Aiken J, Roudier E, Ciccone J, Drouin G, Stromberg A, Vojnovic J, Olfert IM, 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. 2016 Mar;30(3):1120-34. doi: 10.1096/fj.15-276964. Epub 2015 Nov 17.
Significance of the research:
In skeletal muscle tissue, the capillary network matches oxygen and nutrient delivery to the metabolic needs of active muscle cells. Angiogenesis, the growth of new capillaries, therefore plays a key role in muscle adaptation to exercise training. One bout of intense exercise is a powerful stimulus for the release of various pro-angiogenic factors, in particular the Vascular Endothelial Growth Factor-A (VEGF). We have recently demonstrated that Murine Double Minute-2 (Mdm2) was indispensable for exercise-induced VEGF production and angiogenesis in rodent skeletal muscle (Roudier et al. 2012). The current publication suggests that the "classical" representation of Mdm2 as an upstream regulator of VEGF-A expression might in fact be much more complex. We show that in response to a single bout of exercise, both VEGF-A and Mdm2 that is phosphorylated on its serine 166 (p-Ser166-Mdm2) are strongly induced in both rodent and human skeletal muscle, and that this response was impaired in VEGF-A myofibre-specific knockout mice. This suggested that VEGF-A may be an essential regulator of Mdm2 phosphorylation in vivo in response to a single bout of exercise. In order to explore this relationship further, using both primary human and rodent endothelial cells, we demonstrate that recombinant VEGF-A protein stimulates p-Ser166-Mdm2. This recombinant VEGF-A stimulation significantly elevated endothelial cell migration, however, this effect was entirely abrogated by the Mdm2 antagonist Nutlin-3a, suggesting that VEGF-driven endothelial cell migration is dependent on Mdm2 activity. To further study the functional impact of p-Ser166-Mdm2 in endothelial cells, primary human endothelial cells were infected with lentiviruses carrying plasmids encoding for either wild-type Mdm2 (WT-Mdm2) or a mimetic of Mdm2 phosphorylation on serine 166 (S166D-Mdm2). Overexpression of S166D-Mdm2 protein greatly enhances endothelial cell migration, indicating that Mdm2 activation (i.e. phosphorylation) promotes the pro-angiogenic activity of endothelial cells. Mdm2 can interact with the transcription factors HIF-1α, p53 and FoxO1 that regulate both pro- and anti-angiogenic molecules. Using co-immunoprecipitation, we show increased Mdm2 protein binding to FoxO1, but not HIF-1α or p53, in the S166D-Mdm2 compared to WT-Mdm2 overexpressing cells. This corresponded to a reduction in the mRNA expression of hallmark anti-angiogenic FoxO1 transcriptional targets (p27, sprouty-2, TSP-1) and an increased VEGF-A to TSP-1 mRNA ratio, thus promoting a pro-angiogenic environment. These findings clearly establish a novel pro-angiogenic role of p-Ser166-Mdm2 in human skeletal muscle and endothelial cells.