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

Abstract

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.

Neuroprotective and neurorestorative effects of thymosin β4 treatment initiated 6 hours after traumatic brain injury in rats

“…4,13,64 A candidate extracellular receptor of high affinity for Tβ4 is the β subunit of cell surface-located ATP synthase, which would allow extracellular Tβ4 to signal via a purinergic receptor to promote endothelial cell migration. 19 Tβ4 induces endothelial progenitor cell migration via the PI3K/Akt/eNOS signal transduction pathway. 51 Internalization of exogenous Tβ4 may be responsible for the antiapoptotic effects of exogenous Tβ4 on human corneal epithelial cells 24 Internalization of the exogenous Tβ4 followed by activation of integrin-linked kinase and Akt seem to be the major mechanisms that promote the survival of cardiomyocytes.…”

New insights regarding the regulation of chemotaxis by nucleotides, adenosine, and their receptors

“…Although there has been debate regarding its contribution to extracellular ATP synthesis [78], F1F0 synthase appears to be an important positive regulator of endothelial cell migration [79][80][81]. Interestingly, F1F0 synthase is also the major endothelial binding site for angiostatin, a potent inhibitor of angiogenesis and endothelial cell migration [82,83].…”

Thymosin beta 4 treatment improves left ventricular function after myocardial infarction and is related to Up-regulation of chitinase 3-like-1 in mice

“…Recently it was reported that TB4 increases extracellular ATP synthesis and possibly induces purine receptormediated endothelial cell migration [3]. This encouraged us to screen plasma samples for ATP and ADP and for activities of soluble purine-converting enzymes.…”

“…The activation of the Akt pathway decreases cell necrosis and apoptosis as well as inflammation after ischemic insult in the heart [2]. Recently TB4 was linked to increased cell surface ATP production by acting through extracellular ATP synthase and it was speculated that the increase in local ATP production could influence cell behavior through purinergic signaling [3].…”