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Harvey R. Herschman - One of the best experts on this subject based on the ideXlab platform.
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Historical Aspects of COX-2
2003Co-Authors: Harvey R. HerschmanAbstract:The prostanoids and the leukotrienes are the two major subclasses of the bioactive compounds known as eicosanoids. These hormones are derived from C20 fatty acids. Although a variety of polyunsaturated fatty acids (PUFAs) can serve as precursors to the eicosanoids, the bulk of the prostanoids and leukotrienes are derived from arachidonic acid (AA). Precursor AA does not exist free in cells; AA is present in membrane bound glycerophospholipids. When cells receive an appropriate stimulus, either a secretory or a cellular phospholipase is activated to cleave AA from the membrane phospholipid pool (Fig. 1). The free AA liberated by ligand-stimulated phospholipase activation can then serve as substrate for the formation either of prostanoids or leukotrienes. The Prostaglandin Synthase/cyclooxygenase (COX) enzyme carries out a two-step reaction. In the first step, AA is subjected to a bis oxygenation COX reaction that results in the formation of Prostaglandin G2 (PGG2). This COX reaction is rapidly followed by a hydroperoxidase reaction, occurring at a distinct site on the Prostaglandin Synthase/COX enzyme, to convert PGG2 to PGH2. PGH2 is the common intermediate for the synthesis of the various Prostaglandins (e.g., PGE2, PGF2α, PGD2, etc.), the prostacyclins, and the thromboxanes. The specific nature of the Prostaglandins produced in various cell types depends on the presence of specific Prostaglandin Synthases (e.g., Prostaglandin E2 Synthase, Prostaglandin D2 Synthase, etc.); each of these enzymes uses as substrate the common PGH2 produced by (COX) from free AA. Alternatively, the free AA released by ligand-activated phospholipases can serve as substrate for the lipoxygenase pathway, leading to the formation of the leukotrienes.
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Regulation of COX-2 Expression in Fibroblasts, Osteoblasts, Mast Cells, and Macrophages
Advances in Prostaglandin and Leukotriene Research, 2001Co-Authors: Harvey R. Herschman, David J. Wadleigh, S T ReddyAbstract:The Prostaglandin Synthase/cyclooxygenase (COX) enzymes catalyze two reactions. The cyclooxygenase activity of these enzymes first converts free arachidonic acid, released from membrane lipids by a phospholipase, to PGG2. This intermediate is then converted to PGH2 by the COX hydroperoxidase activity. PGH2 is the precursor for all prostanoids-Prostaglandins, thromboxanes and prostacyclins.
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Recent Progress in the Cellular and Molecular Biology of Prostaglandin Synthesis
Trends in Cardiovascular Medicine, 1998Co-Authors: Harvey R. HerschmanAbstract:Abstract The discovery of an inducible form of Prostaglandin Synthase initiated a reexamination of the biochemical pathways for ligand-induced Prostaglandin synthesis. As a result, new pharmaceutical agents with potential activity against pain, fever, chronic and acute inflammation, cardiovascular disorders, and colon cancer have been developed and are currently under intense investigation. Careful biochemical and pharmacologic studies of the differences between the constitutive and inducible Prostaglandin Synthase enzymes have suggested a previously unexpected mechanism for some of the therapeutic effects of aspirin. Identification of a new phospholipase, and recognition of its role in mast cell Prostaglandin production and in transcellular Prostaglandin synthesis, have identified additional potential targets for pharmacologic intervention in inflammation and other Prostaglandin-mediated disorders.
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Prostaglandin Synthase 1 and Prostaglandin Synthase 2 are coupled to distinct phospholipases for the generation of Prostaglandin d2 in activated mast cells
Journal of Biological Chemistry, 1997Co-Authors: Srinivasa T Reddy, Harvey R. HerschmanAbstract:Abstract Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of Prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by Prostaglandin Synthase 1 (PGS1) and Prostaglandin Synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.
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Prostaglandin Synthase-1 and Prostaglandin Synthase-2 are coupled to distinct phospholipases for the generation of Prostaglandin D2 in activated mast cells.
Journal of Biological Chemistry, 1997Co-Authors: Srinivasa T Reddy, Harvey R. HerschmanAbstract:Abstract Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of Prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by Prostaglandin Synthase 1 (PGS1) and Prostaglandin Synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.
Srinivasa T Reddy - One of the best experts on this subject based on the ideXlab platform.
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Prostaglandin Synthase 1 and Prostaglandin Synthase 2 are coupled to distinct phospholipases for the generation of Prostaglandin d2 in activated mast cells
Journal of Biological Chemistry, 1997Co-Authors: Srinivasa T Reddy, Harvey R. HerschmanAbstract:Abstract Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of Prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by Prostaglandin Synthase 1 (PGS1) and Prostaglandin Synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.
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Prostaglandin Synthase-1 and Prostaglandin Synthase-2 are coupled to distinct phospholipases for the generation of Prostaglandin D2 in activated mast cells.
Journal of Biological Chemistry, 1997Co-Authors: Srinivasa T Reddy, Harvey R. HerschmanAbstract:Abstract Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of Prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by Prostaglandin Synthase 1 (PGS1) and Prostaglandin Synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.
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FUNCTION AND REGULATION OF Prostaglandin Synthase-2
Advances in Experimental Medicine and Biology, 1997Co-Authors: Harvey R. Herschman, Srinivasa T ReddyAbstract:The prostanoids (Prostaglandins, prostacyclins, thromboxanes) are one subset of arachidonic-acid derived lipid mediators of inflammation. The leukotrienes constitute the second major class of arachidonic-acid derived lipid mediators. A variety of inflammatory signals (including exogenous mediators such as endotoxin/lipopoly-saccharide and endogenous mediators such as inflammatory cytokines) stimulate cellular phospholipase activities. As a result, arachidonic acid is released from phospholipid membrane stores. This free arachidonic acid is converted either (i) to leukotrienes via the lipoxygenase pathway(s), or (ii) to prostanoids. The key enzyme in prostanoid biosynthesis is Prostaglandin Synthase (PGS), also known as cyclooxygenase (COX). PGS/COX converts free arachidonate first to Prostaglandin G2 (PGG2) by a cyclooxygenase reaction, and then -in a second enzymatic step- to Prostaglandin H2 (PGH2) by a hydroperoxidase reaction. PGH2 is then converted to the various Prostaglandins (PGE2, PGD2, PGF2a, etc), prostacyclin, (PGI2) or thromboxane by cell-type restricted synthetases that use PGH2 as substrate. The various prostanoids have a wide range of positive and negative immuno-modulatory effects on lymphoid, myeloid, and endothelial cells. New pharmacologic agents that modulate the activity of enzymes involved in prostanoid synthesis or the expression of the genes encoding these enzymes will provide improved directions in the management of acute and chronic inflammatory diseases.
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Gastrin-releasing peptide-induced expression of Prostaglandin Synthase-2 in Swiss 3T3 cells.
Prostaglandins, 1997Co-Authors: J. Randolph Hecht, Srinivasa T Reddy, Harvey R. Herschman, Javier Duque, John H. Walsh, Lee W. SliceAbstract:Prostaglandins, produced in response to mitogens and cytokines, are potent modulators of gastrointestinal physiology and pathophysiology. We investigated modulation of Prostaglandin Synthase 2 (PGS-2) expression by the gastrin-releasing peptide (GRP) receptor in Swiss 3T3 cells. PGS-2 mRNA expression in Swiss 3T3 cells was determined by Northern blot analysis. PGS-2 protein expression in Swiss 3T3 cells was measured by Western blot analysis. GRP caused a transient induction of PGS-2 mRNA in Swiss 3T3 cells that resulted in GRP-dependent expression of PGS-2 protein. Transcriptional activation of PGS-2 by GRP was independent of de novo protein synthesis and was not affected by pertussis toxin. Comparison of signaling pathways used by PMA or EGF to those used by GRP showed that PGS-2 induction by GRP increased under conditions that inhibit PKC activity. Dexamethasone, which blocks PMA and EGF induction of PGS-2, also inhibited GRP-induced accumulation of PGS-2 mRNA. These results show that PGS-2 expression in Swiss 3T3 cells is not only controlled by PKC and receptor tyrosine kinase pathways but also by G-protein coupled receptor signaling pathways.
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Transcellular Prostaglandin Production following Mast Cell Activation Is Mediated by Proximal Secretory Phospholipase A and Distal Prostaglandin Synthase 1
Journal of Biological Chemistry, 1996Co-Authors: Srinivasa T Reddy, Harvey R. HerschmanAbstract:Abstract Prostaglandins mediate many biological processes. Arachidonic acid, the common precursor for all Prostaglandins, is released from membrane phospholipids by both secretory and cytoplasmic forms of phospholipase A. Free arachidonate is converted to Prostaglandin H, the common precursor to all prostanoids, by Prostaglandin Synthase. Both mitogen-induced Prostaglandin synthesis in fibroblasts and endotoxin-induced Prostaglandin synthesis in macrophages require expression of the inducible Prostaglandin Synthase-2; arachidonate released in these contexts is unavailable to Prostaglandin Synthase-1 constitutively present in fibroblasts or macrophages. In contrast to the results for fibroblasts and macrophages, Prostaglandin synthesis by activated mast cells is mediated by Prostaglandin Synthase-1. Mast cell activation also provokes release of secretory phospholipase A (sPLA). We now demonstrate that sPLA released from activated mast cells can mobilize arachidonate from distal Swiss 3T3 cells. This arachidonate is then used by Prostaglandin Synthase-1 present in 3T3 cells for Prostaglandin synthesis. We thus distinguish two pathways for Prostaglandin synthesis: (i) an intracellular pathway by which arachidonate released following ligand stimulation is made available only to Prostaglandin Synthase-2, and (ii) a transcellular pathway by which sPLA of proximal cells mobilizes, in distal cells, arachidonate available to Prostaglandin Synthase-1. Molecular and pharmacologic approaches to modulating Prostaglandin-mediated events will differ for these two pathways.
D A Kujubu - One of the best experts on this subject based on the ideXlab platform.
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Transforming growth factor β1 augments mitogen‐induced Prostaglandin synthesis and expression of the TIS10/Prostaglandin Synthase 2 gene both in swiss 3T3 cells and in murine embryo fibroblasts
Journal of Cellular Physiology, 1994Co-Authors: Rebecca S. Gilbert, Srinivasa T Reddy, Weilin Xie, D A Kujubu, Steven Luner, Harvey R. HerschmanAbstract:Transforming growth factor-beta (TGF-beta), a potent cytokine, modulates a wide variety of biological responses. Among its actions, TGF-beta can augment Prostaglandin synthesis in several cell types. Although TGF-beta alone has no effect on Prostaglandin production in Swiss 3T3 cells, we find that TGF-beta augments the ability of tetradecanoyl phorbol acetate (TPA) or serum to stimulate PGE2 production. The TIS10 gene is a primary response gene encoding a second form of Prostaglandin Synthase (PGS), the rate-limiting enzyme in the biosynthesis of Prostaglandins, thrornboxanes, and prostacyclins from arachidonic acid. TIS10/PGS-2 expression is induced by mitogens in Swiss 3T3 cells. TGF-beta also augments mitogen-induced synthesis and accumulation of TIS10/PGS-2 protein and induction of TIS10/PGS-2 message in Swiss 3T3 cells. In contrast, TGF-beta has little or no effect on the level of PGS-1 (EC1.14.99.1) message, either alone or in concert with TPA or serum. TGF-beta concentrations in the range of 0.01–0.10 ng/ml (0.4–4.0 pM) maximally enhance mitogen induction of TIS10/PGS-2 message. TPA-induced accumulation of unspliced TIS10/PGS-2 transcript is augmented by TGF-beta, suggesting that this cytokine exerts its effect on expression of the TIS10/PGS-2 gene by transcriptional regulation. TGF-beta also augments TPA-induced Prostaglandin production, TIS10/PGS-2 antigen accumulation, and TIS10/PGS-2 message induction in primary cultures of mouse embryo fibroblasts. Dexamethasone attenuates TGF-beta enhancement of all these mitogen-induced responses: PGE2 accumulation, appearance of TIS10/PGS-2 protein and message, and accumulation of TIS10/PGS-2 unprocessed transcript. © 1994 wiley-Liss, Inc.
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Expression of the protein product of the Prostaglandin Synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells.
The Journal of biological chemistry, 1993Co-Authors: D A Kujubu, S T Reddy, Bradley S. Fletcher, Harvey R. HerschmanAbstract:Abstract TIS10/PGS-2 encodes a Prostaglandin Synthase (PGS) distinct from the previously described enzyme PGS-1 (EC 1.14.99.1). We have now generated antipeptide antisera, directed to an amino acid sequence unique to the murine TIS10/PGS-2 protein, which specifically recognize the TIS10/PGS-2 antigen. TIS10/PGS-2 protein was undetectable in quiescent Swiss 3T3 cells. The level of TIS10/PGS-2 protein peaked between 6 and 8 h following phorbol ester stimulation of cells, then declined to basal levels after 18-24 h. Synthesis of TIS10/PGS-2 protein was dramatically increased in the second hour following mitogen stimulation and remained elevated for several hours. The half-life of the TIS10/PGS-2 protein was 4 h. Immunofluorescence studies demonstrated a perinuclear and cytoplasmic localization of the TIS10/PGS-2 antigen. As expected, detection of induced TIS10/PGS-2 antigen was dependent on protein synthesis. Metabolically labeled TIS10/PGS-2 protein migrated as a 71/73-kDa doublet following immunoprecipitation. Dexamethasone blocked both the TPA- and serum-induced appearance of TIS10/PGS-2 antigen. These studies demonstrate the existence of a mitogen-inducible, glucocorticoid-inhibitable, immunologically distinct Prostaglandin Synthase protein.
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Chromosomal organization of the inducible and constitutive Prostaglandin Synthase/cyclooxygenase genes in mouse.
Genomics, 1993Co-Authors: Ping Zi Wen, Harvey R. Herschman, D A Kujubu, Bradley S. Fletcher, Craig H. Warden, Aldons J. LusisAbstract:Two distinct Prostaglandin Synthase/cyclooxygenase (PGS/COS) enzymes have recently been recognized. One (EC 1.14.00.1) is largely constitutive and has been characterized in a variety of species, whereas the other (also known as TIS10) is inducible by mitogens and inhibitable by glucocorticoids. Along with activation of phospholipase A2, the latter PGS/COS is likely to mediate ligand-induced Prostaglandin production in a variety of cell types. The two enzymes have similar gene structures and activities. There is accumulating evidence that PGS/COX activity may play a role in inflammatory diseases. For example, PGS/COX expression is upregulated in inflammatory joint diseases and is genetically controlled. As part of an effort to examine genetic factors regulating PGS/COX expression and the possible involvement of the enzymes in mouse models of inflammatory disorders, we report here the chromosomal mapping of the genes for the constitutive and regulated PGS/COX enzymes. We will refer to the constitutive enzyme as Pgs-1 and the inducible enzyme as Pgs-2. 14 refs., 1 tab.
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Dexamethasone inhibits mitogen induction of the TIS10 Prostaglandin Synthase/cyclooxygenase gene.
The Journal of biological chemistry, 1992Co-Authors: D A Kujubu, Harvey R. HerschmanAbstract:Abstract Glucocorticoids block the induced secretion of Prostaglandins in a variety of biological contexts. We have identified a primary response gene, TIS10, which encodes a mitogen-inducible Prostaglandin Synthase/cyclooxygenase in Swiss 3T3 cells. TIS10 is distinct from Prostaglandin Synthase/cyclooxygenase. (EC 1.14.99.1), previously cloned from mouse, man, and sheep. Dexamethasone blocks Prostaglandin E2 synthesis by 3T3 cells in response to tetradecanoylphorbol acetate. Dexamethasone also blocks both phorbol ester- and forskolin-induced TIS10 mRNA accumulation. In contrast, phorbol esters, forskolin, and dexamethasone have little or no effect on the levels of Prostaglandin Synthase/cyclooxygenase mRNA in 3T3 cells. Moreover, dexamethasone does not inhibit induction of TIS8/egr-1, another primary response gene. Inhibition of the synthesis of TIS10 Prostaglandin Synthase/cyclooxygenase may be a principal mechanism by which glucocorticoids block Prostaglandin synthesis and secretion.
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dexamethasone inhibits mitogen induction of the tis10 Prostaglandin Synthase cyclooxygenase gene
Journal of Biological Chemistry, 1992Co-Authors: D A Kujubu, Harvey R. HerschmanAbstract:Abstract Glucocorticoids block the induced secretion of Prostaglandins in a variety of biological contexts. We have identified a primary response gene, TIS10, which encodes a mitogen-inducible Prostaglandin Synthase/cyclooxygenase in Swiss 3T3 cells. TIS10 is distinct from Prostaglandin Synthase/cyclooxygenase. (EC 1.14.99.1), previously cloned from mouse, man, and sheep. Dexamethasone blocks Prostaglandin E2 synthesis by 3T3 cells in response to tetradecanoylphorbol acetate. Dexamethasone also blocks both phorbol ester- and forskolin-induced TIS10 mRNA accumulation. In contrast, phorbol esters, forskolin, and dexamethasone have little or no effect on the levels of Prostaglandin Synthase/cyclooxygenase mRNA in 3T3 cells. Moreover, dexamethasone does not inhibit induction of TIS8/egr-1, another primary response gene. Inhibition of the synthesis of TIS10 Prostaglandin Synthase/cyclooxygenase may be a principal mechanism by which glucocorticoids block Prostaglandin synthesis and secretion.
Weilin Xie - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation of Prostaglandin Synthase 2 gene expression by platelet-derived growth factor and serum.
Journal of Biological Chemistry, 1996Co-Authors: Weilin Xie, Harvey R. HerschmanAbstract:Abstract Prostaglandin Synthase 2 (PGS2) is an immediate-early gene induced in a variety of cellular contexts. We investigate here the transcriptional activation of the murine PGS2 gene in NIH 3T3 cells, in response to the mitogens platelet-derived growth factor (PDGF) or serum. Site-directed mutagenesis experiments demonstrate that a consensus cyclic AMP response element (CRE) in the murine PGS2 promoter is essential for optimal PGS2 gene expression in response to PDGF or to serum. Overexpression of c-Jun potentiates PDGF- or serum-induced luciferase expression from a reporter construct containing the first 371 nucleotides of the PGS2 promoter. In contrast, overexpression of other transcription factors binding to the CRE element of the PGS2 gene inhibits induction by PDGF or serum. Moreover, positioning the c-Jun activation domain next to the minimal PGS2 promoter via a GAL4 DNA binding site rather than the CRE is sufficient to permit serum or PDGF stimulation of luciferase expression from this modified reporter construct. PDGF or serum treatment both activate c-Jun N-terminal kinase (JNK), the mitogen-activated protein kinase responsible for phosphorylation and activation of c-Jun. Cotransfection of plasmids expressing dominant-negative Ras, Rac1, MEKK-1, or JNK along with the [PGS2][luciferase] reporter prevents induction by PDGF or serum, demonstrating that serum and PDGF induction of the PGS2 gene in NIH 3T3 cells requires activation of a Ras/Rac1/MEKK-1/JNK kinase/JNK signal transduction leading to phosphorylation of c-Jun. Additional cotransfection experiments with plasmids expressing dominant-negative Raf1 and ERK demonstrate that induction of PGS2 gene expression by PDGF and serum also requires activation of a Ras/Raf1/mitogen-activated protein kinase kinase (MAPKK)/ERK signal transduction pathway.
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v-src Induces Prostaglandin Synthase 2 Gene Expression by Activation of the c-Jun N-terminal Kinase and the c-Jun Transcription Factor
Journal of Biological Chemistry, 1995Co-Authors: Weilin Xie, Harvey R. HerschmanAbstract:Abstract A consensus cyclic AMP response element (CRE) in the murine Prostaglandin Synthase-2 (PGS2) promoter is essential for pgs2 gene expression induced by pp60, the v-src oncogene product. In this study, we investigate (i) the transcription factors active at the PGS2 “CRE site” in response to v-src activation and (ii) the signal transduction pathways by which pp60 activates these transcription factors. Transient transfection assays with pgs2 promoter/luciferase reporter chimeric genes suggest that c-Jun mediates v-src-induced pgs2 gene expression. Antibody supershift experiments demonstrate that c-Jun can participate in a complex with the pgs2 promoter CRE site. Moreover, in vitro immunocomplex assays demonstrate that pp60 expression strongly activates c-Jun N-terminal kinase (JNK1) enzyme activity. Serines 63 and 73, the sites of c-Jun phosphorylation by JNK, are essential for v-src-induced, pgs2 promoter-mediated luciferase expression. Cotransfection studies with plasmids expressing wild-type JNK, dominant-negative JNK, and dominant-negative MEKK-1 confirm that activation of the Ras/MEKK-1/JNK/c-Jun pathway is required for v-src-induced pgs2 gene expression. Overexpression of either wild-type ERK-1 or ERK-2 proteins also potentiate v-src-mediated luciferase expression driven by the pgs2 promoter, and expression of dominant-negative mutants of ERK-1, ERK-2, or Raf-1 attenuate this response. Thus, in response to v-src expression, a Ras/MEKK-1/JNK signal transduction pathway activating c-Jun and a Ras/Raf-1/ERK pathway converge to mediate pgs2 gene expression via the CRE site in the pgs2 promoter.
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Inflammation, reproduction, cancer and all that…. The regulation and role of the inducible Prostaglandin Synthase
BioEssays, 1995Co-Authors: Harvey R. Herschman, Weilin Xie, Srinivasa T ReddyAbstract:Discovery of a second, inducible Prostaglandin Synthase provides explanations for many previously puzzling observations, but also raises new questions about prostanoid synthesis. A cis-acting sequence closely related to the cyclic AMP response element has been shown to play a role in both basal and induced Prostaglandin Synthase 2 gene expression. Aspirin and other currently available non-steroidal anti-inflammatory drugs that inhibit Prostaglandin Synthase activity do not effectively discriminate between the inducible Prostaglandin Synthase 2 and constitutive Prostaglandin Synthase 1 enzymes. Identification of a second Prostaglandin Synthase, induced by inflammatory stimuli, initiated a search for isoform-specific inhibitors. Use of Prostaglandin Synthase 2 specific inhibitors and antisense oligonucleotides has led to the suggestion that specific ligands activate alternative pathways of prostanoid production, using one of the Prostaglandin Synthase isoforms preferentially. The coupling mechanisms by which these pathways are activated in response to alternative stimuli should provide additional routes of intervention in prostanoid production.
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v-src induction of the TIS10/PGS2 Prostaglandin Synthase gene is mediated by an ATF/CRE transcription response element.
Molecular and Cellular Biology, 1994Co-Authors: Weilin Xie, Bradley S. Fletcher, Robert D. Andersen, Harvey R. HerschmanAbstract:We recently reported the cloning of a mitogen-inducible Prostaglandin Synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.
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v src induction of the tis10 pgs2 Prostaglandin Synthase gene is mediated by an atf cre transcription response element
Molecular and Cellular Biology, 1994Co-Authors: Weilin Xie, Bradley S. Fletcher, Robert D. Andersen, Harvey R. HerschmanAbstract:We recently reported the cloning of a mitogen-inducible Prostaglandin Synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.
Bradley S. Fletcher - One of the best experts on this subject based on the ideXlab platform.
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v-src induction of the TIS10/PGS2 Prostaglandin Synthase gene is mediated by an ATF/CRE transcription response element.
Molecular and Cellular Biology, 1994Co-Authors: Weilin Xie, Bradley S. Fletcher, Robert D. Andersen, Harvey R. HerschmanAbstract:We recently reported the cloning of a mitogen-inducible Prostaglandin Synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.
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v src induction of the tis10 pgs2 Prostaglandin Synthase gene is mediated by an atf cre transcription response element
Molecular and Cellular Biology, 1994Co-Authors: Weilin Xie, Bradley S. Fletcher, Robert D. Andersen, Harvey R. HerschmanAbstract:We recently reported the cloning of a mitogen-inducible Prostaglandin Synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.
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Platelet-activating factor and retinoic acid synergistically activate the inducible Prostaglandin Synthase gene.
Proceedings of the National Academy of Sciences, 1994Co-Authors: Nicolas G. Bazan, Harvey R. Herschman, Bradley S. Fletcher, Pranab K. MukherjeeAbstract:Abstract Platelet-activating factor (PAF), a potent lipid mediator generated in cell injury and in the inflammatory and immune responses, promotes transcriptional activation of several primary response genes. TIS10/PGS-2 is a primary response gene encoding the inducible form of Prostaglandin Synthase. The inductive effects of PAF and retinoic acid (RA), alone and in combination, were studied with the regulatory region of TIS10/PGS-2 transfected into an exponentially growing glioblastoma-neuroblastoma NG108-15 hybrid in the human SH-SY5Y neuroblastoma or in the NIH 3T3 cell. RA alone exhibited only a small inductive effect. However, in the presence of RA (100 nM), a PAF-dependent (1-50 nM) synergistic activation of luciferase reporter constructs driven by regulatory regions of the TIS10/PGS-2 gene was found. The hetrazepine BN-50730, an antagonist selective for intracellular PAF binding sites, inhibited PAF and RA induction of luciferase from the TIS10/PGS-2 promoter. Thus, the intracellular PAF binding site is involved in TIS10/PGS-2 expression. Induction is rapid, suggesting that the combination of PAF and RA activates a preexisting latent transcription factor(s). Deletion studies restrict the major PAF and RA cis-acting response element of the TIS10/PGS-2 gene to a 70-nucleotide sequence as an intracellular inducer of TIS10/PGS-2 expression. The synergistic effect of RA and PAF represents an unusual convergence of nuclear signaling pathways by which, through the modulation of preexisting transcription factors, specific gene expression can be upregulated. PAF-dependent induction of TIS10/PGS-2 expression may play a role in cell injury, differentiation, inflammation, and immune responses.
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Expression of the protein product of the Prostaglandin Synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells.
The Journal of biological chemistry, 1993Co-Authors: D A Kujubu, S T Reddy, Bradley S. Fletcher, Harvey R. HerschmanAbstract:Abstract TIS10/PGS-2 encodes a Prostaglandin Synthase (PGS) distinct from the previously described enzyme PGS-1 (EC 1.14.99.1). We have now generated antipeptide antisera, directed to an amino acid sequence unique to the murine TIS10/PGS-2 protein, which specifically recognize the TIS10/PGS-2 antigen. TIS10/PGS-2 protein was undetectable in quiescent Swiss 3T3 cells. The level of TIS10/PGS-2 protein peaked between 6 and 8 h following phorbol ester stimulation of cells, then declined to basal levels after 18-24 h. Synthesis of TIS10/PGS-2 protein was dramatically increased in the second hour following mitogen stimulation and remained elevated for several hours. The half-life of the TIS10/PGS-2 protein was 4 h. Immunofluorescence studies demonstrated a perinuclear and cytoplasmic localization of the TIS10/PGS-2 antigen. As expected, detection of induced TIS10/PGS-2 antigen was dependent on protein synthesis. Metabolically labeled TIS10/PGS-2 protein migrated as a 71/73-kDa doublet following immunoprecipitation. Dexamethasone blocked both the TPA- and serum-induced appearance of TIS10/PGS-2 antigen. These studies demonstrate the existence of a mitogen-inducible, glucocorticoid-inhibitable, immunologically distinct Prostaglandin Synthase protein.
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Chromosomal organization of the inducible and constitutive Prostaglandin Synthase/cyclooxygenase genes in mouse.
Genomics, 1993Co-Authors: Ping Zi Wen, Harvey R. Herschman, D A Kujubu, Bradley S. Fletcher, Craig H. Warden, Aldons J. LusisAbstract:Two distinct Prostaglandin Synthase/cyclooxygenase (PGS/COS) enzymes have recently been recognized. One (EC 1.14.00.1) is largely constitutive and has been characterized in a variety of species, whereas the other (also known as TIS10) is inducible by mitogens and inhibitable by glucocorticoids. Along with activation of phospholipase A2, the latter PGS/COS is likely to mediate ligand-induced Prostaglandin production in a variety of cell types. The two enzymes have similar gene structures and activities. There is accumulating evidence that PGS/COX activity may play a role in inflammatory diseases. For example, PGS/COX expression is upregulated in inflammatory joint diseases and is genetically controlled. As part of an effort to examine genetic factors regulating PGS/COX expression and the possible involvement of the enzymes in mouse models of inflammatory disorders, we report here the chromosomal mapping of the genes for the constitutive and regulated PGS/COX enzymes. We will refer to the constitutive enzyme as Pgs-1 and the inducible enzyme as Pgs-2. 14 refs., 1 tab.
