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

By an unknown mechanism, β‐thymosins are extracellular modulators of angiogenesis, inflammation, wound healing, and development. We were interested in identifying β‐thymosin interactors and determining their importance in β‐thymosins signaling in human vein endothelial cells (HUVECs). We performed pulldown experiments with biotinylated thymosin β‐4 (Tβ4) 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 Tβ4 binding to the β subunit of ATP synthase a K D of 12 nM. Blocking antibodies and antagonists (oligomycin, IC 50 ~1.8 µM;piceatannol, IC 50 –1.05 µM;and angiostatin, IC 50 ~2.9 µg/ml) of ATP synthase inhibited the Tβ4‐induced increase in cell surface ATP levels, as measured by luciferase assay, and the Tβ 4‐induced increase in HUVEC migration, as measured by transwell migration assay. Silencing of the ATP‐responsive purinergic receptor P2X4 with siRNA also blocked Tβ4‐induced HUVEC migration in a trans‐well assay. Furthermore, in silico we identified common amphiphilic α‐helical structural similarities between β‐thymosins and the inhibitory factor 1 (IF1), an inhibitor of ATP synthase hydrolysis. In summary, we have identified an extracellular signaling pathway where Tβ4 increases cell surface ATP levels via ATP synthase and have shown further that ATP‐responsive P2X4 receptor is required for Tβ4‐induced HUVEC migration.—Freeman, K. W., Bowman, B. R., Zetter, B. R. Regenerative protein thymosin β‐4 is a novel regulator of purinergic signaling. FASEB J. 25, 907–915 (2011). www.fasebj.org