Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells

2006 ◽  
Vol 290 (1) ◽  
pp. C172-C182 ◽  
Author(s):  
Huiqing Cao ◽  
Nagadhara Dronadula ◽  
Gadiparthi N. Rao
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Dna Synthesis ◽  
Egf Receptor ◽  
Mammalian Target Of Rapamycin ◽  
Dominant Negative ◽  
Independent Manner ◽  
Signaling Axis ◽  
And Migration ◽  
Interfering Rna

To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin suppressed both thrombin-induced VSMC DNA synthesis and migration. Adenovirus-mediated expression of dominant-negative Akt also inhibited thrombin-induced VSMC DNA synthesis and migration. Furthermore, thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Suppression of Fra-1 by its small interfering RNA attenuated both thrombin-induced VSMC DNA synthesis and migration. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner, and neutralizing anti-FGF-2 antibodies inhibited thrombin-stimulated VSMC DNA synthesis and motility. In addition, thrombin stimulated the tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity significantly blocked Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin induces both VSMC DNA synthesis and motility via EGFR-dependent stimulation of PI3K/Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells

2002 ◽  
Vol 283 (2) ◽  
pp. L354-L363 ◽  
Author(s):  
Elena A. Goncharova ◽  
Alaina J. Ammit ◽  
Carla Irani ◽  
Richard G. Carroll ◽  
Andrew J. Eszterhas ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Dna Synthesis ◽  
Vascular Remodeling ◽  
Dominant Negative ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Necessary And Sufficient ◽  
Proliferation And Migration

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% ( P < 0.001; χ2analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.

Abstract 1359: Only the Catalytic p110alpha Isoform Mediates Class IA PI 3-Kinase-Induced Atherogenic Signals in Vascular Smooth Muscle Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Marius Vantler ◽  
Lenard Mustafov ◽  
Evren Caglayan ◽  
Stephan Rosenkranz
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Vascular Smooth Muscle Cells ◽  
Fold Increase ◽  
And Migration ◽  
Induced Apoptosis ◽  
Stimulation Of

Proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMC) are pivotal determinants of the pathogenesis of vascular diseases, which are mainly controlled by growth factor dependent activation of PI 3-Kinase (PI3K). Growth factors like platelet-derived growth factor (PDGF) activate class IA PI3Ks containing one of three p110 catalytic subunits (p110alpha, p110beta, and p110delta). We investigated the specific function of these isoforms for PDGF-controlled proliferation, migration, and apoptosis of VSMC using novel isoform-specific inhibitors. PDGF-dependent proliferation and migration solely depended on p110alpha. Stimulation of VSMC with PDGF-BB (50 ng/ml) mediated a 2.5±0.4 increase ( p <0.05) of DNA-synthesis (BrdU incorporation assay) and induced a 3.4+/−0.7 fold increase ( p <0.05) of VSMC migration (modified Boyden-chamber). Inhibition of p110alpha with PIK075 (1 μ M, Ki=100 nM) completely abrogated PDGF-dependent DNA-synthesis and migration ( p <0,05), whereas inhibitors against p110beta (TGX 221, 1 μ M) or p110delta (IC87114 1 μ M) had no influence. Consistently, PDGF-induced DNA-synthesis and migration were suppressed by siRNA-dependent downregulation of p110alpha ( p <0,05) whereas p110beta or p110delta knockdown had no effect. Interestingly, stimulation of VSMC with PDGF-BB (50 ng/ml) induced anti- or proapoptotic effects depending on the duration of PDGFR activation. Incubation of VSMC with H 2 O 2 (50 μ M, 16h) led to a 2.8±0.7 fold increase ( p >0.05) of apoptosis (Cell Death Detection ELISA). Simultanous addition of PDGF-BB (50 ng/ml) significantly diminished the H 2 O 2 -induced apoptosis (52±7%, p >0.05). In contrast, prestimulation with PDGF-BB 24h prior to the addition of H 2 O 2 led to an increase of H 2 O 2 -induced apoptosis (7.8±1.3, p >0.05). The anti- as well as the proapoptotic effect depended strictly on p110alpha as PIK075 (1 μ M, p <0,05) or p110alpha specific siRNA completely abrogated PDGF-BB-mediated pro- as well as antiapoptotic effects. Our results demonstrate that only the catalytical PI3K subunit p110alpha mediates the growth factor-induced atherogenic responses. Therefore, p110alpha represents an interesting therapeutic target for prevention of atherosclerosis and restenosis formation.

PKC-δ and CaMKII-δ2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

2004 ◽  
Vol 286 (6) ◽  
pp. C1281-C1289 ◽  
Author(s):  
Roman Ginnan ◽  
Paul J. Pfleiderer ◽  
Kevin Pumiglia ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Purinergic Receptor ◽  
Selective Inhibition ◽  
Dominant Negative ◽  
Dependent Protein ◽  
Camkii Activation

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC-δ activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-δ, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC-α activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-α, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-δ2, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-RasN17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC-δ and CaMKII-δ2 coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells.

scholarly journals Rho and Rho Kinase Mediate Thrombin-Stimulated Vascular Smooth Muscle Cell DNA Synthesis and Migration

1999 ◽  
Vol 84 (10) ◽  
pp. 1186-1193 ◽  
Author(s):  
Tammy M. Seasholtz ◽  
Mousumi Majumdar ◽  
Daniel D. Kaplan ◽  
Joan Heller Brown
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Dna Synthesis ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Rho Kinase ◽  
And Migration

scholarly journals Effect of TPA on ion fluxes and DNA synthesis in vascular smooth muscle cells.

1985 ◽  
Vol 101 (2) ◽  
pp. 454-459 ◽  
Author(s):  
N E Owen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Vascular Smooth Muscle Cells ◽  
Egf Receptor ◽  
Phorbol Esters ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
A431 Cells

Previous reports have suggested that phorbol esters can decrease the affinity of epidermal growth factor (EGF) for its cellular receptors. Investigations of the consequences of the interaction between phorbol esters and EGF, however, have been limited to EGF-stimulated Na/H exchange in A431 cells (Whitely, B., D. Cassel, Y.-X. Zuang, and L. Glaser, 1984, J. Cell Biol., 99:1162-1166). In the present study, the effect of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) on EGF-stimulated ion transport and DNA synthesis was determined in cultured vascular smooth muscle cells (A7r5). It was found that TPA stimulated Na/H exchange when added alone (half-maximal stimulatory concentration, 25 nM). However, when cells were pretreated with TPA and then challenged with EGF, TPA significantly inhibited EGF-stimulated Na/H exchange (78%; half-maximal inhibition [Ki] at 2.5 nM). Subsequently the effects of TPA on Na/K/Cl co-transport were measured. TPA was observed to inhibit Na/K/Cl co-transport (half-maximal inhibitory concentration, 50 nM) and also to inhibit EGF-stimulated Na/K/Cl co-transport (100%; Ki at 5 nM). Finally, the effects of TPA on DNA synthesis were assessed. TPA had a modest stimulatory effect on DNA synthesis (half-maximal stimulatory concentration, 6 nM), but had a significant inhibitory effect on EGF-stimulated DNA synthesis (56%; Ki at 5 nM). These findings suggest that the inhibitory effect of TPA on EGF-receptor functions goes beyond previously reported effects on Na/H exchange in A431 cells and extends to EGF-stimulation of Na/K/Cl co-transport and DNA synthesis in vascular smooth muscle cells.

Thrombospondin-1 Induces DNA Synthesis And Migration In Human Vascular Smooth Muscle Cells

1996 ◽  
Vol 24 (3) ◽  
pp. 446S-446S ◽  
Author(s):  
MAHENDRA K. PATEL ◽  
GERARD F. CLUNN ◽  
JOANNE S. LYMN ◽  
ALUN D. HUGHES
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Thrombospondin 1 ◽  
And Migration

17β-Estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level

2006 ◽  
Vol 290 (2) ◽  
pp. H538-H546 ◽  
Author(s):  
Kai Kappert ◽  
Evren Caglayan ◽  
Michael Huntgeburth ◽  
Anselm T. Bäumer ◽  
Jan Sparwel ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Premenopausal Women ◽  
Cellular Responses ◽  
Dominant Negative ◽  
Vsmc Proliferation ◽  
Downstream Signaling ◽  
Pdgf Signaling ◽  
17Β Estradiol ◽  
And Migration

Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17β-estradiol (E2) on β-PDGF receptor (β-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 μM–0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 ± 15.8% and 79.4 ± 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter β-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the β-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-γ, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the β-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.

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