Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

Hannah Knight, Giancarlo Abis, Manpreet Kaur, Hannah L H Green, Susanne Krasemann, Kristin Hartmann, Steven Lynham, James Clark, Lan Zhao, Clemens Ruppert, Astrid Weiss, Ralph T Schermuly, Philip Eaton, Olena Rudyk

Research output: Contribution to journalArticlepeer-review

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.

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.

Original languageEnglish
Pages (from-to)966-988
Number of pages23
JournalCirculation Research
Volume133
Issue number12
DOIs
Publication statusPublished - 8 Dec 2023

Keywords

  • Humans
  • Mice
  • Animals
  • Cyclins/metabolism
  • Pulmonary Arterial Hypertension/metabolism
  • Cysteine/metabolism
  • Endothelial Cells/metabolism
  • Cell Proliferation
  • Pulmonary Artery/metabolism
  • Phosphorylation
  • Cell Cycle Checkpoints
  • Cyclin D/metabolism
  • Cells, Cultured
  • Cyclin-Dependent Kinase 4/genetics

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