Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation
- Circ Res. 2023 Dec 8;133(12):966-988. doi: 10.1161/CIRCRESAHA.122.321836.
- 1. School of Cardiovascular and Metabolic Medicine and Sciences, British Heart Foundation Centre of Research Excellence (H.K., M.K., H.L.H.G., J.C., O.R.), King's College London, United Kingdom.
- 2. Division of Biosciences, Institute of Structural and Molecular Biology, University College London, United Kingdom (G.A.).
- 3. Institute of Neuropathology, University Medical Centre Hamburg-Eppendorf, Germany (S.K., K.H.).
- 4. Proteomics Core Facility, Centre of Excellence for Mass Spectrometry (S.L.), King's College London, United Kingdom.
- 5. National Heart and Lung Institute, Faculty of Medicine, Imperial College London, United Kingdom (L.Z.).
- 6. Universities of Giessen and Marburg Lung Center Giessen Biobank, Justus-Liebig-University Giessen, Germany (C.R.).
- 7. Department of Internal Medicine, Justus-Liebig-University Giessen, Giessen, Member of the German Center for Lung Research (DZL), Germany (A.W., R.T.S.).
- 8. William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom (P.E.).
- # Contributed equally.
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.