Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein

AbstractUrokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP), but independent of the phosphatidylinositol 3 (PI3) kinase pathway, whereas vascular endothelial growth factor (VEGF)–induced antiapoptosis was PI3 kinase dependent. uPA-induced cell survival involved phosphorylation of p21-activated kinase 1 (Pak1) and the IκB kinase α that leads to nuclear factor κB (NF-κB) p52 activation. Indeed, blocking NF-κB activation by using specific NF-κB inhibitors abolished uPA-induced cell survival as it blocked uPA-induced XIAP up-regulation. Furthermore, down-regulating XIAP expression by small interfering RNA (siRNA) significantly reduced uPA-dependent endothelial cell survival. This mechanism is also important for VEGF-induced antiapoptosis because VEGF-dependent up-regulation of XIAP was found defective in uPA−/− endothelial cells. This led us to conclude that uPA is part of a novel NF-κB–dependent cell survival pathway.

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Urokinase (uPA) Induces Endothelial Cell Survival Via the X-Linked Inhibitor of Apoptosis Protein (XIAP).

Blood ◽

10.1182/blood.v110.11.3712.3712 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3712-3712

Author(s):

Gerald W. Prager ◽

Patrick M. Brunner ◽

Judit Mihaly ◽

Yuri Koschelnick ◽

Bernd R. Binder

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Cell Survival ◽

Pi3 Kinase ◽

Endothelial Cell Migration ◽

Inhibitor Of Apoptosis ◽

Inhibitor Of Apoptosis Protein ◽

Xiap Expression ◽

Apoptosis Protein

Abstract uPA plays an important role in angiogenesis: Originally, the urokinase system has been implicated to assist the angiogenic process by it’s proteolytic properties. It is now becoming increasingly evident that uPA additionally elicits many pro-angiogenic responses like differentiation, proliferation and cell migration in a non-proteolytic fashion via induction of intracellular signal transduction. In this study we demonstrate that in endothelial cells uPA protects against apoptosis by transcriptional upregulation of inhibitor of apoptosis proteins (IAPs), among them most prominently the X-linked inhibitor of apoptosis protein (XIAP). In contrast to canonical growth factors, like vascular endothelial growth factor (VEGF), uPA elicits anti-apoptosis independently of the PI3-kinase pathway. uPA-induced cell survival is dependent on the type of extracellular matrix used indicating the involvement of integrin adhesion receptors. Thereby, uPA induces phosphorylation of the CDC42 downstream effector p21-activated kinase 1 (PAK1), which leads to IkappaB kinase alpha (IKKa) phosphorylation, a prerequisite for NFkappaB activation. Blocking NFkappaB by using the specific NFkappaB inhibitor BAY 11–7082 or by adenoviral-mediated overexpression of its inhibitor, IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival. Downregulating XIAP expression by small interfering RNA techniques significantly reduces cell survival efficiencies of uPA in endothelial cells. From these data we conclude that uPA activation, which is a main player in endothelial cell migration and invasion, provides an additional, PI3-kinase independent cell survival mechanism.

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X-Linked Inhibitor of Apoptosis Protein (XIAP) Supports Urokinase (uPA)-Induced Endothelial Cell Survival

Blood ◽

10.1182/blood.v112.11.5461.5461 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5461-5461

Author(s):

Gerald W Prager ◽

Judit Mihaly ◽

Patrick Brunner ◽

Christoph Zielinski ◽

Bernd Binder

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Cell Survival ◽

Endothelial Cell Migration ◽

Migration And Invasion ◽

Inhibitor Of Apoptosis ◽

Inhibitor Of Apoptosis Protein ◽

Endothelial Cell Survival ◽

Apoptosis Protein

Abstract High uPA expressing tumors are associated with poor prognosis. While a direct effect on tumor cell behavior is described, uPA has especially been shown to mediate (tumor-) angiogenesis. Originally, the urokinase system has been implicated to assist the angiogenic process by it’s proteolytic activities. It is now becoming increasingly evident that uPA additionally elicits a whole array pro-angiogenic responses like differentiation, proliferation and cell migration, independent of its proteolytic activity by inducing intracellular signal transduction. Here we show that uPA induces upregulation of inhibitor of apoptosis proteins (IAPs), which protects endothelial cells against apoptosis. Thereby, uPA-induced endothelial cell survival is mediated by transcriptional upregulation the X-linked inhibitor of apoptosis protein (XIAP), because downregulation of XIAP by small interfering RNA techniques significantly reduces uPA mediated cell survival efficiencies of uPA in endothelial cells. The antiapoptotic activity of uPA was dependent on the presence of uPAR and LRP, but independent of the PI3kinase pathway, while VEGF-dependent antiapoptosis is mainly PI3kinase dependent. uPA-induced cell survival is dependent on the type of extracellular matrix on which cells are attached used indicating the involvement of integrin adhesion receptors. TherebyConsistently, uPA induces phosphorylation of the CDC42 downstream effector p21-activated kinase 1 (PAK1), which leads to IkappaB kinase alpha (IKKa) phosphorylation, a prerequisite for NFkappaB activation. As a consequence, p52/p50 but not p65 is are translocated into the nucleus. Blocking NFkappaB by using the specific NFkappaB inhibitor BAY 11–7082 or by adenoviral-mediated overexpression of its inhibitor, IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival. From these data we conclude that uPA, which is a main player in endothelial cell migration and invasion, provides an additional, PI3-kinase independent but NFkappaB dependent cell survival mechanism.

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X-Linked Inhibitor of Apoptosis Protein Is an Important Regulator of Vascular Endothelial Growth Factor–Dependent Bovine Aortic Endothelial Cell Survival

Circulation Research ◽

10.1161/circresaha.107.163667 ◽

2008 ◽

Vol 102 (8) ◽

pp. 896-904 ◽

Cited By ~ 29

Author(s):

Jongmin Kim ◽

Jongbong Park ◽

Seungmin Choi ◽

Sung-Gil Chi ◽

Amy L. Mowbray ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cell ◽

Inhibitor Of Apoptosis ◽

Bovine Aortic Endothelial Cell ◽

Vascular Endothelial ◽

Inhibitor Of Apoptosis Protein ◽

Endothelial Cell Survival ◽

Apoptosis Protein ◽

Important Regulator ◽

Endothelial Growth Factor

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Vascular Endothelial Growth Factor (VEGF) Induces Cell Survival Via an Additional Urokinase (uPA)-Dependent Mechanism.

Blood ◽

10.1182/blood.v104.11.3919.3919 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3919-3919

Author(s):

Gerald W. Prager ◽

Yuri Koshelnick ◽

Judit Mihaly ◽

Patrick Brunner ◽

Bernd R. Binder

Keyword(s):

Endothelial Cell ◽

Growth Factor ◽

Cell Survival ◽

Pi3 Kinase ◽

Endothelial Cell Migration ◽

Inhibitor Of Apoptosis ◽

Dependent Manner ◽

Independent Manner ◽

Apoptosis Protein ◽

Nf Kappab

Abstract VEGF, the most important angiogenic growth factor, is known to induce cell-survival mainly via phosphorylation of the pro-apoptotic proteins BAD, PED/PEA-2 or pro-caspase-9 or inhibition of SAPKs (stress activated kinases). These mechanisms are all dependent on the PI3-kinase/Akt pathway. We could show recently that VEGF also induces pro-uPA activation via the PI3-kinase signaling pathway beside and independent of the transcriptional upregulation of uPA. Active uPA does not only contribute to angiogenesis via its proteolytic properties, but also effectuates itself pro-angiogenic signalling by the induction of endothelial cell migration, proliferation and differentiation. We were interested whether generation of uPA upon VEGF is inducing an additional effect on endothelial cell survival. First, we compared VEGF with urokinase in respect to cell survival in apoptosis assays and observed a pivotal anti-apoptotic effect of both stimuli, dose dependently and dependent on the type of matrix used. In addition, cell survival effects were additive, when both stimuli were added simultaneously. While VEGF-induced cell survival was PI3-kinase dependent, because it could be inhibited by the specific PI3-kinase inhibitor LY294002, the uPA-induced cell survival was PI3-kinase independent. Furthermore, uPA was able to rescue apoptosis induced by PI3-kinase inhibition in VEGF stimulated endothelial cells. From these data we conclude that uPA is indeed inducing an additional - PI3-kinase independent - cell survival mechanism. While VEGF led to a PI3-kinase dependent phosphorylation of Akt, which resulted in CDC42 activation, uPA activated CDC42 and its downstream effectors PAK leading to IKK-1 phosphorylation in a PI3-kinase/Akt independent manner. This indicates that the anti-apoptotic properties of uPA are not Akt, but NF-kappaB mediated. Indeed, when we used adenovirus overexpressing I-kappaB to block the NF-kappaB pathway, uPA was ineffective to support cell survival. In addition VEGF and uPA, both induced a transcriptional upregulation of inhibitor of apoptosis proteins (IAPs) in an NF-kappaB-dependent manner, among them most significantly the X-linked inhibitor of apoptosis protein (XIAP); again VEGF in a PI3-kinase dependent, uPA in a PI3-kinase independent manner. From these data we conclude that VEGF is inducing cell survival in a strictly PI3-kinase dependent manner, on the one hand via its known mitochondrial pathway, but also via the PI3-kinase dependent pro-uPA activation leading to an NFkappa B dependent upregulation of inhibitor of antiapoptosis proteins.

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Vascular Endothelial Genes That Are Responsive to Tumor Necrosis Factor- In Vitro Are Expressed in Atherosclerotic Lesions, Including Inhibitor of Apoptosis Protein-1, Stannin, and Two Novel Genes

Blood ◽

10.1182/blood.v93.10.3418.410k23_3418_3431 ◽

1999 ◽

Vol 93 (10) ◽

pp. 3418-3431 ◽

Cited By ~ 107

Author(s):

Anton J.G. Horrevoets ◽

Ruud D. Fontijn ◽

Anton Jan van Zonneveld ◽

Carlie J.M. de Vries ◽

Jan Wouter ten Cate ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Necrosis ◽

Inhibitor Of Apoptosis ◽

Vascular Endothelial ◽

Inhibitor Of Apoptosis Protein ◽

Atherosclerotic Lesions ◽

Apoptosis Protein ◽

Novel Transcripts ◽

Necrosis Factor

Activation and dysfunction of endothelial cells play a prominent role in patho-physiological processes such as atherosclerosis. We describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by monocyte conditioned medium or tumor necrosis factor-. A minority of the fragments (22/106) represent known genes involved in various processes, including leukocyte trafficking, vesicular transport, cell cycle control, apoptosis, and cellular protection against oxidative stress. Full-length cDNA clones were obtained for five novel transcripts that were induced or repressed more than 10-fold in vitro. These novel human cDNAs CA2_1, CG12_1, GG10_2, AG8_1, and GG2_1 encode inhibitor of apoptosis protein-1 (hIAP-1), homologues of apolipoprotein-L, mouse rabkinesin-6, rat stannin, and a novel 188 amino acid protein, respectively. Expression of 4 novel transcripts is shown by in situ hybridization on healthy and atherosclerotic vascular tissue, using monocyte chemotactic protein-1 as a marker for inflammation. CA2_1 (hIAP-1) and AG8_1 are expressed by endothelial cells and macrophage foam cells of the inflamed vascular wall. CG12_1 (apolipoprotein-L like) was specifically expressed in endothelial cells lining the normal and atherosclerotic iliac artery and aorta. These results substantiate the complex change in the gene expression pattern of vascular endothelial cells, which accompanies the inflammatory reaction of atherosclerotic lesions.

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Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration

Blood ◽

10.1182/blood-2005-11-4447 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1243-1250 ◽

Cited By ~ 174

Author(s):

Benoit Favier ◽

Antoine Alam ◽

Pauline Barron ◽

Jacques Bonnin ◽

Patricia Laboudie ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Survival ◽

Endothelial Cell Migration ◽

Human Endothelial Cell ◽

Human Endothelial Cells ◽

Endothelial Cell Survival ◽

Biological Responses ◽

And Migration ◽

Endothelial Growth Factor

Abstract Neuropilin 2 (NRP2) is a receptor for the vascular endothelial growth factor (VEGF) and the semaphorin (SEMA) families, 2 unrelated ligand families involved in angiogenesis and neuronal guidance. NRP2 specifically binds VEGF-A and VEGF-C, although the biological relevance of these interactions in human endothelial cells is poorly understood. In this study, we show that both VEGF-A and VEGF-C induce the interaction of NRP2 with VEGFR-2. This interaction correlated with an enhancement of the VEGFR-2 phosphorylation threshold. Overexpression of NRP2 in primary human endothelial cells promoted cell survival induced by VEGF-A and VEGF-C. In contrast, SEMA3F, another ligand for NRP2, was able to inhibit human endothelial cell survival and migration induced by VEGF-A and VEGF-C. Moreover, a siRNA targeting specifically NRP2 was a potent inhibitor of human endothelial cell migration induced by VEGF-A and VEGF-C. Thus, our data indicate that NRP2 acts as a coreceptor that enhances human endothelial cell biological responses induced by VEGF-A and VEGF-C.

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Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

Blood ◽

10.1182/blood-2008-02-139725 ◽

2009 ◽

Vol 113 (4) ◽

pp. 856-865 ◽

Cited By ~ 190

Author(s):

Emanuela Rosati ◽

Rita Sabatini ◽

Giuliana Rampino ◽

Antonio Tabilio ◽

Mauro Di Ianni ◽

...

Keyword(s):

Notch Signaling ◽

Cell Survival ◽

Ex Vivo ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Inhibitor Of Apoptosis ◽

Apoptosis Resistance ◽

Inhibitor Of Apoptosis Protein ◽

Xiap Expression ◽

Apoptosis Protein

Abstract Notch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

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