title: DNA-dependent protein kinase (DNA-PK) permits vascular smooth muscle cell proliferation through phosphorylation of the orphan nuclear receptor NOR1.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany.
authors: Senad
authors: S
authors: Medunjanin
authors: Department of Cardiology, Hannover University, Hannover, Germany.
authors: Jan-Marcus
authors: JM
authors: Daniel
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany.
authors: Sönke
authors: S
authors: Weinert
authors: Department of Cardiology, Hannover University, Hannover, Germany.
authors: Jochen
authors: J
authors: Dutzmann
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany.
authors: Frank
authors: F
authors: Burgbacher
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany.
authors: Sarah
authors: S
authors: Brecht
authors: Division of Endocrinology and Molecular Medicine, University of Kentucky College of Medicine, Lexington, KY, USA.
authors: Dennis
authors: D
authors: Bruemmer
authors: Institute of Experimental Internal Medicine, Magdeburg University, Magdeburg, Germany.
authors: Thilo
authors: T
authors: Kähne
authors: Institute of Experimental Internal Medicine, Magdeburg University, Magdeburg, Germany.
authors: Michael
authors: M
authors: Naumann
authors: Department of Cardiology, Hannover University, Hannover, Germany.
authors: Daniel G
authors: DG
authors: Sedding
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany.
authors: Werner
authors: W
authors: Zuschratter
authors: Department of Internal Medicine, Division of Cardiology, Angiologie and Pneumology, Magdeburg University, Magdeburg, Germany r.braun-dullaeus@med.ovgu.de.
authors: Ruediger C
authors: RC
authors: Braun-Dullaeus
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery / Q000187:drug effects / Q000201:enzymology / Q000293:injuries / Q000473:pathology; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D058426:Neointima; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D034622:RNA Interference; D016160:Retinoblastoma Protein / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D054875:Ubiquitination; D066253:Vascular Remodeling / Q000187:drug effects; D057772:Vascular System Injuries / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology
chemical_list: C530104:CCND1 protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; C479458:OSCP1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse
keywords: Atherosclerosis; Cardiovascular diseases; Molecular biology; Signal transduction