STAT-3-dependent cytosolic phospholipase A2 expression is required for thrombin-induced vascular smooth muscle cell motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).     

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.     

Endothelial heparan sulfate is necessary but not sufficient for control of vascular smooth muscle cell growth

The state of the endothelial cell (EC) determines the nature of its control of vascular smooth muscle cell (vSMC) biology. Conditioned medium from postconfluent ECs inhibits vSMC proliferation, whereas subconfluent conditioned medium from the same ECs has a stimulatory effect. We and others have identified confluent endothelial cells' production of heparan sulfate proteoglycans (HSPG) as critical to vSMC growth control. The question that arises is whether the stimulation that is observed with subconfluent cells is from (1) aberrant HSPG production, (2) elaboration of noninhibitory species of HSPG, or (3) production of other factors, such as mitogens, which counteract the inhibitory HSPG to stimulate vSMCs. We studied the relative effects of conditioned medium produced by both subconfluent and postconfluent EC cultures on vSMC growth. Conditioned medium was fractionated into nonproteoglycan (non-PG) and proteoglycan (PG) components by anion-exchange chromatography. The PG fractionation profile and the antiproliferative activity of the HSPGs isolated from both subconfluent and postconfluent EC-conditioned media were similar. However, the HSPG fraction alone could not approach the inhibitory potential of unfractionated conditioned medium from postconfluent EC cultures. Non-PG proteins produced by the endothelial cultures had no effect on vSMC growth on their own. Yet, when they were mixed together with HSPG fractions, from either subconfluent or postconfluent EC cultures, the full growth effects were returned. Non-PG protein fractions from postconfluent cultures with HSPG fractions gave maximal inhibition of vSMC growth, whereas non-PG protein fractions from subconfluent EC cultures with HSPG fractions produced the maximal stimulation. Thus, whereas the net stimulatory or inhibitory effect on vSMC growth of EC-conditioned medium is density dependent, this effect does not result from a difference in the antiproliferative heparan sulfate component but rather from non-PG proteins that interact with the heparan sulfates.     

A requirement for calcium-independent phospholipase A2 in thrombin-induced arachidonic acid release and growth in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A2 (iPLA2). Without affecting its levels, thrombin increased iPLA2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.     

Phosphorylation of cytosolic group IV phospholipase A(2) is necessary but not sufficient for Arachidonic acid release in P388D(1) macrophages

Activation of the cytosolic Group IV phospholipase A(2) (cPLA(2)) by agonists has been correlated with the direct phosphorylation of the enzyme by members of the mitogen-activated protein kinase (MAPK) cascade. Phosphorylation of the cPLA(2) increases the specific activity of the enzyme, thereby stimulating the arachidonic acid release. We show here, however, that conditions that lead to full phosphorylation of the cPLA(2) do not lead to enhanced AA release. As the above observations were made under both Ca(2+)-dependent and Ca(2+)-independent conditions, they emphasize that the current paradigm for activation of the cPLA(2) in cells involving both phosphorylation and Ca(2+) is incomplete and that other factors should be taken into account.     

Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation

Thrombospondin (TS) is an extracellular glycoprotein whose synthesis and secretion by vascular smooth muscle cells (SMC) is regulated by platelet-derived growth factor. We have used a panel of five monoclonal antibodies against TS to determine an essential role for thrombospondin in the proliferation of cultured rat aortic SMC. All five monoclonal antibodies inhibited SMC growth in 3-d and extended cell number assays; the growth inhibition was specific for anti-TS IgG. The effects of one antibody (D4.6) were examined in detail and were found to be reversable and dose dependent. Cells treated with D4.6 at 50 micrograms/ml (which resulted in a greater than 60% reduction in cell number at day 8) were morphologically identical to control cells. D4.6-treated SMC were analyzed by flow cytofluorimetry and were found to be arrested in the G1 phase of the cell cycle. To determine a possible cellular site of action of TS in cell growth, SMC were examined by immunofluorescence using a polyclonal antibody against TS. TS was observed diffusely bound to the cell surface of serum- or platelet-derived growth factor-treated cells. The binding of TS to SMC was abolished in the presence of heparin, which prevents the binding of TS to cell surfaces and inhibits the growth of SMC. Monoclonal antibody D4.6, like heparin, largely abolished cell surface staining of TS but had no detectable effect on the cellular distribution of fibronectin. These results were corroborated by metabolic labeling experiments. We conclude that cell surface-associated TS is functionally essential for the proliferation of vascular SMC, and that this requirement is temporally located in the G1 phase of the cell cycle. Agents that perturb the interaction of TS with the SMC surface, such as heparin, may inhibit SMC proliferation in this manner.     

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.     

Cks1 is required for tumor cell proliferation but not sufficient to induce hematopoietic malignancies

The Cks1 component of the SCF(Skp2) complex is necessary for p27(Kip1) ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27(Kip1) levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27(Kip1) levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27(Kip1). To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.     

Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces

Summary The membrane lipid composition of Saccharomyces was manipulated by growing cells anaerobically with or without ergosterol and unsaturated fatty acid. Cells low in ergosterol but enriched in unsaturated fatty acid residues on membrane phospholipids produced high concentrations, 13–15.5% w/v, of ethanol at substrate conversion efficiencies of around 90%.     

Perinuclear localization of cytosolic phospholipase A(2)alpha is important but not obligatory for coupling with cyclooxygenases

In response to Ca(2+) signaling, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) translocates from the cytosol to the perinuclear membrane, where downstream eicosanoid-synthetic enzymes, such as cyclooxygenase (COX), are localized. Although the spatiotemporal perinuclear colocalization of cPLA(2)alpha and COXs has been proposed to be critical for their functional coupling leading to prostanoid production, definitive evidence for this paradigm has remained elusive. To circumstantiate this issue, we took advantage of a chimeric cPLA(2)alpha mutant harboring the C2 domain of protein kinase Calpha, which translocates to the plasma membrane following cell activation. Transfection analyses of the native or chimeric cPLA(2)alpha in combination with COX-1 or COX-2 revealed that, even though the arachidonate-releasing capacities of native and mutant cPLA(2)alpha were comparable, prostaglandin production by mutant cPLA(2)alpha was markedly impaired as compared with that by native cPLA(2)alpha. We thus conclude that the perinuclear localization of cPLA(2)alpha is preferential, even if not obligatory, for efficient coupling with COXs.     

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.     

PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Activation of EphB2 and its ligands promotes vascular smooth muscle cell proliferation.

EphB2 and its ligands regulate interactions between endothelial and mesenchymal cells in developing arteries. In adult arteries, the relationship between smooth muscle cells and overlying intact endothelium is responsible for maintaining the health of the vessel. Heparin inhibits vascular smooth muscle cell growth in culture and intimal hyperplasia following endothelial denudation. Using gene microarrays, we identified the tyrosine kinase receptor EphB2 as being differentially expressed in response to continuous intravenous heparin administration in the rabbit model of arterial injury. EphB2 protein levels increased in cultured bovine vascular smooth muscle cells following serum stimulation and were decreased in a dose-dependent fashion by heparin. Fc chimeras of the binding domain of the EphB2 ligands blocked the formation of the EphB2 ligand-receptor complex and reduced growth of serum-stimulated vascular smooth muscle cells in a dose-dependent fashion. Activation of the ligand by an Fc chimera to EphB2 followed a parabolic dose-response growth curve, indicating growth stimulation until the chimera begins to compete with native receptors. Co-administration of EphB2/Fc chimera with heparin shifted the dose-response curve to the right. These data indicate a possible new route of Heparin's antiproliferative effect and a role of EphB2 and its ligands in vascular smooth muscle cell proliferation.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.