title: Phosphorylation of murine double minute-2 on Ser166 is downstream of VEGF-A in exercised skeletal muscle and regulates primary endothelial cell migration and FoxO gene expression.
abstract: We demonstrated in a previous study that murine double minute (Mdm)-2 is essential for exercise-induced skeletal muscle angiogenesis. In the current study, we investigated the mechanisms that regulate Mdm2 activity in response to acute exercise and identified VEGF-A as a key stimulator of Mdm2 phosphorylation on Ser(166) (p-Ser166-Mdm2). VEGF-A and p-Ser166-Mdm2 protein levels were measured in human and rodent muscle biopsy specimens after 1 bout of exercise. VEGF-A-dependent Mdm2 phosphorylation was demonstrated in vivo in mice harboring myofiber-specific deletion of VEGF-A (mVEGF(-/-)) and in vitro in primary human and rodent endothelial cells (ECs). Exercise increased VEGF-A and p-Ser166-Mdm2 protein levels respectively by 157 and 68% in human muscle vs. pre-exercise levels. Similar results were observed in exercised rodent muscles compared to sedentary controls; however, exercise-induced Mdm2 phosphorylation was significantly attenuated in mVEGF(-/-) mice. Recombinant VEGF-A elevated p-Ser166-Mdm2 by 50-125% and stimulated migration by 33% in ECs when compared to untreated cells, whereas the Mdm2 antagonist Nutlin-3a abrogated VEGF-driven EC migration. Finally, overexpression of a Ser166-Mdm2 phosphorylation mimetic increased EC migration, increased Mdm2 to FoxO1 binding (+55%), and decreased FoxO1-dependent gene expression compared with ECs overexpressing WT-Mdm2. Our results suggest that VEGF-mediated Mdm2 phosphorylation on Ser(166) is a novel proangiogenic pathway within the skeletal muscle.
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Aiken
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA eroudier@yorku.ca.
authors.forename: Emilie
authors.initials: E
authors.lastname: Roudier
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Ciccone
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Genevieve
authors.initials: G
authors.lastname: Drouin
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Stromberg
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Jovana
authors.initials: J
authors.lastname: Vojnovic
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: I Mark
authors.initials: IM
authors.lastname: Olfert
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Tara
authors.initials: T
authors.lastname: Haas
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gustafsson
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Grenier
authors.affiliation: *Faculty of Health, School of Kinesiology and Health Science, Angiogenesis Research Group, York University, Toronto, Ontario, Canada; Department of Surgery, Université de Sherbrooke, Sherbrooke, Québec, Canada; Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; and Center for Cardiovascular and Respiratory Sciences and Division of Exercise Physiology, West Virginia University, Morgantown, West Virginia, USA.
authors.forename: Olivier
authors.initials: O
authors.lastname: Birot
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D051858:Forkhead Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D008297:Male; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010805:Physical Conditioning, Animal / Q000502:physiology*; D010879:Piperazines / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D051858:Forkhead Transcription Factors; D007093:Imidazoles; D010879:Piperazines; D042461:Vascular Endothelial Growth Factor A; D012694:Serine; C482205:nutlin 3; D051736:Proto-Oncogene Proteins c-mdm2
keywords: Mdm2; angiogenesis; physical exercise