Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion.

The binding of neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial cells (ECs) presents special requirements in the regulation of intercellular adhesion. ECs that are stimulated by certain agonists, including thrombin and cytokines (tumor necrosis factor alpha, interleukin-1), generate molecular signals that induce the adhesion of PMNs (endothelial cell-dependent neutrophil adhesion). Our experiments demonstrate that the mechanism of binding induced by thrombin is distinct from that induced by the cytokines based on the time courses, the requirement for protein synthesis, and differential binding of HL60 promyelocytic leukemia cells to ECs activated by the two classes of agonists. The rapid EC-dependent PMN adhesion (initiated in minutes) that occurs when the ECs are stimulated by thrombin is temporally coupled with the accumulation of platelet-activating factor, a biologically active phosphoglyceride that remains associated with ECs and that activates PMNs by binding to a cell surface receptor. A portion of the newly synthesized platelet-activating factor (PAF) is on the EC surface, as demonstrated by experiments in which the rate of hydrolysis of PAF synthesized by activated ECs was accelerated by extracellular PAF acetylhydrolase. When ECs were treated with exogenous PAF they became adhesive for PMNs; the PMN binding was prevented by incubating the ECs with PAF acetylhydrolase or by treating the PMNs with competitive PAF receptor antagonists. Thus PAF associated with the EC plasma membrane induces PMN binding, an observation supported by experiments in which PAF in model membranes (liposomes) stimulated rapid PMN adhesion to ECs and to cell-free surfaces. In addition, competitive antagonists of the PAF receptor inhibited the binding of PMNs to ECs activated by thrombin and other rapidly acting agonists, but not to ECs activated by tumor necrosis factor alpha, indicating that PAF that is endogenously synthesized by ECs can mediate neutrophil adhesion. These experiments demonstrate a novel mechanism by which a cell-associated phospholipid, PAF, can serve as a signal for an intercellular adhesive event.

Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.

The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.

Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils.

The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC-associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins.

Integrin-dependent control of translation: engagement of integrin alphaIIbbeta3 regulates synthesis of proteins in activated human platelets.

Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell-cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin alphaIIbbeta3 by fibrinogen and platelets deficient in this integrin, we found that alphaIIbbeta3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin alphaIIbbeta3 is engaged by a conformation-altering antibody against integrin alphaIIbbeta3. Thus, outside-in signals delivered by integrin alphaIIbbeta3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin alpha2beta1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression.

Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis.

Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1beta precursor (pro-IL-1beta). Unexpectedly, the mRNA for IL-1beta and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro-IL-1beta protein, a response that is abolished by translational inhibitors. A portion of the IL-1beta is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of beta3 integrin engagement markedly attenuated the synthesis of IL-1beta, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.

Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.

Highly purified human thrombin stimulates the adherence of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC). When Indium-labeled PMNs were incubated with primary monolayers of cultured human umbilical vein EC, the basal adherence was 10 +/- 1% of the PMNs at 5 min. Addition of thrombin (2 U/ml) increased the mean adherence to 42 +/- 15%. Enhanced neutrophil adherence in response to thrombin was confirmed by experiments with unlabeled leukocytes, examined by phase contrast and scanning electron microscopy. The action of thrombin was on the EC, since it did not directly stimulate PMN adhesiveness when measured by aggregation or by adherence to nylon fiber columns. Furthermore, enhanced neutrophil adherence occurred when endothelial monolayers were treated with thrombin and washed before adding 111Indium (111In)-labeled PMNs. Thrombin that had been inactivated with antithrombin III and heparin did not enhance neutrophil adherence. Prothrombin, Factor Xa, and fibrinogen were also ineffective. The stimulated adherence of PMNs was maximal 5 min after incubation of the EC with thrombin, and decreased thereafter. The response was dose-dependent, with half-maximal stimulation at 0.2-0.25 U thrombin/ml. The enhanced PMN adherence caused by thrombin may result in part from the production of platelet-activating factor (PAF) by the stimulated EC since thrombin-stimulated EC synthesize PAF with a time course and concentration dependence that are similar to the time and concentration relationships for thrombin-stimulated PMN adherence, PAF itself promoted neutrophil adherence to the EC monolayers, and pretreatment of PMNs with PAF decreased the adherence stimulated by thrombin and PAF, but not adherence stimulated by N-formylmethionyl-leucyl-phenylalanine and C5a fragments, which indicates specific desensitization of PAF-mediated adherence. These studies demonstrate the endothelial cell-dependent stimulation of PMN adherence by thrombin, a novel mechanism of enhanced leukocyte adherence that may be important in interactions between the coagulation and inflammatory systems.

Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate.

Cultured human endothelial cells synthesize prostacyclin (PGI2), a potent inhibitor of platelet function, when stimulated with histamine, bradykinin, or ATP. Paradoxically, we report that these agonists also induced the rapid and sustained synthesis of platelet-activating factor (PAF) by endothelial cells. In fact, the synthesis of this potent activator of platelets and neutrophils was induced by stimulation of the same receptor subtype that induced PGI2 synthesis: stimulation of a histamine H1 or a bradykinin B2 receptor induced both PAF and PGI2 synthesis. However, two physiologically important differences exist between the production of PAF and PGI2 by endothelial cells. The synthesis of PGI2 proceeded for only 7.5 min before the abrupt termination of synthesis, whereas the synthesis of PAF was clearly detectable even 45 min after stimulation. Although maximal accumulation of PAF occurred after 10-15 min of stimulation, the prolonged synthesis resulted in the presence of PAF for up to 1 h after stimulation. Secondly, whereas PGI2 was released from the cell monolayer, PAF remained cell-associated without significant release to the external medium. Endothelial cell-generated PAF, therefore, does not function as a hormone. The prolonged association of this potent activator of platelets and neutrophils with endothelial cells may mediate some of the inflammatory properties of histamine and bradykinin. It may also be a factor in the formation of a thrombogenic vascular surface, an event suggested to play a primary role in the pathogenesis of thrombosis and atherosclerosis.

Retrograde inflammatory signaling from neutrophils to endothelial cells by soluble interleukin-6 receptor alpha.

Endothelial cells initiate the inflammatory response by recruiting and activating leukocytes. IL-6 is not an agonist for this, but we found soluble IL-6 receptor alpha-subunit (IL-6Ralpha), with their constitutive IL-6 synthesis, stimulated endothelial cells to synthesize E-selectin, intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, IL-6, and IL-8, and to bind neutrophils. Neutrophils express significant amounts of IL-6Ralpha and upon stimulation shed it: this material activates endothelial cells through a newly constituted IL-6 receptor. Retrograde signaling from PMN activated in the extravascular compartment to surrounding endothelial cells will recruit more and a wider variety of leukocytes. The limiting signal is a soluble receptor, not a cytokine.

Proliferation-dependent changes in release of arachidonic acid from endothelial cells.

Stimulation of endothelial cells resulted in release of arachidonic acid from phospholipids. The magnitude of this response decreased as the cells became confluent and the change coincided with a decrease in the percentage of cells in growth phases (G2+M); this was not a consequence of time in culture or a factor in the growth medium. Preconfluent cells released approximately 30% of arachidonic acid; confluent cells released only 6%. The decreasing release of arachidonic acid was demonstrated using metabolic labeling, mass measurements of arachidonic acid, and measurement of PGI2. The decrease was not due to a changing pool of arachidonic acid, and mass measurements showed no depletion of arachidonic acid. Release from each phospholipid and from each phospholipid class decreased with confluence. Conversion of confluent cells to the proliferative phenotype by mechanical wounding of the monolayer caused increased release of arachidonic acid. Potential mechanisms for these changes were investigated using assays of phospholipase activity. Phospholipase A2 activity changed in concert with the alteration in release, a consequence of changes in phosphorylation of the enzyme. The increased release of arachidonic acid from preconfluent, actively dividing cells may have important physiologic implications and may help elucidate mechanisms regulating release of arachidonic acid.

The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation. Differential induction of GM-CSF, IL-8, and E-selectin expression.

Brown recluse spider (Loxosceles reclusa) venom induces severe dermonecrotic lesions. The mechanism for this is unknown but presents an interesting paradox: necrosis is completely dependent on the victim's neutrophils, yet neutrophils are not activated by the venom. We show Loxosceles venom is a potent, but disjointed, endothelial cell agonist. It weakly induced E-selectin expression, but not intercellular adhesion molecule-1 or IL-6 expression, yet significantly stimulated release of IL-8 and large amounts of GM-CSF by 4 h. In contrast, TNF strongly induced all of these, except for GM-CSF. PMN bound to E-selectin on venom-activated endothelial cells, apparently via counterreceptors different from those that bind E-selectin on TNF alpha-activated monolayers. Notably, PMN bound venom-activated monolayers only at intercellular junctions, did not polarize, and completely failed to migrate beneath the monolayer. Despite this, bound PMN demonstrated increased intracellular Ca2+ levels and secreted primary and secondary granule markers. The latter event was suppressed by sulfones used to treat envenomation. We have defined a new endothelial cell agonist, Loxosceles venom, that differentially stimulates the inflammatory response of endothelial cells. This, in turn, leads to a dysregulated PMN response where adhesion and degranulation are completely dissociated from shape change and transmigration.

Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation.

Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.

Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces.

In acute inflammatory responses, selectins mediate initial rolling of neutrophils (PMNs) along the endothelial surface. This is followed by tight adhesion that requires activation-dependent up-regulation of CD11/CD18 integrins on PMNs. For emigration to occur, the initial bonds that are established at the endothelial surface must be disengaged. We show that activation of PMNs results in their detachment from P-selectin, a glycoprotein expressed at the surface of inflamed endothelium that mediates initial tethering of PMNs. Loosening of the bond occurs when PMNs are activated by platelet-activating factor, which is coexpressed with P-selectin, or by other signaling molecules. The time course of reduced adhesion to P-selectin, when compared to up-regulation of CD11/CD18 integrins, suggests that "bond trading" may occur as activated PMNs transmigrate in vivo. Activation of PMNs did not alter binding of fluid-phase P-selectin, indicating that the ligand(s) for P-selectin is not shed or internalized. Using microspheres coated with P-selectin, we found that ligands for P-selectin were randomly distributed over the surfaces of rounded, unactivated PMNs. An antibody against P-selectin glycoprotein ligand-1 (PSGL-1) completely inhibited binding of P-selectin-coated beads suggesting that P-selectin glycoprotein ligand-1 is the critical binding site in this assay. In contrast to the dispersed pattern on unactivated PMNs, the ligands for P-selectin were localized on the uropods of activated, polarized cells. Pretreating PMNs with cytochalasin D before activation prevented the change in cell shape, the redistribution of binding sites for P-selectin-coated beads, and the decrease in cellular adhesiveness for P-selectin. These experiments indicate that the distribution of ligands for P-selectin is influenced by cellular activation and by cytoskeletal interactions, and that redistribution of these ligands may influence adhesive interactions. Activation of PMNs may cause loosening or disengagement of bonds between P-selectin and its ligands, facilitating transendothelial migration.

Hydrogen peroxide stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion.

Oxidant-induced damage to the intima of pulmonary and systemic vessels is thought to be an important mechanism of injury in a variety of syndromes of vascular damage. Hydrogen peroxide (H2O2) is an active oxygen metabolite that may induce intimal injury by cytolytic attack or by inducing biochemical and functional alterations in the endothelial cells (EC); however, mechanisms involved in noncytolytic perturbation of EC are largely unknown. We found that H2O2 stimulated the synthesis of platelet-activating factor (PAF) by primary cultures of bovine pulmonary artery endothelium (BPAEC) and by human umbilical vein endothelium (HUVEC). In each cell type the incorporation of [3H]acetate into [3H-acetyl]PAF was concentration- and time-dependent and was temporally dissociated from severe plasma membrane disruption and cytolytic cell injury; the newly synthesized PAF remained associated with the EC. H2O2 caused permeabilization of EC to 45Ca2+ and an increase in intracellular Ca2+, suggesting that a transmembrane Ca2+ flux is the signal that initiates PAF synthesis. H2O2 also induced the endothelial cell-dependent adhesion of neutrophils to HUVEC monolayers. This response was rapid, with an onset within minutes and a subsequent time course that paralleled the time course of PAF accumulation, and was dependent on extracellular Ca2+ but not on de novo protein synthesis. These studies demonstrate that H2O2 can induce two rapid activation responses of endothelium, PAF synthesis and EC-dependent neutrophil adhesion, events that may be important in physiologic and pathologic inflammation.

Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.

Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules.

Oncostatin M is a member of the IL-6 family of cytokines that is primarily known for its effects on cell growth. Endothelial cells have an abundance of receptors for oncostatin M, and may be its primary target. We determined if oncostatin M induces a key endothelial cell function, initiation of the inflammatory response. We found that subcutaneous injection of oncostatin M in mice caused an acute inflammatory reaction. Oncostatin M in vitro stimulated: (a) polymorphonuclear leukocyte (PMN) transmigration through confluent monolayers of primary human endothelial cells; (b) biphasic PMN adhesion through rapid P-selectin expression, and delayed adhesion mediated by E-selectin synthesis; (c) intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 accumulation; and (d) the expression of PMN activators IL-6, epithelial neutrophil activating peptide-78, growth-related cytokine alpha and growth-related cytokine beta without concomitant IL-8 synthesis. The nature of the response to oncostatin M varied with concentration, suggesting high and low affinity oncostatin M receptors independently stimulated specific responses. Immunohistochemistry showed that macrophage-like cells infiltrating human aortic aneurysms expressed oncostatin M, so it is present during a chronic inflammatory reaction. Therefore, oncostatin M, but not other IL-6 family members, fulfills Koch's postulates as an inflammatory mediator. Since its effects on endothelial cells differ significantly from established mediators like TNFalpha, it may uniquely contribute to the inflammatory cycle.

Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein.

Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10(-2) U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL.

Activated platelets signal chemokine synthesis by human monocytes.

Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions.

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking.

Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.