Journal Article

DNA-dependent protein kinase (DNA-PK) permits vascular smooth muscle cell proliferation through phosphorylation of the orphan nuclear receptor NOR1

Senad Medunjanin, Jan-Marcus Daniel, Sönke Weinert, Jochen Dutzmann, Frank Burgbacher, Sarah Brecht, Dennis Bruemmer, Thilo Kähne, Michael Naumann, Daniel G. Sedding, Werner Zuschratter and Ruediger C. Braun-Dullaeus

in Cardiovascular Research

Published on behalf of European Society of Cardiology

Volume 106, issue 3, pages 488-497
Published in print June 2015 | ISSN: 0008-6363
Published online April 2015 | e-ISSN: 1755-3245 | DOI: https://dx.doi.org/10.1093/cvr/cvv126
DNA-dependent protein kinase (DNA-PK) permits vascular smooth muscle cell proliferation through phosphorylation of the orphan nuclear receptor NOR1

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Aims

Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC.

Methods and results

Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated up-regulation of NOR1 protein as well as the cell-cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1, and hyperphosphorylation of the retinoblastoma protein were prevented by DNA-PK inhibition. Co-immunoprecipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in post-transcriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury.

Conclusion

DNA-PK directly phosphorylates NOR-1 and, this way, modulates SMC proliferation. These data add to our understanding of vascular remodelling processes and opens new avenues for treatment of vascular proliferative diseases.

Keywords: Cardiovascular diseases; Atherosclerosis; Molecular biology; Signal transduction

Journal Article.  5173 words.  Illustrated.

Subjects: Cardiovascular Medicine

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