Regenerative protein thymosin β-4 is a novel regulator of purinergic signaling

By an unknown mechanism, beta-thymosins are extracellular modulators of angiogenesis, inflammation, wound healing, and development. We were interested in identifying beta-thymosin interactors and determining their importance in beta-thymosins signaling in human vein endothelial cells (HUVECs). We performed pulldown experiments with biotinylated thymosin beta-4 (T beta 4) in comparison to neutravidin beads alone and used mass spectrometric analysis to identify differentially interacting proteins. By this method, we identified F1-F0 ATP synthase, a known target of antiangiogenic angiostatin. By surface plasmon resonance, we determined for T beta 4 binding to the beta subunit of ATP synthase a K-D of 12 nM. Blocking antibodies and antagonists (oligomycin, IC50 similar to 1.8 mu M; piceatannol, IC50 similar to 1.05 mu M; and angiostatin, IC50 similar to 2.9 mu g/ml) of ATP synthase inhibited the T beta 4-induced increase in cell surface ATP levels, as measured by luciferase assay, and the T beta 4-induced increase in HUVEC migration, as measured by transwell migration assay. Silencing of the ATP-responsive purinergic receptor P2X4 with siRNA also blocked T beta 4-induced HUVEC migration in a transwell assay. Furthermore, in silico we identified common amphiphilic alpha-helical structural similarities between beta-thymosins and the inhibitory factor 1 (IF1), an inhibitor of ATP synthase hydrolysis. In summary, we have identified an extracellular signaling pathway where T beta 4 increases cell surface ATP levels via ATP synthase and have shown further that ATP-responsive P2X4 receptor is required for T beta 4-induced HUVEC migration.-Freeman, K. W., Bowman, B. R., Zetter, B. R. Regenerative protein thymosin beta-4 is a novel regulator of purinergic signaling. FASEB J. 25, 907-915 (2011). www.fasebj.org