uPA and uPAR shRNA inhibit angiogenesis via enhanced secretion of SVEGFR1 independent of GM-CSF but dependent on TIMP-1 in endothelial and glioblastoma cells.

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.

Raghu H ，Nalla AK ，Gondi CS ，Gujrati M ，Dinh DH ，Rao JS ... -  《-》

Suppression of uPA and uPAR attenuates angiogenin mediated angiogenesis in endothelial and glioblastoma cell lines.

Raghu H ，Lakka SS ，Gondi CS ，Mohanam S ，Dinh DH ，Gujrati M ，Rao JS ... -  《PLoS One》

Specific knockdown of uPA/uPAR attenuates invasion in glioblastoma cells and xenografts by inhibition of cleavage and trafficking of Notch -1 receptor.

Raghu H ，Gondi CS ，Dinh DH ，Gujrati M ，Rao JS ... -  《Molecular Cancer》

Interleukin-4 and granulocyte-macrophage colony-stimulating factor mediates the upregulation of soluble vascular endothelial growth factor receptor-1 in RAW264.7 cells-a process in which p38 mitogen-activated protein kinase signaling has an important role

Soluble vascular endothelial growth factor receptor-1 (sVEGFR1) antagonizes angiogenesis by inhibiting the biological function of vascular endothelial growth factor (VEGF). Immature dendritic cells (imDCs) express high levels of sVEGFR1 during development and are antiangiogenic. This study aimed to investigate the changes in VEGFR1, sVEGFR1, and VEGF levels during the development of imDCs and explore the underlying signaling mechanisms. To model the differentiation of imDCs from monocytes, RAW264.7 cells, a murine monocyte/macrophage cell line, were stimulated by interleukin-4 (IL-4; 10 ng/mL, 20 ng/mL, and 40 ng/mL) and/or by granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/mL, 20 ng/mL, and 50 ng/mL) and/or pretreated by the p38 inhibitor SB203580. The levels of VEGFR1, sVEGFR1, and VEGF were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot, and enzyme-linked immunosorbent assay (ELISA). IL-4 increased the VEGFR1 mRNA and sVEGFR1 levels in RAW264.7 (p < 0.05). This increase was inhibited by SB203580. Granulocyte-macrophage colony-stimulating factor increased the sVEGFR1 levels, but it had no significant effect on VEGFR1 mRNA levels. SB203580 decreased the expression of VEGFR1 mRNA induced by GM-CSF, whereas sVEGFR1 was unaffected. IL-4 had a greater effect on sVEGFR1 levels, compared to GM-CSF. IL-4 and GM-CSF increased sVEGFR1 levels, but did not significantly effect VEGF expression, and led to the antiangiogenesis properties of monocytes. p38 Mitogen-activated protein kinase signaling has an important role in the process.

Xia L ，Dong Z ，Zhang Y ，Zhang X ，Song X ，Sun M ，Hu Y ，Liu S ，Wang K ，Qu X ，Wei F ... -  《-》

siRNA-mediated simultaneous downregulation of uPA and its receptor inhibits angiogenesis and invasiveness triggering apoptosis in breast cancer cells.

Subramanian R ，Gondi CS ，Lakka SS ，Jutla A ，Rao JS ... -  《INTERNATIONAL JOURNAL OF ONCOLOGY》