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Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.
- Source :
-
Circulation research [Circ Res] 2023 Dec 08; Vol. 133 (12), pp. 966-988. Date of Electronic Publication: 2023 Nov 13. - Publication Year :
- 2023
-
Abstract
- 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.<br />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.<br />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.<br />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.<br />Competing Interests: Disclosures None.
- Subjects :
- Humans
Mice
Animals
Cysteine metabolism
Endothelial Cells metabolism
Cell Proliferation
Pulmonary Artery metabolism
Phosphorylation
Cell Cycle Checkpoints
Cyclin D metabolism
Cells, Cultured
Cyclin-Dependent Kinase 4 genetics
Cyclin-Dependent Kinase 4 metabolism
Cyclins metabolism
Pulmonary Arterial Hypertension metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1524-4571
- Volume :
- 133
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Circulation research
- Publication Type :
- Academic Journal
- Accession number :
- 37955182
- Full Text :
- https://doi.org/10.1161/CIRCRESAHA.122.321836