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

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

References

[1] Libby P. The changing landscape of atherosclerosis. Nature 2021; 592(7855): 524–533

[2] Björkegren JLM, Lusis AJ. Atherosclerosis: recent developments. Cell 2022; 185(10): 1630–1645

[3] Hetherington I, Totary-Jain H. Anti-atherosclerotic therapies: milestones, challenges, and emerging innovations. Mol Ther 2022; 30(10): 3106–3117

[4] Basatemur GL, Jørgensen HF, Clarke MCH, Bennett MR, Mallat Z. Vascular smooth muscle cells in atherosclerosis. Nat Rev Cardiol 2019; 16(12): 727–744

[5] Miano JM, Fisher EA, Majesky MW. Fate and state of vascular smooth muscle cells in atherosclerosis. Circulation 2021; 143(21): 2110–2116

[6] Golforoush P, Yellon DM, Davidson SM. Mouse models of atherosclerosis and their suitability for the study of myocardial infarction. Basic Res Cardiol 2020; 115(6): 73

[7] Swiatlowska P, Sit B, Feng Z, Marhuenda E, Xanthis I, Zingaro S, Ward M, Zhou X, Xiao Q, Shanahan C, Jones GE, Yu CH, Iskratsch T. Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching. Sci Adv 2022; 8(15): eabm3471

[8] Borgo C, D’Amore C, Sarno S, Salvi M, Ruzzene M. Protein kinase CK2: a potential therapeutic target for diverse human diseases. Signal Transduct Target Ther 2021; 6(1): 183

[9] Herrmann J, Soares SM, Lerman LO, Lerman A. Potential role of the ubiquitin-proteasome system in atherosclerosis: aspects of a protein quality disease. J Am Coll Cardiol 2008; 51(21): 2003–2010

[10] Demasi M, Laurindo FR. Physiological and pathological role of the ubiquitin-proteasome system in the vascular smooth muscle cell. Cardiovasc Res 2012; 95(2): 183–193

[11] Grootaert MOJ, Finigan A, Figg NL, Uryga AK, Bennett MR. SIRT6 protects smooth muscle cells from senescence and reduces atherosclerosis. Circ Res 2021; 128(4): 474–491

[12] Ye M, Guo X, Wang H, Wang Y, Qian X, Deng H, Wang W, Yang S, Ni Q, Chen J, Lv L, Zhao Y, Xue G, Li Y, Zhang L. Mutual regulation between β-TRCP mediated REST protein degradation and Kv1.3 expression controls vascular smooth muscle cell phenotype switch. Atherosclerosis 2020; 313: 102–110

[13] Lange SM, Armstrong LA, Kulathu Y. Deubiquitinases: from mechanisms to their inhibition by small molecules. Mol Cell 2022; 82(1): 15–29

[14] Mevissen TE, Hospenthal MK, Geurink PP, Elliott PR, Akutsu M, Arnaudo N, Ekkebus R, Kulathu Y, Wauer T, El Oualid F, Freund SM, Ovaa H, Komander D. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 2013; 154(1): 169–184

[15] Clague MJ, Urbé S, Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function. Nat Rev Mol Cell Biol 2019; 20(6): 338–352

[16] Liao Y, Yang M, Wang K, Wang Y, Zhong B, Jiang N. Deubiquitinating enzyme OTUB1 in immunity and cancer: good player or bad actor?. Cancer Lett 2022; 526: 248–258

[17] Mulas F, Wang X, Song S, Nishanth G, Yi W, Brunn A, Larsen PK, Isermann B, Kalinke U, Barragan A, Naumann M, Deckert M, Schlüter D. The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13. Cell Mol Immunol 2021; 18(6): 1512–1527

[18] Koschel J, Nishanth G, Just S, Harit K, Kröger A, Deckert M, Naumann M, Schlüter D. OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation. Cell Death Differ 2021; 28(7): 2257–2275

[19] Bonacci T, Suzuki A, Grant GD, Stanley N, Cook JG, Brown NG, Emanuele MJ. Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates. EMBO J 2018; 37(16): e98701

[20] Luo Q, Wu X, Zhao P, Nan Y, Chang W, Zhu X, Su D, Liu Z. OTUD1 Activates caspase-independent and caspase-dependent apoptosis by promoting AIF nuclear translocation and MCL1 degradation. Adv Sci (Weinh) 2021; 8(8): 2002874

[21] Yang GS, Zheng B, Qin Y, Zhou J, Yang Z, Zhang XH, Zhao HY, Yang HJ, Wen JK. Salvia miltiorrhiza-derived miRNAs suppress vascular remodeling through regulating OTUD7B/KLF4/NMHC IIA axis. Theranostics 2020; 10(17): 7787–7811

[22] Karunarathna U, Kongsema M, Zona S, Gong C, Cabrera E, Gomes AR, Man EP, Khongkow P, Tsang JW, Khoo US, Medema RH, Freire R, Lam EW. OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance. Oncogene 2016; 35(11): 1433–1444

[23] Zhu D, Xu R, Huang X, Tang Z, Tian Y, Zhang J, Zheng X. Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1. Cell Death Differ 2021; 28(6): 1773–1789

[24] Jahan AS, Biquand E, Muñoz-Moreno R, Le Quang A, Mok CK, Wong HH, Teo QW, Valkenburg SA, Chin AWH, Man Poon LL, Te Velthuis A, García-Sastre A, Demeret C, Sanyal S. OTUB1 is a key regulator of RIG-I-dependent immune signaling and is targeted for proteasomal degradation by influenza A NS1. Cell Rep 2020; 30(5): 1570–1584.e6

[25] Liu S, Jiang X, Cui X, Wang J, Liu S, Li H, Yang J, Zhang C, Zhang W. Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis. Cell Death Dis 2021; 12(4): 385

[26] Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods 2009; 6(5): 359–362

[27] Shin D, Bhattacharya A, Cheng YL, Alonso MC, Mehdipour AR, van der Heden van Noort GJ, Ovaa H, Hummer G, Dikic I. Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection. Elife 2020; 9: e58277

[28] Schubert AF, Nguyen JV, Franklin TG, Geurink PP, Roberts CG, Sanderson DJ, Miller LN, Ovaa H, Hofmann K, Pruneda JN, Komander D. Identification and characterization of diverse OTU deubiquitinases in bacteria. EMBO J 2020; 39(15): e105127

[29] Biros E, Gäbel G, Moran CS, Schreurs C, Lindeman JH, Walker PJ, Nataatmadja M, West M, Holdt LM, Hinterseher I, Pilarsky C, Golledge J. Differential gene expression in human abdominal aortic aneurysm and aortic occlusive disease. Oncotarget 2015; 6(15): 12984–12996

[30] Herhaus L, Al-Salihi M, Macartney T, Weidlich S, Sapkota GP. OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nat Commun 2013; 4(1): 2519

[31] Li Y, Yang JY, Xie X, Jie Z, Zhang L, Shi J, Lin D, Gu M, Zhou X, Li HS, Watowich SS, Jain A, Yun Jung S, Qin J, Cheng X, Sun SC. Preventing abnormal NF-κB activation and autoimmunity by Otub1-mediated p100 stabilization. Cell Res 2019; 29(6): 474–485

[32] Zhu Q, Fu Y, Li L, Liu CH, Zhang L. The functions and regulation of Otubains in protein homeostasis and diseases. Ageing Res Rev 2021; 67: 101303

[33] Ruiz-Serrano A, Monné Rodríguez JM, Günter J, Sherman SPM, Jucht AE, Fluechter P, Volkova YL, Pfundstein S, Pellegrini G, Wagner CA, Schneider C, Wenger RH, Scholz CC. OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control. FASEB J 2021; 35(12): e22039

[34] Cheng A, Grant CE, Noble WS, Bailey TL. MoMo: discovery of statistically significant post-translational modification motifs. Bioinformatics 2019; 35(16): 2774–2782

[35] Baumer Y, McCurdy S, Alcala M, Mehta N, Lee BH, Ginsberg MH, Boisvert WA. CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis. Atherosclerosis 2017; 256: 105–114

[36] Goetzl EJ, Schwartz JB, Mustapic M, Lobach IV, Daneman R, Abner EL, Jicha GA. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease. FASEB J 2017; 31(8): 3689–3694

[37] He C, Medley SC, Hu T, Hinsdale ME, Lupu F, Virmani R, Olson LE. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis. Nat Commun 2015; 6(1): 7770

[38] Papadopoulos N, Lennartsson J. The PDGF/PDGFR pathway as a drug target. Mol Aspects Med 2018; 62: 75–88

[39] Iglesias-Gato D, Chuan YC, Jiang N, Svensson C, Bao J, Paul I, Egevad L, Kessler BM, Wikström P, Niu Y, Flores-Morales A. OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo. Mol Cancer 2015; 14(1): 8

[40] Han X, Ren C, Lu C, Qiao P, Yang T, Yu Z. Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis. Cell Death Differ 2022; 29(9): 1864–1873

[41] Goncharov T, Niessen K, de Almagro MC, Izrael-Tomasevic A, Fedorova AV, Varfolomeev E, Arnott D, Deshayes K, Kirkpatrick DS, Vucic D. OTUB1 modulates c-IAP1 stability to regulate signalling pathways. EMBO J 2013; 32(8): 1103–1114

[42] Martínez-Férriz A, Ferrando A, Fathinajafabadi A, Farràs R. Ubiquitin-mediated mechanisms of translational control. Semin Cell Dev Biol 2022; 132: 146–154

[43] Takayama Y, May P, Anderson RG, Herz J. Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor β (PDGFR β). J Biol Chem 2005; 280(18): 18504–18510

[44] Wang X, Li Y, He M, Kong X, Jiang P, Liu X, Diao L, Zhang X, Li H, Ling X, Xia S, Liu Z, Liu Y, Cui CP, Wang Y, Tang L, Zhang L, He F, Li D. UbiBrowser 2.0: a comprehensive resource for proteome-wide known and predicted ubiquitin ligase/deubiquitinase-substrate interactions in eukaryotic species. Nucleic Acids Res 2022; 50(D1): D719–D728

[45] Li Z, Chen S, Jhong JH, Pang Y, Huang KY, Li S, Lee TY. UbiNet 2.0: a verified, classified, annotated and updated database of E3 ubiquitin ligase-substrate interactions. Database (Oxford) 2021; 2021: baab010

[46] Xu H, Zhou J, Lin S, Deng W, Zhang Y, Xue Y. PLMD: an updated data resource of protein lysine modifications. J Genet Genomics 2017; 44(5): 243–250

[47] Wiener R, Zhang X, Wang T, Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 2012; 483(7391): 618–622

[48] Zhang Y, Wang Y, Zhang L, Xia L, Zheng M, Zeng Z, Liu Y, Yarovinsky T, Ostriker AC, Fan X, Weng K, Su M, Huang P, Martin KA, Hwa J, Tang WH. Reduced platelet miR-223 induction in Kawasaki disease leads to severe coronary artery pathology through a miR-223/PDGFRβ vascular smooth muscle cell axis. Circ Res 2020; 127(7): 855–873

[49] Reddi AL, Ying G, Duan L, Chen G, Dimri M, Douillard P, Druker BJ, Naramura M, Band V, Band H. Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation. J Biol Chem 2007; 282(40): 29336–29347

[50] Hamid T, Xu Y, Ismahil MA, Rokosh G, Jinno M, Zhou G, Wang Q, Prabhu SD. Cardiac mesenchymal stem cells promote fibrosis and remodeling in heart failure: role of PDGF signaling. JACC Basic Transl Sci 2022; 7(5): 465–483

[51] Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol 2008; 28(5): 812–819

[52] Saldana M, VanderVorst K, Berg AL, Lee H, Carraway KL. Otubain 1: a non-canonical deubiquitinase with an emerging role in cancer. Endocr Relat Cancer 2019; 26(1): R1–R14

[53] Yap C, Mieremet A, de Vries CJM, Micha D, de Waard V. Six shades of vascular smooth muscle cells illuminated by KLF4 (Krüppel-like factor 4). Arterioscler Thromb Vasc Biol 2021; 41(11): 2693–2707

[54] Durham AL, Speer MY, Scatena M, Giachelli CM, Shanahan CM. Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness. Cardiovasc Res 2018; 114(4): 590–600

[55] Sun XX, Challagundla KB, Dai MS. Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1. EMBO J 2012; 31(3): 576–592

[56] Jean-Charles PY, Wu JH, Zhang L, Kaur S, Nepliouev I, Stiber JA, Brian L, Qi R, Wertman V, Shenoy SK, Freedman NJ. USP20 (ubiquitin-specific protease 20) inhibits TNF (tumor necrosis factor)-triggered smooth muscle cell inflammation and attenuates atherosclerosis. Arterioscler Thromb Vasc Biol 2018; 38(10): 2295–2305

[57] Moll HP, Lee A, Minussi DC, da Silva CG, Csizmadia E, Bhasin M, Ferran C. A20 regulates atherogenic interferon (IFN)-γ signaling in vascular cells by modulating basal IFNβ levels. J Biol Chem 2014; 289(45): 30912–30924

[58] Karvinen H, Rutanen J, Leppänen O, Lach R, Levonen AL, Eriksson U, Ylä-Herttuala S. PDGF-C and -D and their receptors PDGFR-α and PDGFR-β in atherosclerotic human arteries. Eur J Clin Invest 2009; 39(4): 320–327

[59] Rorsman C, Tsioumpekou M, Heldin CH, Lennartsson J. The ubiquitin ligases c-Cbl and Cbl-b negatively regulate platelet-derived growth factor (PDGF) BB-induced chemotaxis by affecting PDGF receptor β (PDGFRβ) internalization and signaling. J Biol Chem 2016; 291(22): 11608–11618

[60] Sarri N, Wang K, Tsioumpekou M, Castillejo-López C, Lennartsson J, Heldin CH, Papadopoulos N. Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRβ and affect PDGF-BB-induced STAT3 signalling. Cell Mol Life Sci 2022; 79(2): 85

[61] Newman AAC, Serbulea V, Baylis RA, Shankman LS, Bradley X, Alencar GF, Owsiany K, Deaton RA, Karnewar S, Shamsuzzaman S, Salamon A, Reddy MS, Guo L, Finn A, Virmani R, Cherepanova OA, Owens GK. Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms. Nat Metab 2021; 3(2): 166–181

[62] Guo X, Nie L, Esmailzadeh L, Zhang J, Bender JR, Sadeghi MM. Endothelial and smooth muscle-derived neuropilin-like protein regulates platelet-derived growth factor signaling in human vascular smooth muscle cells by modulating receptor ubiquitination. J Biol Chem 2009; 284(43): 29376–29382

[63] Childs BG, Baker DJ, Wijshake T, Conover CA, Campisi J, van Deursen JM. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 2016; 354(6311): 472–477

[64] Gomez D, Baylis RA, Durgin BG, Newman AAC, Alencar GF, Mahan S, St Hilaire C, Müller W, Waisman A, Francis SE, Pinteaux E, Randolph GJ, Gram H, Owens GK. Interleukin-1β has atheroprotective effects in advanced atherosclerotic lesions of mice. Nat Med 2018; 24(9): 1418–1429

[65] Fredman G, Kamaly N, Spolitu S, Milton J, Ghorpade D, Chiasson R, Kuriakose G, Perretti M, Farokzhad O, Tabas I. Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice. Sci Transl Med 2015; 7(275): 275ra20

RIGHTS & PERMISSIONS

Higher Education Press