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The actin binding site on thymosin ß₄ promotes angiogenesis¹

DEBORAH PHILP, THOMAS HUFF^*, YONG SONG GHO, EWALD HANNAPPEL^* and HYNDA K. KLEINMAN²

Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA;
^* Institute of Biochemistry, University of Erlangen-Nuremberg, Erlangen, Germany; and
Graduate School of East-West Medical Science, Kyunghee University, Yong In, Korea

²Correspondence: NIH, NIDCR, 30/433, 30 Convent Dr. MSC-4370, Bethesda, MD 20892, USA. E-mail: hkleinman{at}dir.nidcr.nih.gov

SPECIFIC AIMS

Thymosin ß₄, a ubiquitous 43 amino acid, 5 kDa polypeptide, is angiogenic and can promote endothelial cell migration and adhesion, tubule formation, aortic ring sprouting, and angiogenesis. Using naturally occurring thymosin ß₄ proteolytic fragments and synthetic peptides, the present study determined which portion of the thymosin ß₄ peptide is essential for its angiogenic activity.

PRINCIPAL FINDINGS

1. Actin-mediated HUVEC cell adhesion to thymosin ß₄
Actin has been reported to be on the cell surface of endothelial cells and to be important in angiogenin-mediated angiogenesis. Since thymosin ß₄ contains an actin binding domain, we added actin to the cell adhesion assay and found that soluble actin blocked adhesion to thymosin ß₄ substrates (Table 1 A). As a control, soluble actin had no effect on laminin-1-mediated adhesion. These data demonstrate that actin can block thymosin ß₄-mediated adhesion and suggest that the actin binding site on thymosin ß₄ is important in cell binding to thymosin ß₄.

2. Actin blocks aortic ring vessel sprouting in the presence of thymosin ß₄
Thymosin ß₄ has been shown to promote vessel sprouting from rat aorta and from pig coronary artery rings in a dose-dependent manner. We find that actin has angiogenic activity in this model system (Table 1 B). Actin has also been found to promote angiogenesis in the CAM assay (Y. S. Gho, unpublished results). Neither thymosin ß₄ nor actin was as active as the ECGS-positive control in promoting sprouting. Thymosin ß₄-mediated sprouting was significantly blocked by the addition of exogenous actin (Table 1 B). The effect of exogenous actin on thymosin ß₄-mediated angiogenesis is specific since exogenous actin did not inhibit ECGS-mediated vessel sprouting. Some increase is observed in sprouting when actin and ECGS are added together. These molecules are both angiogenic and their affects appear to be additive. These data demonstrate that thymosin ß₄ promotes angiogenesis ex vivo and that exogenous actin blocks this activity.

3. Identification of the actin binding domain on thymosin ß₄ for its angiogenic activity
We next determined the active site on thymosin ß₄ for endothelial cell migration and vessel sprouting using various synthetic peptides and proteolytic fragments of thymosin ß₄. Synthetic and naturally occurring thymosin ß₄ fragments were active in both assays (Table 2 and data not shown). Thymosin ß₄-sulfoxide was also active. Synthetic peptides and proteolytic fragments containing either the carboxyl-terminal part or all of the actin binding sequence (Tß₄^1–26, Tß₄^13–23, Tß₄^7–43, Tß₄^13–43, Tß₄^10–28, Tß₄^20–43, and Tß₄^17–23) were generally highly active in the assays. In contrast, synthetic peptides and proteolytic fragments lacking the actin binding sequence (Tß₄^1–6 and Tß₄^13–19) were inactive with Tß₄^1–15, showing very weak activity in only the sprouting assay. The smallest active synthetic peptide, Tß₄^17–23, contained seven amino acids, was fully active for migration and sprouting, and was able to block attachment to thymosin ß₄ (Table 2 , Fig. 1 ). All other members of the ß-thymosins (ß₄-sulfoxide, ß₄ Ala, ß₉, and ß₁₀) with a conserved actin binding domain were also active (Table 2) . The actin binding domain of thymosin ß₁₅ contained within amino acids 13-23 was active in the sprouting assay. These data demonstrate that the actin binding site contained within thymosin ß₄ is active for angiogenesis. This site appears to be the major active domain since the seven amino acid actin binding peptide blocks adhesion to thymosin ß₄. Related family members with sequence homology are also predicted to have this activity.

View larger version (17K): [in this window] [in a new window]   Figure 1. Endothelial cells adhere to thymosin ß4, and aortic ring sprouting of vessels is increased in its presence. The actin binding peptide from thymosin ß4 promotes endothelial cell migration and aortic ring sprouting, like the parent molecule. When the actin binding peptide LKKTETQ is added to the assay, adhesion to thymosin ß4 is blocked. Soluble actin blocks thymosin ß4-induced adhesion and sprouting, suggesting that actin is the cell surface ligand for thymosin ß4.

CONCLUSIONS

Thymosin ß₄ has been found to promote angiogenesis. Here we have localized the motif for this activity to the central actin binding domain using seven different synthetic peptides and six different proteolytic fragments. Peptides and fragments containing the entire actin binding domain (LKKTETQ) were active for endothelial cell migration and vessel sprouting, whereas those lacking this sequence were inactive. A seven amino acid peptide, Tß₄^17–23 (LKKTETQ), was the smallest peptide tested that was able to duplicate the activity of the intact molecule (Fig. 1) . Actin has been shown to promote angiogenesis (Y. S. Gho and H. K. Kleinman, unpublished data), and it showed activity in our ex vivo vessel sprouting assay. When added together with thymosin ß₄, instead of further increasing sprouting activity, reduced activity was observed. Exogenous actin blocked cell adhesion to thymosin ß₄, but not to laminin-1. These data suggest that thymosin ß₄ interacts with endothelial cells to initiate angiogenesis via binding of the central actin binding domain to cell surface actin. We found that this actin binding seven amino acid peptide blocked cell adhesion to thymosin ß₄ in a dose-dependent manner.

Actin is an abundant protein present in all cells. It is important in maintaining cell structure and regulating cell motility. Actin has previously been found to be an endothelial cell surface molecule that functions as a ligand for another adhesive angiogenic molecule, angiogenin. Actin, however, has not been definitively demonstrated on the cell surface. Only one paper has shown this, and more work needs to be done. The receptor for thymosin ß₄ is not known, but thymosin ß₄ binding to cells and internalization have been demonstrated. Similar to the work described here, soluble exogenous actin blocked the angiogenic activity of angiogenin in the chick chorioallantoic membrane assay. Furthermore, antagonistic peptides that block the binding of angiogenin to actin inhibit its activity. Both thymosin ß₄ and angiogenin increase protease activity. Peptides antagonistic to the angiogenin receptor that block its binding to actin block protease activity and tumor growth and metastasis in vivo. What remains to be determined is whether actin is the cell surface receptor for these actin binding molecules or if another ligand(s) is present.

Within the ß-thymosin family, the actin binding motif is highly conserved. Most of the heterogeneity of these molecules occurs at the carboxyl terminus. Based on the conservation of the actin binding sequence, it was not surprising that the other members of the ß-thymosin family tested were active for endothelial cell migration and vessel sprouting. Thymosin ß₁₅ is the only ß-thymosin possessing an amino acid substitution within the expected actin binding domain LKKTNT instead of LKKTET. Thymosin ß₁₅ is increased in prostate cancer and is reported to be important in cell migration. It is possible that thymosin ß₁₅ acts not only by increasing cell migration but also by increasing angiogenesis, which would contribute to the malignant potential of these tumor cells. Other investigators have shown that thymosin ß₄ levels are elevated in metastatic lesions; when transfected into tumor cells, malignancy in vitro and in vivo is increased. Furthermore, the sulfoxide of thymosin ß₄ has been described to possess anti-inflammatory activity. It likely acts in malignancy to increase migration and angiogenesis and to reduce immune surveillance, but this has yet to be determined.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0121fje; doi: 10.1096/fj.03-0121fje

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