Regenerative protein thymosin beta-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 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.
Vascular Biology Program and Department of Surgery, Children's Hospital, Boston, MA 02115, USA.
Kevin W
KW
Freeman
Brian R
BR
Bowman
Bruce R
BR
Zetter
D000595:Amino Acid Sequence; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D058848:Receptors, Purinergic P2X4 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013947:Thymosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D014945:Wound Healing / Q000502:physiology; D000097795:ATPase Inhibitory Protein; D000099201:Thymosin beta(4)
D011506:Proteins; D006180:Proton-Translocating ATPases; D058848:Receptors, Purinergic P2X4; D013947:Thymosin; D000097795:ATPase Inhibitory Protein; D000099201:Thymosin beta(4)