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

2004 ◽  
Vol 279 (44) ◽  
pp. 46122-46128 ◽  
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.

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

1998 ◽  
Vol 330 (2) ◽  
pp. 1009-1014 ◽  
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.

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

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 6023-6033 ◽  
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.

scholarly journals Transactivation of Platelet-Derived Growth Factor Receptor α by the GTPase-Deficient Activated Mutant of Gα12

2006 ◽  
Vol 26 (1) ◽  
pp. 50-62 ◽  
Author(s):  
Rashmi N. Kumar ◽  
Ji Hee Ha ◽  
Rangasudhagar Radhakrishnan ◽  
Danny N. Dhanasekaran
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Signaling Pathway ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT The GTPase-deficient, activated mutant of Gα12 (Gα12Q229L, or Gα12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor α (PDGFRα) in Gα12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Gα12QL stimulates the functional expression of PDGFRα and demonstrate that the expression of PDGFRα by Gα12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Gα12QL or the activation of Gα12-coupled receptors stimulates the expression of PDGFRα in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRα in Gα12QL-transformed cells. Analysis of the functional consequences of the Gα12-PDGFRα signaling axis indicates that Gα12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Gα12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRα- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRα-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Gα12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRα attenuated Gα12-mediated neoplastic transformation of NIH 3T3 cells.

scholarly journals Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

1995 ◽  
Vol 15 (12) ◽  
pp. 6777-6784 ◽  
Author(s):  
C A Pickett ◽  
A Gutierrez-Hartmann
Keyword(s):  
Signal Transduction ◽  
Transcription Factors ◽  
Growth Factor ◽  
Map Kinase ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Dose Dependent

We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types.

Vasoconstriction: a new activity for platelet-derived growth factor

Science ◽  
1986 ◽  
Vol 232 (4746) ◽  
pp. 87-90 ◽  
Author(s):  
BC Berk ◽  
RW Alexander ◽  
TA Brock ◽  
MA Gimbrone ◽  
RC Webb
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Rat Aorta ◽  
Platelet Derived Growth Factor ◽  
Free Calcium ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.

Kinetics and Regulation of Tyrosine Phosphorylation of the Platelet Derived Growth Factor Receptor

1989 ◽  
Vol 75 (4) ◽  
pp. 362-366
Author(s):  
Lilia Alberghina ◽  
Renata Zippel ◽  
Enzo Martegani ◽  
Emmapaola Sturani
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Growth Conditions ◽  
Platelet Derived Growth Factor ◽  
Dependent Manner ◽  
Pdgf Receptor ◽  
Receptor Complexes ◽  
Phosphorylated Form

Platelet derived growth factor (PDGF) interaction with the cells induces rapid tyrosine phosphorylation of the PDGF receptor in a dose dependent manner. At 37 °C phosphorylation of the receptor is followed by its dephosphorylation and internalization. It is observed that the higher the ligand concentration, the more transient is the response, and the observed kinetics are explained by a simple kinetic model. At 4 °C the phosphorylated form of the receptor is more stable; however, if PDGF is dissociated from the cell surface-associated ligand-receptor complexes, the receptors are rapidly dephosphorylated, indicating that phosphatases specific for phosphotyrosine groups are very active within the cells. In fact, addition of orthovanadate stabilizes the phosphorylated form of the receptor and helps in recognizing possible physiological substrates of the PDGF receptor kinase. The expression of PDGF receptors on the cell surface has been investigated under different growth conditions: a positive correlation exists between the amount of PDGF receptors and the duplication times of exponentially growing cultures. Moreover, during exponential growth the PDGF receptors are scarcely expressed, and their number increases reaching a maximal value when the population enters the stationary phase.

Patterns of tyrosine phosphorylation differ in vascular hypertrophy and hyperplasia

1994 ◽  
Vol 267 (6) ◽  
pp. C1674-C1681 ◽  
Author(s):  
S. Ali ◽  
G. W. Dorn
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Growth Response ◽  
Thromboxane A2 ◽  
The Other ◽  
Platelet Derived Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Gtpase Activating Protein ◽  
Herbimycin A

Vascular smooth muscle cells (VSMC) undergo hypertrophy when exposed to thromboxane A2 and hyperplasia when exposed to phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor (PDGF). Each of these three agonists stimulate rapid tyrosine phosphorylation of numerous VSMC proteins. The current studies were undertaken to identify proteins that are specifically tyrosine phosphorylated in one or the other growth response. All three agonists increased the phosphotyrosine content of multiple proteins. In Western analysis of phosphotyrosine immunoprecipitates, the hyperplastic agents PDGF and PMA increased tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), GTPase-activating protein (GAP), and phosphatidylinositol-3-kinase (PI-3-kinase), while the hypertrophic agonist thromboxane failed to tyrosine-phosphorylate either of these three substrates. Tyrosine kinase inhibition with herbimycin A (5 microM) prevented agonist-stimulated tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase. In growth studies, herbimycin A inhibited PMA- and PDGF-induced hyperplasia but not thromboxane-stimulated hypertrophy. These results indicate that tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase are specific responses for VSMC hyperplasia but not thromboxane-stimulated hypertrophy.

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