15(S)-hydroxyeicosatetraenoic acid–induced angiogenesis requires Src-mediated Egr-1–dependent rapid induction of FGF-2 expression

Abstract To understand the mechanisms underlying 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE]–induced angiogenesis, we studied the role of Egr-1. 15(S)-HETE induced Egr-1 expression in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Blockade of Egr-1 via forced expression of its dominant-negative mutant attenuated 15(S)-HETE–induced HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. 15(S)-HETE–induced Egr-1 expression requires Src activation. In addition, adenovirus-mediated expression of dominant-negative mutant of Src blocked 15(S)-HETE's effects on migration and tube formation of HDMVECs and Matrigel plug angiogenesis. 15(S)-HETE induced fibroblast growth factor-2 (FGF-2) expression rapidly via Src-mediated production of Egr-1. Cloning and mutational analysis of FGF-2 promoter revealed that Egr-1 binding site proximal to transcription start site is required for 15(S)-HETE–induced FGF-2 expression. Neutralizing antibody-mediated suppression of FGF-2 function also attenuated the effects of 15(S)-HETE on HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. Furthermore, in contrast to wild-type mice, 12/15-LOX−/− mice exhibited decreased Matrigel plug angiogenesis in response to AA, which was rescued by 15(S)-HETE. On the basis of these observations, we conclude that 15(S)-HETE–induced angiogenesis requires Src-mediated Egr-1–dependent rapid induction of FGF-2. These findings may suggest that 15(S)-HETE could be a potential endogenous regulator of pathologic angiogenesis associated with atherosclerosis and restenosis.

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The 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires Janus kinase 2-signal transducer and activator of transcription-5B–dependent expression of interleukin-8

Blood ◽

10.1182/blood-2008-10-183210 ◽

2009 ◽

Vol 113 (23) ◽

pp. 6023-6033 ◽

Cited By ~ 15

Author(s):

Sergey Y. Cheranov ◽

Dong Wang ◽

Venkatesh Kundumani-Sridharan ◽

Manjula Karpurapu ◽

Qiuhua Zhang ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tube Formation ◽

Dominant Negative ◽

Janus Kinase ◽

Time Dependent ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Signal Transducer ◽

Matrigel Plug ◽

Negative Mutant

Abstract To understand the molecular basis underlying 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)–induced angiogenesis, we have studied the role of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling. The 15(S)-HETE stimulated tyrosine phosphorylation of Jak2 in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). Inhibition of Jak2 activation via adenovirus-mediated expression of its dominant-negative mutant attenuated 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. Similarly, 15(S)-HETE activated tyrosine phosphorylation of STAT-5B in a time-dependent manner. Dominant-negative mutant-mediated interference of STAT-5B activation suppressed 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. The 15(S)-HETE induced interleukin-8 (IL-8) expression in Jak2-STAT-5B–dependent manner in HRMVECs. In addition, neutralizing anti–IL-8 antibodies reduced 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. Cloning and Transfac analysis of IL-8 promoter revealed the presence of 1 putative STAT-binding sequence at −476 nt, and electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed the binding of STAT-5B to this site in response to 15(S)-HETE. Mutational analysis showed that STAT binding site is essential for 15(S)-HETE–induced IL-8 promoter activity. Together, these observations suggest that 15(S)-HETE–induced angiogenesis requires Jak2-STAT-5B–dependent expression of IL-8.

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An essential role for SRC-activated STAT-3 in 14,15-EET–induced VEGF expression and angiogenesis

Blood ◽

10.1182/blood-2007-11-126680 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5581-5591 ◽

Cited By ~ 79

Author(s):

Sergey Y. Cheranov ◽

Manjula Karpurapu ◽

Dong Wang ◽

Baolin Zhang ◽

Richard C. Venema ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Vegf Expression ◽

Matrigel Plug

Abstract To understand the molecular mechanisms underlying 14,15-epoxyeicosatrienoic acid (14,15-EET)–induced angiogenesis, here we have studied the role of signal transducer and activator of transcription-3 (STAT-3). 14,15-EET stimulated the tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus in human dermal microvascular endothelial cells (HDMVECs). Adenovirus-mediated delivery of dominant negative STAT-3 substantially inhibited 14,15-EET–induced HDMVEC migration, and tube formation and Matrigel plug angiogenesis. 14,15-EET activated Src, as measured by its tyrosine phosphorylation and blockade of its activation by adenovirus-mediated expression of its dominant negative mutant, significantly attenuated 14,15-EET–induced STAT-3 phosphorylation in HDMVECs and the migration and tube formation of these cells and Matrigel plug angiogenesis. 14,15-EET induced the expression of vascular endothelial cell growth factor (VEGF) in a time- and Src-STAT-3–dependent manner in HDMVECs. Transfac analysis of VEGF promoter revealed the presence of STAT-binding elements and 14,15-EET induced STAT-3 binding to this promoter in vivo, and this interaction was inhibited by suppression of Src-STAT-3 signaling. Neutralizing anti-VEGF antibodies completely blocked 14,15-EET–induced HDMVEC migration and tube formation and Matrigel plug angiogenesis. These results reveal that Src-dependent STAT-3–mediated VEGF expression is a major mechanism of 14,15-EET–induced angiogenesis.

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An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility

Journal of Biological Chemistry ◽

10.1074/jbc.m406922200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 46122-46128 ◽

Cited By ~ 35

Author(s):

Indira Neeli ◽

Zhimin Liu ◽

Nagadhara Dronadula ◽

Z. Alex Ma ◽

Gadiparthi N. Rao

Keyword(s):

Smooth Muscle ◽

Growth Factor ◽

Tyrosine Phosphorylation ◽

Dominant Negative ◽

Platelet Derived Growth Factor ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Cytosolic Phospholipase ◽

Negative Mutant

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A2(cPLA2). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA2in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA2prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA2rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA2signaling axis.

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Targeting of GLUT6 (formerly GLUT9) and GLUT8 in rat adipose cells

Biochemical Journal ◽

10.1042/bj3580517 ◽

2001 ◽

Vol 358 (2) ◽

pp. 517-522 ◽

Cited By ~ 51

Author(s):

Ivonne LISINSKI ◽

Annette SCHÜRMANN ◽

Hans-Georg JOOST ◽

Samuel W. CUSHMAN ◽

Hadi AL-HASANI

Keyword(s):

Plasma Membrane ◽

Constitutive Expression ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Wild Type ◽

Subcellular Targeting ◽

Intracellular Compartments ◽

Adipose Cells ◽

Negative Mutant

The subcellular targeting of the two recently cloned novel mammalian glucose transporters, GLUT6 {previously referred to as GLUT9 [Doege, Bocianski, Joost and Schürmann (2000) Biochem. J. 350, 771–776]} and GLUT8, was analysed by expression of haemagglutinin (HA)-epitope-tagged GLUTs in transiently transfected primary rat adipose cells. Similar to HA-GLUT4, both transporters, HA-GLUT6 and HA-GLUT8, were retained in intracellular compartments in non-stimulated cells. In contrast, mutation of the N-terminal dileucine motifs in both constructs led to constitutive expression of the proteins on the plasma membrane. Likewise, when endocytosis was blocked by co-expression of a dominant-negative mutant of the dynamin GTPase, wild-type HA-GLUT6 and HA-GLUT8 accumulated on the cell surface. However, in contrast with HA-GLUT4, no translocation of HA-GLUT6 and HA-GLUT8 to the plasma membrane was observed when the cells were stimulated with insulin, phorbol ester or hyperosmolarity. Thus GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells, but are unresponsive to stimuli that induce translocation of GLUT4.

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Biochemical and Biological Functions of the N-Terminal, Noncatalytic Domain of Extracellular Signal-Regulated Kinase 2

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.249-259.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 249-259 ◽

Cited By ~ 38

Author(s):

Scott T. Eblen ◽

Andrew D. Catling ◽

Marcela C. Assanah ◽

Michael J. Weber

Keyword(s):

Kinase Activity ◽

Mutational Analysis ◽

Kinase Domain ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Transcriptional Responses ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal

ABSTRACT Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are important components in signal transduction pathways involved in many cellular processes, including cell differentiation and proliferation. These proteins consist of a central kinase domain flanked by short N- and C-terminal noncatalytic domains. While the regulation of ERK2 by sequences within the kinase domain has been extensively studied, little is known about the small regions outside of the kinase domain. We performed mutational analysis on the N-terminal, noncatalytic domain of ERK2 in an attempt to determine its role in ERK2 function and regulation. Deleting or mutating amino acids 19 to 25 (ERK2-Δ19-25) created an ERK2 molecule that could be phosphorylated in response to growth factor and serum stimulation in a MEK (mitogen-activated protein kinase kinase or ERK kinase)-dependent manner but had little kinase activity and was unable to bind to MEK in vivo. Since MEK acts as a cytoplasmic anchor for the ERKs, the lack of a MEK interaction resulted in the aberrant nuclear localization of ERK2-Δ19-25 mutants in serum-starved cells. Assaying these mutants for their ability to affect ERK signaling revealed that ERK2-Δ19-25 mutants acted in a dominant-negative manner to inhibit transcriptional signaling through endogenous ERKs to an Elk1-responsive promoter in transfected COS-1 cells. However, ERK2-Δ19-25 had no effect on the phosphorylation of RSK2, an ERK2 cytoplasmic substrate, whereas a nonactivatable ERK (T183A) that retained these sequences could inhibit RSK2 phosphorylation. These results suggest that the N-terminal domain of ERK2 profoundly affects ERK2 localization, MEK binding, kinase activity, and signaling and identify a novel dominant-negative mutant of ERK2 that can dissociate at least some transcriptional responses from cytoplasmic responses.

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Exogenous C2-ceramide activates c-fos serum response element via Rac-dependent signalling pathway

Biochemical Journal ◽

10.1042/bj3301009 ◽

1998 ◽

Vol 330 (2) ◽

pp. 1009-1014 ◽

Cited By ~ 18

Author(s):

Byung-Chul KIM ◽

Jae-Hong KIM

Keyword(s):

Phospholipase A2 ◽

Reporter Gene ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Response Element ◽

Serum Response ◽

Negative Mutant

Ceramide is an important regulatory molecule implicated in a variety of biological processes in response to stress and cytokines. To understand the signal transduction pathway of ceramide to the nucleus, in the present study, we examined whether C2-ceramide, a cell permeable ceramide, activates c-fos serum response element (SRE). Treatment of Rat-2 fibroblast cells with C2-ceramide caused the stimulation of c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner by transient transfection analysis. Next, we examined the role of Rho family GTPases in the ceramide-induced signalling to SRE activation. By reporter gene analysis following transient transfections with various plasmids expressing a dominant negative mutant form of Cdc42, Rac1 or RhoA, C2-ceramide-induced SRE activation was shown to be selectively repressed by pEXV-RacN17 encoding a dominant negative mutant of Rac1, suggesting that Rac activity is essential for the signalling cascade of ceramide to the nucleus. In a further study to analyse the downstream mediator of Rac in the ceramide-signalling pathway, we observed that either pretreatment with mepacrine, a potent and specific inhibitor of phospholipase A2, or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide repressed the C2-ceramide-induced SRE activation selectively, implying a critical role of cPLA2 in C2-ceramide-induced signalling to nucleus. Consistent with these results, the translocation of cPLA2 protein as well as the release of arachidonic acid, a principal product of phospholipase A2, was rapidly induced by the addition of C2-ceramide in a Rac-dependent manner. Together, our findings suggest the critical role of ‘Rac and subsequent activation of phospholipase A2’ in ceramide-signalling to nucleus.

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