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Stephen M Prescott

Publications and source records attributed to Stephen M Prescott.

At least 37 records · Page 2Linked to original sources
PubMed2003

Regulation of COX-2 transcription in a colon cancer cell line by Pontin52/TIP49a.

Cyclooxygenase-2 (COX-2) is expressed early in colon carcinogenesis and is known to play a crucial role in the progress of the disease. Here we show that the regulation of the expression of this enzyme in a colon cancer cell line, and in patients, is associated with overexpression of the Wnt pathway-associated proteins, Pontin52/TIP49a and LEF-1. Recently shown to be essential for transformation via the c-Myc pathway, Pontin52/TIP49a promotes COX-2 expression in tissue culture and is overexpressed in colon cancer tissue, co-localizing with COX-2 expression in transformed tissue, relative to paired normal tissue.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

Molecular characterization of the constitutive expression of the plasma platelet-activating factor acetylhydrolase gene in macrophages.

Plasma platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase that inactivates platelet-activating factor (PAF) and PAF-like lipids to generate products with little or no biological activity. The levels of circulating PAF-AH correlate with several disease syndromes. We previously reported that mediators of inflammation regulate the expression of the human PAF-AH gene at the transcriptional level. In the present paper, we characterize the constitutive expression of plasma PAF-AH using the mouse gene as a model system, and we report comparative results obtained using human and mouse promoter constructs. We first cloned, sequenced and analysed the promoter region of the murine plasma PAF-AH (mPAF-AH) gene and found that this gene lacks a canonical TATA box. We demonstrated that the cis -elements required for basal transcription are localized within the -316 to -68 bp region. In vitro band-shift and supershift assays showed that Sp1 and Sp3 transcription factors from RAW264.7 and J774A.1 macrophage nuclear extracts bound strongly to a distal GC-rich site within -278/-243 [specificity protein (Sp-A)] and to a proximal TC-rich motif within -150/-114 (Sp-B). In addition, we observed weak binding to a GA-rich site within -110/-82 (Sp-C). The regions containing Sp-B and Sp-C are highly conserved between the human and mouse genes. Forced expression of Sp1 or Sp3 in Sp-lacking Drosophila SL2 cells induced markedly the activity of the exogenous mPAF-AH promoter in a dose-dependent manner, and this induction was dependent on the presence of intact Sp-A and Sp-B. Interestingly, we found that the Sp1- and Sp3-associated DNA-binding activities increased during the maturation of primary human monocytes into macrophages in cell culture. These results demonstrate that Sp1 and Sp3 are key factors that contribute to the basal, constitutive transcription of the plasma PAF-AH gene in macrophages.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2003

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes.

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.

Animals↗
PubMed2003

Protein kinase C alpha phosphorylates and negatively regulates diacylglycerol kinase zeta.

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGK zeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGK zeta and found that PKC alpha phosphorylated DGK zeta on serines within the MARCKS PSD in vitro and in vivo. DGK zeta also coimmunoprecipitated with PKC alpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGK zeta S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGK zeta or a control mutant (DGK zeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKC alpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGK zeta, but not DGK zeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKC alpha attenuates DGK zeta activity. Supporting this, we found that cells expressing DGK zeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGK zeta S/N that could not be phosphorylated. Taken together, these results indicate that PKC alpha phosphorylates DGK zeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.

Binding Sites↗
PubMed2003

Regulation of cyclooxygenase-2 expression by the translational silencer TIA-1.

The cyclooxygenase-2 (COX-2) enzyme catalyzes the rate-limiting step of prostaglandin formation in inflammatory states, and COX-2 overexpression plays a key role in carcinogenesis. To understand the mechanisms regulating COX-2 expression, we examined its posttranscriptional regulation mediated through the AU-rich element (ARE) within the COX-2 mRNA 3'-untranslated region (3'UTR). RNA binding studies, performed to identify ARE-binding regulatory factors, demonstrated binding of the translational repressor protein TIA-1 to COX-2 mRNA. The significance of TIA-1-mediated regulation of COX-2 expression was observed in TIA-1 null fibroblasts that produced significantly more COX-2 protein than wild-type fibroblasts. However, TIA-1 deficiency did not alter COX-2 transcription or mRNA turnover. Colon cancer cells demonstrated to overexpress COX-2 through increased polysome association with COX-2 mRNA also showed defective TIA-1 binding both in vitro and in vivo. These findings implicate that TIA-1 functions as a translational silencer of COX-2 expression and support the hypothesis that dysregulated RNA-binding of TIA-1 promotes COX-2 expression in neoplasia.

Animals↗
PubMed2003

Association of diacylglycerol kinase zeta with protein kinase C alpha: spatial regulation of diacylglycerol signaling.

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKzeta inhibits PKCalpha activity and that DGK activity is required for this inhibition. We also show that DGKzeta directly interacts with PKCalpha in a signaling complex and that the binding site in DGKzeta is located within the catalytic domain. Because PKCalpha can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKzeta, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKzeta and PKCalpha and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKzeta mutant that was unable to bind PKCalpha did not inhibit PKCalpha activity. Together, our results suggest that DGKzeta spatially regulates PKCalpha activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCalpha-mediated DGKzeta phosphorylation.

3T3 Cells↗
PubMed2003

Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects. This bioactive phospholipid mediates processes as diverse as wound healing, physiological inflammation, apoptosis, angiogenesis, reproduction and long-term potentiation. Recent progress has demonstrated the participation of MAP kinase signaling pathways as modulators of the two critical enzymes, phospholipase A2 and acetyltransferase, involved in the remodeling pathway of PAF biosynthesis. The unregulated production of structural analogs of PAF by non-specific oxidative reactions has expanded this superfamily of signaling molecules to include "PAF-like" lipids whose mode of action is identical to that of authentic PAF. The action of members of this family is mediated by the PAF receptor, a G protein-coupled membrane-spanning molecule that can engage multiple signaling pathways in various cell types. Inappropriate activation of this signaling pathway is associated with many diseases in which inflammation is thought to be one of the underlying features. Inactivation of all members of the PAF superfamily occurs by a unique class of enzymes, the PAF acetylhydrolases, that have been characterized at the molecular level and that terminate signals initiated by both regulated and unregulated PAF production.

Acetyltransferases↗
PubMed2003

Cell-cell interactions: leukocyte-endothelial interactions.

Interactions of leukocytes with endothelial cells are early events in acute and chronic inflammation, immune surveillance of tissues, and wound defense and repair. In contrast with their requisite roles in host defense, dysregulated leukocyte-endothelial interactions mediate inflammatory tissue injury, thrombosis, and other pathologic sequelae. Recent observations also identify dysregulated leukocyte-endothelial interactions in neoplasia and sickle cell vasculopathy. Leukocyte interactions with inflamed endothelial cells are mediated by selectins, signaling molecules that include lipids and chemokines, integrins and their ligands, and junctional molecules. They provide multiple checkpoints for regulation in physiologic inflammation and hemostasis and for dysregulation in pathologic syndromes. Neutrophil-endothelial encounters illustrate a multistep paradigm for inflammatory cell-cell interactions and provide the basis for multiple variations on the central themes.

Animals↗
PubMed2002

Outside-in signals delivered by matrix metalloproteinase-1 regulate platelet function.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix proteins. These enzymes are implicated in a variety of physiological and pathological events characterized by extracellular matrix remodeling. Recent studies suggest that MMPs may have a signaling capacity, but direct evidence supporting this concept is lacking. In the present study, we demonstrate that outside-in signals delivered by exogenous MMP-1 (interstitial collagenase) markedly increase the number of tyrosine-phosphorylated proteins in platelets. Active MMP-1 also targets beta(3) integrins to areas of cell contact and primes platelets for aggregation. Examination of the endogenous enzyme demonstrated that activated platelets process latent MMP-1 into its active form. Neutralization of MMP-1 activity with MMP inhibitors or specific blocking antibodies markedly attenuates agonist-induced phosphorylation of intracellular proteins, movement of beta(3) integrins to cell contact points, and intercellular aggregation. The finding that MMP-1 is rapidly activated in platelets and controls functional responses identifies a new role for this metalloproteinase as a signaling molecule that regulates thrombotic events.

Antigens, CD↗
PubMed2002

Sol Sherry lecture in thrombosis: molecular events in acute inflammation.

The inflammatory response is characterized by a multistep molecular interaction between "signaling" cells, such as endothelial cells, and "responding" cells, such as neutrophils and monocytes. In the first step, selectins produced by signaling cells mediate the tethering of responding cells at sites of inflammation. Subsequently, an additional mediator expressed by signaling cells activates the tethered responding cells. Under pathological conditions, the same mechanism is invoked in inappropriate ways: (1) by prolonged presentation of selectins on the cell surface and (2) by the unregulated production of oxidized phospholipids that mimic the normal secondary signaling molecule, platelet-activating factor (PAF). The enzyme PAF acetylhydrolase (PAF-AH) inactivates PAF and oxidized phospholipids and constitutes an "off" switch that suppresses inflammation. Inhibition of normal PAF-AH function or inactivating mutations of the PAF-AH gene can lead to increased susceptibility to inflammatory disease. These studies have relevance to atherosclerosis and thrombosis, because inflammation is a central feature of both.

Acute Disease↗
PubMed2002

Monocyte chemoattractant protein-1 and 5-lipoxygenase products recruit leukocytes in response to platelet-activating factor-like lipids in oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (LDL) contains inflammatory agents, including oxidatively fragmented phospholipids that activate the platelet-activating factor (PAF) receptor, but in vivo events caused by these pathologically generated agents are not well defined. Injection of PAF-like lipids derived from oxidized LDL, or C(4)-PAF that is a major PAF-like lipid in these particles, into the pleural cavity of mice resulted in rapid monocyte, neutrophil, and eosinophil accumulation. Increased numbers of intracellular lipid bodies in these cells show they were in an inflammatory environment. Leukocyte recruitment was abolished by a PAF receptor antagonist, as expected. PAF-like lipids induced 5-lipoxygenase expression in leukocytes, mRNA expression for monocyte chemoattractant protein-1 (MCP-1) and other chemokines, synthesis of MCP-1, and leukotriene B(4). The 5-lipoxygenase inhibitor zileuton impaired neutrophil influx, while MCP-1 had a more global role, as determined with MCP-1(-/-) mice. The lack of MCP-1 abrogated leukocyte accumulation and lipid body formation both in vivo and in vitro and chemokine transcription in vivo, and reduced in vivo leukotriene B(4) production. Thus, PAF-like phospholipids in oxidized LDL induce an inflammatory infiltrate through the PAF receptor, chemokine transcription, lipid body formation, and 5-lipoxygenase expression in leukocytes. MCP-1 has a key role in this inflammatory response, and 5-lipoxygenase products are essential for neutrophil recruitment into the inflamed pleural cavity.

Animals↗
PubMed2002

Retinoic acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2.

Bacterial lipopolysaccharides (LPS) induce expression of multiple genes in endothelial cells, which are critical cellular effectors in various pathologic syndromes. Using subtractive hybridization to identify genes that are differentially induced in human endothelial cells treated with LPS, we found that retinoic acid-inducible gene I (RIG-I) is induced in endothelial cells stimulated with LPS. RIG-I encodes a protein belonging to the DExH-box family which has diverse roles in regulation of gene expression and cellular functions. Cyclooxygenase-2 (COX-2) is also induced in endothelial cells by LPS. Overexpression of RIG-I selectively upregulated expression of COX-2 and also induced COX-2 promoter activity. RIG-I is an inducible gene in stimulated endothelial cells that may have important roles in vascular pathology by virtue of its ability to regulate expression of the COX-2 gene product.

Cell Line↗
PubMed2002

Many actions of cyclooxygenase-2 in cellular dynamics and in cancer.

Cyclooxygenase-2 (COX-2) is the inducible isoform of cyclooxygenase, the enzyme that catalyzes the rate-limiting step in prostaglandin synthesis from arachidonic acid. Various prostaglandins are produced in a cell type-specific manner, and they elicit cellular functions via signaling through G-protein coupled membrane receptors, and in some cases, through the nuclear receptor PPAR. COX-2 utilization of arachidonic acid also perturbs the level of intracellular free arachidonic acid and subsequently affects cellular functions. In a number of cell and animal models, induction of COX-2 has been shown to promote cell growth, inhibit apoptosis and enhance cell motility and adhesion. The mechanisms behind these multiple actions of COX-2 are largely unknown. Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is a key step in carcinogenesis. Overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and inhibition of the COX-2 pathway results in reduction in tumor incidence and progression. Therefore, the potential for application of non-steroidal anti-inflammatory drugs as well as the recently developed COX-2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years. Inhibition of COX-2 promises to be an effective approach in the prevention and treatment of cancer, especially colorectal cancer.

Animals↗
PubMed2002

Activation of vascular cells by PAF-like lipids in oxidized LDL.

The components of inflammation, including macrophages, cytokines and lipid inflammatory mediators, have a role in atherosclerosis. A key lipid mediator in regulated, physiologic inflammation is platelet-activating factor (PAF). PAF activates cells, including monocytes, through a single molecularly characterized receptor, the PAF receptor (PAFR), at exceedingly low concentrations. The PAFR recognizes the short residue, an acetate residue, at the 2-position of the phospholipid, and this sharp specificity precludes receptor activation by other related phosphatidylcholines. Oxidation of low-density lipoproteins (LDLs) is an early and causal step in atherosclerosis that generates inflammatory compounds leading to foam cell formation. One class of oxidatively generated inflammatory compounds are phospholipids that structurally mimic PAF, the PAF-like lipids. Oxidation of LDLs fragments and derivatizes the fatty acid residues at the 2-position of the phosphatidylcholines that comprise the shell of LDLs, an event that allows certain oxidized phospholipids to interact with and activate the PAFR. We know that these products activate polymorphonuclear leukocytes, but because the function of the PAFR differs among cells, we do not know if monocytes or platelets themselves respond to PAF-like lipids. Here, we show that PAF-like lipids from oxidized LDLs are potent and serve as specific agonists for all cells that express the PAFR.

Blood Vessels↗
PubMed2002

The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis.

OBJECTIVES: To review the platelet-activating factor (PAF) signaling system, its regulation, and its dysregulation in acute inflammation and thrombosis and in syndromes that involve these cascades, including sepsis. DATA SOURCES: A summary of published literature from MEDLINE search files and published reviews. DATA EXTRACTION, SYNTHESIS, AND SUMMARY: PAF, a phospholipid signaling molecule, transmits outside-in signals to intracellular transduction systems and effector mechanisms in a variety of cell types, including key cells of the innate immune and hemostatic systems: neutrophils, monocytes, and platelets. Thus, the PAF signaling system is a point of convergence at which injurious stimuli can trigger and amplify both acute inflammatory and thrombotic cascades. The biological activities of PAF are regulated by several precise mechanisms that, together, constrain and control its action in physiologic inflammation. Unregulated synthesis of PAF or defects in the mechanisms that limit its biological activities have the potential to cause pathologic inflammation and thrombosis. In addition, nonenzymatic generation of oxidized phospholipids that are recognized by the PAF receptor can trigger inflammatory and thrombotic events. There is evidence that the PAF signaling system is dysregulated in sepsis, shock, and traumatic injury and that interruption or termination of its effector responses leads to beneficial outcomes. Plasma PAF acetylhydrolase, an enzyme that hydrolyzes PAF and structurally related oxidized phospholipids, yielding products that are no longer recognized by the PAF receptor, may be a particularly important signal terminator. CONCLUSION: The PAF signaling system can trigger inflammatory and thrombotic cascades, amplify these cascades when acting with other mediators, and mediate molecular and cellular interactions (cross talk) between inflammation and thrombosis. Evidence from in vitro experiments, studies of experimental animals, and clinical observations in humans indicates that the PAF signaling system is important in sepsis and other syndromes of inflammatory injury and that therapeutic strategies to interrupt or terminate signaling via the PAF signaling system may be useful in these conditions.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2002

Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving beta 2 integrins and selectin ligands.

Leukocyte adhesion deficiency (LAD) syndromes are failures of innate host defenses against bacteria, fungi, and other microorganisms resulting from defective tethering, adhesion, and targeting of myeloid leukocytes to sites of microbial invasion. LAD I and variant LAD I syndromes are caused by mutations that impair expression or function of integrins of the beta 2 class (CD11/CD18 integrins, or "leukocyte" integrins). In contrast, subjects with LAD II have similar clinical features but intact leukocyte integrin expression and function. The molecular basis for LAD II is defective glycosylation of ligands on leukocytes recognized by the selectin family of adhesion molecules as well as defective glycosylation of other glycoconjugates. The defect has recently been attributed to mutations in a novel fucose transporter localized to the Golgi apparatus. Establishing the molecular basis for LAD syndromes has generated insights into mechanisms of leukocyte accumulation relevant to a broad variety of immunodeficiency syndromes as well as to diseases and disorders of unregulated inflammation that result in tissue damage.

CD18 Antigens↗