Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor-2 expression by suppressing Sp1-dependent DNA binding in human endothelial cells.

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

Post-translational modifications of Abutilon mosaic virus movement protein (BC1) in fission yeast.

The movement protein (MP) of Abutilon mosaic virus (AbMV, Geminiviridae) exhibited a complex band pattern upon gel electrophoresis indicating its post-translational modification when expressed in AbMV-infected plants or, ectopically, in fission yeasts. High-resolution separation according to charge and molecular weight in acetic acid/urea polyacrylamide or sodium dodecyl sulphate polyacrylamide gels followed by western blot analysis revealed a pattern of AbMV MP from infected plants more related to that from fission yeast than from bacteria. For this reason, expression in fission yeast was established as an experimental system to study post-translational modifications of AbMV MP. Metabolic labelling with 32P-orthophosphate confirmed a phosphorylation of all MP variants suggesting multiple phosphorylation sites. Treatment with calf intestinal alkaline phosphatase removed this label completely, but was unable to eliminate all protein bands with lower electrophoretic mobility. Thus, multiple phosphorylations contribute to the complex migration behaviour of MP, but additional post-translational modifications may occur as well.

PPARalpha activators inhibit vascular endothelial growth factor receptor-2 expression by repressing Sp1-dependent DNA binding and transactivation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known antiinflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. In this study, PPARalpha agonists are found to inhibit endothelial VEGFR2 expression, whereas predominant PPARgamma ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. Inhibitory effects of PPARalpha agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between base pairs -60 and -37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1-containing complex formation to this sequence is decreased by PPARalpha treatment, indicating that VEGFR2 gene expression is inhibited by repressing Sp1 site-dependent DNA binding and transactivation. Our coimmunoprecipitation experiments revealed enhanced protein interactions between PPARalpha and Sp1 on PPARalpha activation, thus constituting a probable mechanism by which PPARalpha activators decrease Sp-dependent binding activity to the VEGFR2 promoter. Hence, molecular mechanisms by which PPARs modulate the rate of gene transcription may include direct interactions between specific transcription factors and PPARs that ultimately result in reduced DNA binding to their respective response elements.

Fluid shear stress-induced transcriptional activation of the vascular endothelial growth factor receptor-2 gene requires Sp1-dependent DNA binding.

Hemodynamic forces play a fundamental role in the regulation of endothelial cell survival. As signaling via the vascular endothelial growth factor (VEGF) receptor-2 pathway has been previously demonstrated to impact endothelial cell survival, we hypothesized that laminar shear stress may facilitate survival in part by inducing VEGF receptor-2 expression. This study shows a time- and dose-dependent upregulation of endothelial VEGF receptor-2 expression by fluid shear stress in microvascular and large-vessel derived endothelial cells. A functional analysis of the 5'-regulatory region of the VEGF receptor-2 promoter localized the shear stress-response element to a sequence between bp -60 and -37 that encompasses two adjacent consensus Sp1 transcription factor binding sites. Constitutive and shear stress-inducible Sp1-dependent complexes are bound to this element, indicating that fluid shear stress-induced transcriptional activation of the VEGF receptor-2 gene requires Sp1-dependent DNA binding. Together, these results suggest that biomechanical stimulation may lead to endothelial cell survival by upregulating VEGF receptor-2 expression.

Down-regulation of vascular endothelial growth factor receptor 2 is a major molecular determinant of proteasome inhibitor-mediated antiangiogenic action in endothelial cells.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. This system controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. Recent evidence also established the importance of the proteasome in tumor development, showing antitumor and antiangiogenic actions by using selective inhibitors in vivo. As signaling via the vascular endothelial growth factor receptor 2 (VEGFR2) pathway is critical for angiogenic responses to occur, we explored whether antiangiogenic effects due to proteasome inhibition were partly mediated through decreased endothelial VEGFR2 expression. This study shows that different proteasome inhibitors blocked VEGFR2 expression in a time-dependent and concentration-dependent manner. This blockade was paralleled by the respective inhibition of the formation of capillary-like structures and endothelial cell migration. In contrast, neither tie-2 nor VEGFR1 expression was significantly affected by proteasome inhibitor treatment. The suppressive effects on VEGFR2 expression were not conveyed by increased shedding or a decrease in protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. In line with this conclusion, proteasome inhibition significantly suppressed VEGFR2 mRNA accumulation. In addition, inhibitor treatment considerably decreased the transcriptional activity of 5' deletional VEGFR2 promoter gene constructs. Proteasome inhibition-mediated repression was controlled by a GC-rich region that harbored one consensus Sp1-binding site. Subsequent EMSA analyses showed decreased constitutive Sp1-dependent DNA binding in response to proteasome inhibition. In addition, we could show that proteasome inhibitors reduced VEGFR2 mRNA stability. Therefore, VEGFR2 expression may constitute a critical molecular target of proteasome inhibitors that may mediate their antiangiogenic effects in vivo.

Microtubule-targeted drugs inhibit VEGF receptor-2 expression by both transcriptional and post-transcriptional mechanisms.

Cytoskeletal polymers control a wide range of cellular functions, including proliferation, migration, and gene expression. As changes in endothelial cell shape and motility are required to form vascular networks, we hypothesized that disassembly of actin filaments or microtubules may impact endothelial vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) expression as a critical determinant of angiogenesis. We therefore investigated the effect of actin filament- and microtubule-disrupting agents on VEGFR1 and VEGFR2 expression by endothelial cells. Microtubule (MT) disassembly greatly inhibited endothelial VEGFR2 expression, whereas VEGFR1 expression levels remained largely unchanged. These suppressive effects were neither conveyed by increased VEGFR2 shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms account for the observed effects. In line with this conclusion, MT disruption significantly suppressed endothelial VEGFR2 mRNA accumulation. The treatment considerably decreased transcriptional activity of 5'-deletional VEGFR2 promoter gene constructs. MT disruption-mediated repression was conveyed by a GC-rich region harboring two consensus Sp1-binding sites. Electrophoretic mobility-shift assay analysis demonstrated that constitutive Sp1-dependent DNA binding is decreased by MT disassembly. In addition, we provide evidence for additional post-transcriptional regulatory mechanisms, as the VEGFR2 mRNA half-life is significantly reduced by MT-disrupting agents. Hence, both inhibition of the rate of gene transcription and increased mRNA turnover represent critical molecular mechanisms by which MT disruption inhibits VEGFR2 expression.