Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor κB-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-Kinase activity in breast cancer cells

Tumor growth and metastasis are multifaceted processes that mainly involve cell adhesion, proteolytic degradation of the extracellular matrix, and cell migration. Syk is a member of a tyrosine kinase family that is expressed mostly in hematopoietic cells. Syk is expressed in cell lines of epithelial origin, but its function in these cells remains unknown. Here we report that Syk is expressed in MCF-7 cells but not in MDA-MB-231 cells. The overexpression of wild type Syk kinase but not kinase-negative Syk suppressed cell motility and inhibited the activation of phosphatidylinositol (PI) 3'-kinase in MDA-MB-231 cells. In contrast, when Syk-specific antisense S-oligonucleotide but not the sense S-oligonucleotide was transfected to MCF-7 cells the level of PI 3'-kinase activity as well as cell motility were increased. The MDA-MB-231 cells transfected with wild type Syk cDNA followed by treatment with piceatannol, a Syk inhibitor, enhanced cell motility and PI 3'-kinase activity. Pervanadate, a phosphotyrosine phosphatase inhibitor, induced PI 3'-kinase activity and stimulated the interaction between the inhibitor of nuclear factor κBα (IκBα) and the p85α domain of PI 3'-kinase through tyrosine phosphorylation of the IκBα, which ultimately resulted in nuclear factor κB (NFκB) activation. Pervanadate had no effect on the activation of Syk in these cells. However, Syk suppressed the NFκB transcriptional activation and interaction between IκBα and PI 3'-kinase by inhibiting the tyrosine phosphorylation of IκBα. Syk, PI 3'-kinase inhibitors, and NFκB inhibitory peptide inhibited urokinase type plasminogen activator (uPA) secretion and cell motility in these cells. To our knowledge, this is the first report that Syk suppresses the cell motility and inhibits the PI 3'-kinase activity and uPA secretion by blocking NFκB activity through tyrosine phosphorylation of IκBα. These data further demonstrate a functional molecular link between Syk-regulated PI 3'-kinase activity and NFκB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells

Tyrosine kinase, p56<SUP>lck</SUP>-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways

We have recently reported that tyrosine kinase, p56lck regulates cell motility and nuclear factor &#954;B-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of I&#954;B&#945; following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56lck and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56lck in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells

Integrin signaling is critical for pathological angiogenesis

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis

The angiogenic response is dictated by β3 integrin on bone marrow–derived cells

Angiogenesis is dependent on the coordinated action of numerous cell types. A key adhesion molecule expressed by these cells is the αvβ3 integrin. Here, we show that although this receptor is present on most vascular and blood cells, the key regulatory function in tumor and wound angiogenesis is performed by β3 integrin on bone marrow–derived cells (BMDCs) recruited to sites of neovascularization. Using knockin mice expressing functionally stunted β3 integrin, we show that bone marrow transplantation rescues impaired angiogenesis in these mice by normalizing BMDC recruitment. We demonstrate that αvβ3 integrin enhances BMDC recruitment and retention at angiogenic sites by mediating cellular adhesion and transmigration of BMDCs through the endothelial monolayer but not their release from the bone niche. Thus, β3 integrin has the potential to control processes such as tumor growth and wound healing by regulating BMDC recruitment to sites undergoing pathological and adaptive angiogenesis

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2

β3 phosphorylation of platelet αIIbβ3 is crucial for stability of arterial thrombus and microparticle formation in vivo

Abstract Background It is well accepted that functional activity of platelet integrin αIIbβ3 is crucial for hemostasis and thrombosis. The β3 subunit of the complex undergoes tyrosine phosphorylation shown to be critical for outside-in integrin signaling and platelet clot retraction ex vivo. However, the role of this important signaling event in other aspects of prothrombotic platelet function is unknown. Method Here, we assess the role of β3 tyrosine phosphorylation in platelet function regulation with a knock-in mouse strain, where two β3 cytoplasmic tyrosines are mutated to phenylalanine (DiYF). We employed platelet transfusion technique and intravital microscopy for observing the cellular events involved in specific steps of thrombus growth to investigate in detail the role of β3 tyrosine phosphorylation in arterial thrombosis in vivo. Results Upon injury, DiYF mice exhibited delayed arterial occlusion and unstable thrombus formation. The mean thrombus volume in DiYF mice formed on collagen was only 50% of that in WT. This effect was attributed to DiYF platelets but not to other blood cells and endothelium, which also carry these mutations. Transfusion of isolated DiYF but not WT platelets into irradiated WT mice resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion times. DiYF platelets exhibited reduced adhesion to collagen under in vitro shear conditions compared to WT platelets. Decreased platelet microparticle release after activation, both in vitro and in vivo, were observed in DiYF mice compared to WT mice. Conclusion β3 tyrosine phosphorylation of platelet αIIbβ3 regulates both platelet pro-thrombotic activity and the formation of a stable platelet thrombus, as well as arterial microparticle release

The pY747 peptide has no effect on β3^−/− or DiYF mice.

<p><b>a</b>) pY747 peptide does not inhibit VEGF-induced aortic ring growth from β3^−/− mice. Mouse aortic rings were embedded in matrigel in the presence of 40 ng/mL of VEGF and 40 µM of peptides as indicated. <b>b</b>) Quantification of aortic ring assay as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031071#pone-0031071-g004" target="_blank">Fig. 4a</a>. <b>c</b>) pY747 could not inhibit bFGF-induced aortic ring growth, Mouse aortic rings were isolated from wild type (WT), β3^−/−, and DiYF mice and embedded in matrigel in the presence of 40 ng/mL of VEGF, 20 ng/mL of bFGF or pY747 peptides as indicated. Aortic rings were incubated for 3 days for wild type and β3^−/− aortic rings and 4 days for DiYF aortic rings (longer incubation was used to obtain visible aortic sprouting which is diminished in these mice). <b>d</b>) pY747 peptide does not inhibit angiogenesis in DiYF mice. Peptides' effect on <i>in vivo</i> angiogenesis in wild type mice and DiYF mice was tested as described. <b>e</b>) Quantification of blood vessels in matrigel plus assay as indicated.</p