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 p...

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Veröffentlicht in:The FASEB journal 2011-03, Vol.25 (3), p.907-915
Hauptverfasser: Freeman, Kevin W, Bowman, Brian R, Zetter, Bruce R
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Sprache:eng
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Zusammenfassung: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 KD of 12 nM. Blocking antibodies and antagonists (oligomycin, IC₅₀ ~1.8 μM; piceatannol, IC₅₀ ~1.05 μM; and angiostatin, IC₅₀ ~2.9 μg/ml) of ATP synthase inhibited the Tβ4-induced increase in cell surface ATP levels, as measured by luciferase assay, and the Tβ4-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 transwell 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.
ISSN:0892-6638
1530-6860
DOI:10.1096/fj.10-169417