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane for 2.5 h. Then, the membranes were blocked using 5% milk in PBS-T and probed with primary and secondary antibodies for the indicated time points. Finally, an ECL detection kit was used to develop the immunoreactive bands, and the membrane was exposed to X-ray films (Kodak). This assay was performed as described previously (36Liao Y. Liu Y. Xia X. Shao Z. Huang C. He J. Jiang L. Tang D. Liu J. Huang H. Targeting GRP78-dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy.Theranostics. 2020; 10: 3366-3381Crossref PubMed Scopus (21) Google Scholar). Briefly, antibody and Dynabeads (Invitrogen) were mixed in a reaction for 16 to 24 h. The extracted proteins were mixed with the Dynabeads carrying antibody and incubated for 1 h. The protein was separated from the Dynabeads via centrifugation and evaluated by Western blot analysis. This assay was performed as described previously (37Xia X. Huang C. Liao Y. Liu Y. He J. Guo Z. Jiang L. Wang X. Liu J. Huang H. Inhibition of USP14 enhances the sensitivity of breast cancer to enzalutamide.J. Exp. Clin. Cancer Res. 2019; 38: 220Crossref PubMed Scopus (35) Google Scholar). The treated cells were washed with PBS and fixed with 4% paraformaldehyde. Then, the cells were washed with PBS, followed by permeabilization with 0.5% Triton-X for 5 min and blocking with 5% bovine serum albumin (Sigma). Subsequently, the cells were incubated with a primary antibody at 4 °C and anti-IgG H&L (mouse or rabbit) secondary antibody for 1 h. DAPI was used for nuclear staining, and images were captured by a confocal microscope (Leica TCS SP8). Mouse carotid artery tissues were sliced into 5-μm-thick sections at the optimal cutting temperature and incubated with anti-USP10 (NBP2-01452, Novus Biological, 1:100), anti-α-smooth muscle actin (α-SMA, ab7817, Abcam, 1:200), and anti-Skp2 (15010-1-AP, Proteintech, 1:50), respectively. The plasmids in this study (Flag-USP10 and Myc-Skp2) were purchased from GeneChem. This assay was performed as reported previously (38Xia X. Xu Q. Liu M. Chen X. Liu X. He J. Hu T. Yu C. Huang H. Liu S. Liu N. Deubiquitination of CD36 by UCHL1 promotes foam cell formation.Cell Death Dis. 2020; 11: 636Crossref PubMed Scopus (5) Google Scholar). The adherent cells were incubated in a mixture containing RPMI opti-MEM (Gibco), P3000, plasmids, and Lipofectamine 3000 for 15 min. After 6 h, fresh medium was replaced for an additional 48 h. Furthermore, siRNA (Santa Cruz) was employed to silence the targeted gene. Briefly, the cells were seeded into plates or dishes for 24 h. The mixture containing RPMI opti-MEM, Lipofectamine RNAiMAX, and siRNA was incubated for 15 min and added to adherent cells for 48 h. C57BL/6 female mice were provided for this experiment and approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The mice were divided into three groups: sham, control, and treatment. Briefly, a third of the animals were treated with intraperitoneal injection of Spautin-1 (20 mg/kg) or adeno-associated virus by intravenous injection. After 3 days, mice were anesthetized with chloral hydrate, and then the left common carotids proximal to the carotid bifurcation were ligated using 6-0 silk. A similar procedure was performed as control with hydration medium. The sham was not ligated. After 21 days, animals were anesthetized, and the carotid arteries were excised and embedded in paraffin. The carotid arteries were sectioned for HE staining. The carotid sample was collected and fixed with 4% paraformaldehyde overnight. The samples were embedded in paraffin blocks, sliced into 5-μm-thick sections, and subjected to HE staining. For IHC staining, samples were subjected to microwave-based antigen retrieval in 10 mM sodium citrate buffer. Then, 0.3% hydrogen peroxide was applied to inactivate the endogenous peroxide activity for 10 min. Sections were washed with PBS three times, followed by incubation with primary antibodies and secondary antibodies. A standard ABC-peroxidase system and DAB peroxidase substrate kit were used to evaluate the staining intensity. Lentivirus overexpressing Skp2, USP10, or containing control vector was purchased from GeneChem. Cells were seeded into 6-well plates for 24 h. The mixture of polybrene, lentiviruses, and medium was added to cells for 48 h for subsequent analyses. The data are presented as mean ± standard deviation (SD) from three independent experiments. Unpaired Student's t test or one-way analysis of variance (ANOVA) was used to estimate the statistical probabilities where appropriate. GraphPad Prism 8.0 and SPSS 16.0 were used for statistical analysis. p < 0.05 indicated statistical significance. All the data and material supporting the conclusions were included in the main paper. This article contains supporting information. The authors declare that they have no conflicts of interest with the contents of this article. We thank Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University for flow cytometry analysis. The study was supported by the National Natural Science Foundation of China ( 82170416 , 81873474 , 81570259 ), the Natural Science Foundation of Guangdong ( 2021A1515011387 ), the Science and Technology Program of Guangzhou ( 202002030344 ), Bureau of Education of Guangzhou Municipality ( 14CXY03 ), Guangzhou health and family planning science and technology project ( 20201A011082 ), the Key Medical Disciplines and Specialties Program of Guangzhou (2021–2023). X. X., X. L., R. C., Q. X., Z. L., J. G., Y. J., T. H., C. Y., B. D., H. H., W. O., S. L., and N. L. conceptualization; X. X., X. L., R. C., Q. X., Z. L., J. G., Y. J., T. H., C. Y., B. D., H. H., W. O., S. L., and N. L. data curation; S. L. and N. L. funding acquisition; X. X., X. L., R. C., Q. X., Z. L., J. G., Y. J., T. H., C. Y., B. D., H. H., W. O., S. L., and N. L. methodology; S. L. and N. L. supervision; X. X., S. L., and N. L. writing—original draft; S. L. and N. L. writing—review and editing. 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