Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation
BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH.
METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH.
RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling.
CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.
Medienart: |
E-Artikel |
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Erscheinungsjahr: |
2023 |
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Erschienen: |
2023 |
Enthalten in: |
Zur Gesamtaufnahme - volume:133 |
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Enthalten in: |
Circulation research - 133(2023), 12 vom: 08. Dez., Seite 966-988 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Knight, Hannah [VerfasserIn] |
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Links: |
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Anmerkungen: |
Date Completed 16.12.2023 Date Revised 14.03.2024 published: Print-Electronic Citation Status MEDLINE |
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doi: |
10.1161/CIRCRESAHA.122.321836 |
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funding: |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
NLM364468394 |
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500 | |a Citation Status MEDLINE | ||
520 | |a BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH | ||
520 | |a METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH | ||
520 | |a RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling | ||
520 | |a CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH | ||
650 | 4 | |a Journal Article | |
650 | 4 | |a Research Support, Non-U.S. Gov't | |
650 | 4 | |a cell cycle | |
650 | 4 | |a cell proliferation | |
650 | 4 | |a hypertension, pulmonary | |
650 | 4 | |a myocytes, smooth muscle | |
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700 | 1 | |a Abis, Giancarlo |e verfasserin |4 aut | |
700 | 1 | |a Kaur, Manpreet |e verfasserin |4 aut | |
700 | 1 | |a Green, Hannah L H |e verfasserin |4 aut | |
700 | 1 | |a Krasemann, Susanne |e verfasserin |4 aut | |
700 | 1 | |a Hartmann, Kristin |e verfasserin |4 aut | |
700 | 1 | |a Lynham, Steven |e verfasserin |4 aut | |
700 | 1 | |a Clark, James |e verfasserin |4 aut | |
700 | 1 | |a Zhao, Lan |e verfasserin |4 aut | |
700 | 1 | |a Ruppert, Clemens |e verfasserin |4 aut | |
700 | 1 | |a Weiss, Astrid |e verfasserin |4 aut | |
700 | 1 | |a Schermuly, Ralph T |e verfasserin |4 aut | |
700 | 1 | |a Eaton, Philip |e verfasserin |4 aut | |
700 | 1 | |a Rudyk, Olena |e verfasserin |4 aut | |
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