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Phosphorylation of murine double minute‐2 on Ser 166 is downstream of VEGF‐A in exercised skeletal muscle and regulates primary endothelial cell migration and FoxO gene expression
Author(s) -
Aiken Julian,
Roudier Emilie,
Ciccone Joseph,
Drouin Genevieve,
Stromberg Anna,
Vojnovic Jovana,
Olfert I. Mark,
Haas Tara,
Gustafsson Thomas,
Grenier Guillaume,
Birot Olivier
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.15-276964
Subject(s) - phosphorylation , mdm2 , angiogenesis , skeletal muscle , foxo1 , endocrinology , medicine , chemistry , biology , cancer research , microbiology and biotechnology , gene , protein kinase b , biochemistry
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.—Aiken, J., Roudier, E., Ciccone, J., Drouin, G., Stromberg, A., Vojnovic, J., Olfert, I. M., Haas, T., Gustafsson, T., Grenier, G., Birot, O., 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. FASEB J. 30, 1120–1134 (2016). www.fasebj.org