Busceti Carla L, Marchitti Simona, Bianchi Franca, Di Pietro Paola, Riozzi Barbara, Stanzione Rosita, Cannella Milena, Battaglia Giuseppe, Bruno Valeria, Volpe Massimo, Fornai Francesco, Nicoletti Ferdinando, Rubattu Speranza
IRCCS NEUROMED - Istituto Neurologico Mediterraneo Pozzilli, Italy.
IRCCS NEUROMED - Istituto Neurologico MediterraneoPozzilli, Italy; Department of Physiology and Pharmacology, Sapienza University of RomeRome, Italy.
Front Pharmacol. 2017 Mar 14;8:111. doi: 10.3389/fphar.2017.00111. eCollection 2017.
Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs.
Dkk-3是Wnt信号通路分泌抑制剂Dickkopf蛋白家族的成员，已被证明可促进血管生成。目前尚不清楚这种作用的潜在机制。在这里，我们使用培养的人脐静脉内皮细胞（HUVECs）来研究转化生长因子-β（TGF-β）和血管内皮生长因子（VEGF）在Dkk-3血管生成作用中的参与情况。通过逆转录聚合酶链反应（RTPCR）、免疫印迹、免疫细胞化学和酶联免疫吸附测定（ELISA）评估，向HUVECs中添加人重组Dkk-3肽（1或10 ng/ml）6或12小时可提高细胞内和细胞外VEGF蛋白水平。细胞外VEGF水平的增加与血管内皮生长因子受体2（VEGFR2）的激活有关。VEGFR2的药理学阻断消除了Dkk-3诱导的内皮细胞管形成，表明VEGF是Dkk-3血管生成作用的分子参与者。此外，Dkk-3增强了Smad1/5/8的磷酸化，并将Smad4募集到VEGF基因启动子上，表明Dkk-3激活了激活素受体样激酶1（ALK1）受体，导致VEGF的转录激活。这种机制有助于Dkk-3介导的VEGF表达增加和内皮细胞管形成，因为这两种作用都被小干扰RNA（siRNA）介导的ALK1敲低所消除。总之，我们发现Dkk-3激活ALK1以刺激VEGF产生并诱导HUVECs中的血管生成。
