Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras 26504, Greece.
J Cell Biochem. 2011 Jun;112(6):1532-43. doi: 10.1002/jcb.23066.
Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.
多效蛋白（PTN）是一种肝素结合生长因子，在肿瘤生长和血管生成中起着重要作用。我们之前已经表明，为了使 PTN 诱导内皮细胞迁移，需要与α（ν）β（3）整合素及其受体蛋白酪氨酸磷酸酶β/ζ（RPTPβ/ζ）结合。在本研究中，我们表明，对应于 PTN 羧基末端区域的最后 25 个氨基酸的合成肽（PTN（112-136））抑制了体内鸡胚绒毛尿囊膜（CAM）试验中的血管生成以及 PTN 诱导的人内皮细胞迁移和管状形成。PTN（112-136）抑制了 PTN 与α（ν）β（3）整合素的结合，并且如表面等离子体共振（SPR）测量所示，特异性地与β（3）的细胞外域的特异性环相互作用。此外，它像中和α（ν）β（3）-选择性抗体的作用一样，消除了 PTN 诱导的 FAK Y397 磷酸化。PTN（112-136）不影响 PTN 与内皮细胞中 RPTPβ/ζ 的结合，并且以与 PTN 相似的程度诱导β（3）Y773 磷酸化和 ERK1/2 激活。该作用通过 siRNA 下调 RPTPβ/ζ 或通过 c-src 抑制来抑制，表明 PTN（112-136）可能与 RPTPβ/ζ 相互作用。NMR 光谱研究表明，PTN（112-136）在室温下具有构象灵活性且不存在任何二级结构元素的特征，尽管生物活性肽段 123-132 可能在较低温度下采用确定的结构。总的来说，我们的数据表明，尽管 PTN（112-136）诱导了由 PTN 触发的一些信号通路，但它通过抑制 PTN 与α（ν）β（3）整合素的结合来抑制 PTN 诱导的血管生成活性。
