Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL 61605, USA.
Mol Oncol. 2012 Feb;6(1):33-47. doi: 10.1016/j.molonc.2011.11.008. Epub 2011 Nov 30.
The uPA/uPAR system is known to play a critical role in angiogenesis of glioblastoma. Previously, we have shown that shRNA against uPA and uPAR attenuates angiogenesis by blocking nuclear translocation of angiogenin, inhibition of angiopoietin/Tie2 signaling, and regulating several other pro-angiogenic, angiostatic and anti-angiogenic molecules. Further analysis revealed that GM-CSF, a pleiotropic cytokine, was significantly inhibited in U87MG and 4910 co-cultures with endothelial cells transfected with shRNA against uPA and uPAR. The role of the uPA/uPAR system in this process is not completely understood. Analysis of tumor conditioned medium of U87MG, 4910 and HMECs transfected with shRNA against uPA or uPAR alone or in combination (pU2) revealed inhibition of GM-CSF-enhanced secretion of SVEGFR1 as shown by Western blotting and ELISA. Moreover, phosphorylation of JAK2 and STAT5, the downstream effectors of GM-CSF signaling, was also inhibited in all three cell lines. Phosphorylation at Tyr 166 position of the GM-CSFRβ subunit, the signal activating subunit of the GM-CSF receptor, was inhibited in HMEC, U87MG and 4910 cells. Further analysis revealed that shRNA against uPA and/or uPAR increased secretion of TIMP-1, which is known to enhance SVEGFR1 secretion in endothelial cells. Moreover, addition of purified uPA (with and without GM-CSF) activated JAK2/STAT5 signaling in HMEC. Exogenous addition of SVEGFR1 to pU2 tumor conditioned medium enhanced inhibition of VEGF-induced endothelial capillary tube formation as assessed by an in vitro angiogenesis assay. To determine the significance of these events in vivo, nude mice with pre-established tumors treated with shRNA against uPA and/or uPAR showed decreased levels of GM-CSF and increased levels of SVEGFR1 and TIMP-1 when compared with controls. Enhanced secretion of SVEGFR1 by puPA, puPAR and pU2 in endothelial and GBM cells was mediated indirectly by MMP-7 and augmented by ectodomain shedding of VEGFr1 by tyrosine phosphorylation at the 1213 position. Taken together, these results suggest that the uPA/uPAR system could prove beneficial as an indirect target for inhibition of angiogenesis in glioblastoma.
尿激酶型纤溶酶原激活物（uPA）/尿激酶型纤溶酶原激活物受体（uPAR）系统在胶质母细胞瘤的血管生成中起着关键作用。此前，我们已经表明，针对 uPA 和 uPAR 的 shRNA 通过阻断血管生成素的核易位、抑制血管生成素/Tie2 信号通路以及调节其他几种促血管生成、抗血管生成和抗血管生成分子，可减弱血管生成。进一步分析显示，在与转染针对 uPA 和 uPAR 的 shRNA 的内皮细胞共培养的 U87MG 和 4910 中，粒细胞-巨噬细胞集落刺激因子（GM-CSF）受到显著抑制。uPA/uPAR 系统在这一过程中的作用尚不完全清楚。分析单独或联合转染针对 uPA 或 uPAR 的 shRNA 的 U87MG、4910 和 HMECs 的肿瘤条件培养基表明，GM-CSF 增强的 SVEGFR1 分泌受到抑制，Western 印迹和 ELISA 均显示出这一点。此外，GM-CSF 信号通路的下游效应子 JAK2 和 STAT5 的磷酸化在这三种细胞系中也受到抑制。GM-CSF 受体的信号激活亚基 GM-CSFRβ 亚基 Tyr166 位的磷酸化在 HMEC、U87MG 和 4910 细胞中受到抑制。进一步分析显示，针对 uPA 和/或 uPAR 的 shRNA 增加了 TIMP-1 的分泌，TIMP-1 已知可增强内皮细胞中 SVEGFR1 的分泌。此外，添加纯化的 uPA（有或没有 GM-CSF）可激活 HMEC 中的 JAK2/STAT5 信号通路。在体外血管生成测定中，将 SVEGFR1 添加到 pU2 肿瘤条件培养基中可增强对 VEGF 诱导的内皮毛细血管形成的抑制作用。为了确定这些事件在体内的意义，与对照组相比，用针对 uPA 和/或 uPAR 的 shRNA 处理预先建立肿瘤的裸鼠显示 GM-CSF 水平降低，SVEGFR1 和 TIMP-1 水平升高。uPA、uPAR 和 pU2 在内皮细胞和 GBM 细胞中对 SVEGFR1 的增强分泌是间接通过 MMP-7 介导的，并且通过 1213 位酪氨酸磷酸化增强了 VEGFr1 的外显子脱落。总之，这些结果表明，uPA/uPAR 系统作为胶质母细胞瘤血管生成的间接靶点可能是有益的。
