Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of athero... more Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of atherosclerotic tissues and is regulated by nuclear transcriptional factors including nuclear factor-κB (NF-κB). We constructed a reporter gene assay to measure NF-κB-dependent ...
Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important... more Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [ "C]arachidonic acid, synthesized prostacyclin (€'GIe) together with prostaglandins Eo, Dp, and Fp,. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGIz synthesis recovered rapidly with a TI,, of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGEe, PGDp, and PGF,, synthesis paralleled that of PGIo, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGIe synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells. The material was recovered in protein following delipidation of serum, in the impermeate following filtration through molecular filters, and also in the infranatant after removal of lipoproteins by ultracentrifugation. Activity was not duplicated by a-tocopherol, estradiol, or insulin, or by platelet (PDGF) and fibroblast (FGF) growth factors. However, nanogram quantities of purified mouse epidermal growth factor (EGF) effectively replaced the biological activity of 10% serum. Maximum stimulation occurred at an EGF concentration, in serum-free chemically defined medium, of approximately 40 ngh1.m These studies indicate that synthesis of cyclooxygenase in many cells requires EGE In aspirin-inactivated cells, EGF induces rapid restoration of the enzyme by a cycloheximide-sensitive process that does not require synthesis of new mRNA. Refractoriness to PG-releasing stimuli develops due to inactivation of cyclooxygenase; EGF may also be involved in regeneration of prostaglandin synthetase in vivo. -Bailey, J. M., B. Muza, medium; 15-HETE, 15-hydroxyeicosatetraenoic acid; RB cells, rat aorta smooth muscle cells; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein.
We present evidence supporting the hypothesis that locally produced platelet derived growth facto... more We present evidence supporting the hypothesis that locally produced platelet derived growth factor (PDGF) B-like polypeptides, as well as heparin binding growth factor-1 (HBGF-1), are involved in stimulating the pronounced hyperplasia of rheumatoid synovial stromal fibroblastlike cells. Explanted rheumatoid synovial tissues in vitro spontaneously secreted, in a time dependent manner, mitogenic activity for rheumatoid synoviocytes that was neutralizable by anti-PDGF antibody. PDGF B/c-sis mRNA transcripts were detected in synovium from patients with rheumatoid arthritis (RA) (n = 5). Spontaneous PDGF B-like synthesis was detected by immunoprecipitation of radiolabeled PDGF B-like polypeptides secreted by explanted tissues. Furthermore, rheumatoid synovial tissues, particularly macrophage-like cells, immunostained specifically with anti-PDGF B chain. The extent and intensity of staining and mononuclear cell infiltration were highly correlated. Immunostaining of osteoarthritic and normal synovial tissues was significantly less than RA synovium. PDGF-B immunostaining of synovial specimens previously characterized for expression of HBGF-1, the precursor of acidic fibroblast growth factor (aFGF), revealed that the extent and intensity of expression of HBGF-1 and PDGF-B were highly correlated.
Advances in prostaglandin, thromboxane, and leukotriene research
Our studies are designed to determine which amino acid residues are involved in catalyzing the cy... more Our studies are designed to determine which amino acid residues are involved in catalyzing the cyclooxygenase and hydroperoxidase activities of prostaglandin endoperoxide (PGG/H) synthase. We have deduced from complementary (c)DNAs the amino acid sequences of the sheep and mouse PGG/H synthases, and a portion of the human PGG/H synthase. These enzymes have amino acid sequences which are about 90% identical. Sequence similarities with putative heme binding regions of myeloperoxidase and thyroid peroxidase suggest that the sequence TI(L)WLREHNRV of PGG/H synthase contains the histidine (His309) which is the proximal heme ligand; the distal heme ligand may be His226 which is found in the sequence 222-KALGH-226. Using site-directed mutagenesis, we have replaced Ser530, the serine residue which is acetylated by aspirin, with Ala530 and with Asn530; the Ala530 mutant has both cyclooxygenase and hydroperoxidase activity, while the Asn530 mutant lacks cyclooxygenase activity but retains hydroperoxidase activity. These results establish that the hydroxyl group of Ser530 is not essential for catalysis or substrate binding and suggest that a bulky group at position 530, such as that introduced by aspirin acetylation, prevents arachidonate binding to the cyclooxygenase active site. Finally, we have found that tetranitromethane causes irreversible inactivation of cyclooxygenase activity and that the enzyme is protected from inactivation when ibuprofen is included in the reaction mixture. These results suggest that there is an essential tyrosine at the active site of PGG/H synthase.
Prostanoids, produced by diverse cell types, modulate a variety of pathophysiological processes. ... more Prostanoids, produced by diverse cell types, modulate a variety of pathophysiological processes. The rate of prostanoid synthesis is determined, in part, by the levels of prostanoid-synthetic enzymes, such as cyclooxygenase (Cox), a rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. While two Cox genes have been identified, Cox-2 is unique because it is highly induced in response to cell activation processes including inflammation. W e have studied the effect of interleukin-la (IL-la), a proinflammatory cytokine that facilitates its actions in part by inducing the synthesis of prostanoids, on the expression of Cox-2 in a human cell line (ECV304) and demonstrated that IL-la induces a sustained increase in the expression of the Cox-2 mRNA as well as the functional enzyme. Three mechanistically distinct inhibitors of translation stimulated the expression of the Cox-2 mRNA and potentiated the effect of IL-la. Furthermore, IL-la induced rapid but transient activation of Cox-2 transcription and, in the absence of transcription, prolonged the half-life of the Cox-2 mRNA. Together, these data suggest that post-transcriptional mechanisms are important in the sustained induction of the Cox-2 mRNA and that IL-la may increase the stability of the Cox-2 transcript.
Prostaglandins and thromboxanes, collectively known as prostanoids (PGs), arc produced by biologi... more Prostaglandins and thromboxanes, collectively known as prostanoids (PGs), arc produced by biologic oxidation of arachidonic acid.'PGs are synthesized and sccretcd by a wide variety of cells only when stimulated by a multitude of ccll pcrturbations, ranging from ...
We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, seve... more We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, several cDNA clones that are induced by phorbol myristic acetate in human umbilical vein endothelial cells (HUVEC). One such clone, termed edg-2, was sequenced and was found to encode a human homologue of a Xenopus maternal transcript G10. The deduced amino acid sequence of edg-2 contains a putative nuclear translocation sequence, an N-terminal acidic domain and a cysteine-rich C-terminal domain containing a putative Zinc-finger structure. The structure of edg-2 polypeptide suggests that it may be a nuclear regulator of transcription. The edg-2 mRNA was expressed ubiquitously in cell lines of epithelial and mesenchymal lineages. In addition, the edg-2 polypeptide sequence is highly conserved in evolution and is expressed by lower organisms such as yeast and C. elegans, suggesting that it may be an important regulator of general nuclear function.
The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced du... more The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1.
Journal of Pharmacology and Experimental Therapeutics
We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the... more We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors bel... more The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR␥ has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR␥ ligand, 15-deoxy-⌬ 12,14 -PGJ 2 (15d-PGJ 2 ) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR␥ 1. The PPAR␥ expressed is of functional significance in that two ligands for PPAR␥, 15d-PGJ 2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC.
Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In th... more Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.
The bioactive lipid sphingosine-1-phosphate (SPP) is abundantly formed and released during the ac... more The bioactive lipid sphingosine-1-phosphate (SPP) is abundantly formed and released during the activation of platelets by thrombotic stimuli. Once exported, SPP interacts with the G-protein-coupled receptors (GPCR) of the EDG-1 family. SPP binds to EDG-1 with the dissociation constant of approximately 8 nM and induces signal transduction events such as mitogen-activated protein kinase (MAP kinase) activation, decrease of cAMP levels, remodeling of the actin cytoskeleton, among others. EDG-1 is a prototypical member of a large family of GPCRs that interact with glycero- and sphingolysolipid phosphates, namely, SPP and lysophosphatidic acid (LPA). Three other GPCRs, trivially termed EDG-3, EDG-5, and EDG-8, are also high-affinity receptors for SPP. The four SPP receptor subtypes regulate different intracellular signal transduction pathways. In vascular endothelial cells, cooperative signaling between EDG-1 and EDG-3 subtypes of SPP receptors results in adherens junction assembly, cell survival, morphogenesis into capillary-like networks, and angiogenesis. SPP acts distinctly, albeit cooperatively, with polypeptide angiogenic factors, resulting in the formation of mature neovessels. Thus SPP signaling as an extracellular mediator via the EDG-1 family of GPCRs may be a heretofore unrecognized mechanism for the regulation of angiogenesis and vascular endothelial cell function.
Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of athero... more Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of atherosclerotic tissues and is regulated by nuclear transcriptional factors including nuclear factor-κB (NF-κB). We constructed a reporter gene assay to measure NF-κB-dependent ...
Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important... more Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [ "C]arachidonic acid, synthesized prostacyclin (€'GIe) together with prostaglandins Eo, Dp, and Fp,. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGIz synthesis recovered rapidly with a TI,, of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGEe, PGDp, and PGF,, synthesis paralleled that of PGIo, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGIe synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells. The material was recovered in protein following delipidation of serum, in the impermeate following filtration through molecular filters, and also in the infranatant after removal of lipoproteins by ultracentrifugation. Activity was not duplicated by a-tocopherol, estradiol, or insulin, or by platelet (PDGF) and fibroblast (FGF) growth factors. However, nanogram quantities of purified mouse epidermal growth factor (EGF) effectively replaced the biological activity of 10% serum. Maximum stimulation occurred at an EGF concentration, in serum-free chemically defined medium, of approximately 40 ngh1.m These studies indicate that synthesis of cyclooxygenase in many cells requires EGE In aspirin-inactivated cells, EGF induces rapid restoration of the enzyme by a cycloheximide-sensitive process that does not require synthesis of new mRNA. Refractoriness to PG-releasing stimuli develops due to inactivation of cyclooxygenase; EGF may also be involved in regeneration of prostaglandin synthetase in vivo. -Bailey, J. M., B. Muza, medium; 15-HETE, 15-hydroxyeicosatetraenoic acid; RB cells, rat aorta smooth muscle cells; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein.
We present evidence supporting the hypothesis that locally produced platelet derived growth facto... more We present evidence supporting the hypothesis that locally produced platelet derived growth factor (PDGF) B-like polypeptides, as well as heparin binding growth factor-1 (HBGF-1), are involved in stimulating the pronounced hyperplasia of rheumatoid synovial stromal fibroblastlike cells. Explanted rheumatoid synovial tissues in vitro spontaneously secreted, in a time dependent manner, mitogenic activity for rheumatoid synoviocytes that was neutralizable by anti-PDGF antibody. PDGF B/c-sis mRNA transcripts were detected in synovium from patients with rheumatoid arthritis (RA) (n = 5). Spontaneous PDGF B-like synthesis was detected by immunoprecipitation of radiolabeled PDGF B-like polypeptides secreted by explanted tissues. Furthermore, rheumatoid synovial tissues, particularly macrophage-like cells, immunostained specifically with anti-PDGF B chain. The extent and intensity of staining and mononuclear cell infiltration were highly correlated. Immunostaining of osteoarthritic and normal synovial tissues was significantly less than RA synovium. PDGF-B immunostaining of synovial specimens previously characterized for expression of HBGF-1, the precursor of acidic fibroblast growth factor (aFGF), revealed that the extent and intensity of expression of HBGF-1 and PDGF-B were highly correlated.
Advances in prostaglandin, thromboxane, and leukotriene research
Our studies are designed to determine which amino acid residues are involved in catalyzing the cy... more Our studies are designed to determine which amino acid residues are involved in catalyzing the cyclooxygenase and hydroperoxidase activities of prostaglandin endoperoxide (PGG/H) synthase. We have deduced from complementary (c)DNAs the amino acid sequences of the sheep and mouse PGG/H synthases, and a portion of the human PGG/H synthase. These enzymes have amino acid sequences which are about 90% identical. Sequence similarities with putative heme binding regions of myeloperoxidase and thyroid peroxidase suggest that the sequence TI(L)WLREHNRV of PGG/H synthase contains the histidine (His309) which is the proximal heme ligand; the distal heme ligand may be His226 which is found in the sequence 222-KALGH-226. Using site-directed mutagenesis, we have replaced Ser530, the serine residue which is acetylated by aspirin, with Ala530 and with Asn530; the Ala530 mutant has both cyclooxygenase and hydroperoxidase activity, while the Asn530 mutant lacks cyclooxygenase activity but retains hydroperoxidase activity. These results establish that the hydroxyl group of Ser530 is not essential for catalysis or substrate binding and suggest that a bulky group at position 530, such as that introduced by aspirin acetylation, prevents arachidonate binding to the cyclooxygenase active site. Finally, we have found that tetranitromethane causes irreversible inactivation of cyclooxygenase activity and that the enzyme is protected from inactivation when ibuprofen is included in the reaction mixture. These results suggest that there is an essential tyrosine at the active site of PGG/H synthase.
Prostanoids, produced by diverse cell types, modulate a variety of pathophysiological processes. ... more Prostanoids, produced by diverse cell types, modulate a variety of pathophysiological processes. The rate of prostanoid synthesis is determined, in part, by the levels of prostanoid-synthetic enzymes, such as cyclooxygenase (Cox), a rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. While two Cox genes have been identified, Cox-2 is unique because it is highly induced in response to cell activation processes including inflammation. W e have studied the effect of interleukin-la (IL-la), a proinflammatory cytokine that facilitates its actions in part by inducing the synthesis of prostanoids, on the expression of Cox-2 in a human cell line (ECV304) and demonstrated that IL-la induces a sustained increase in the expression of the Cox-2 mRNA as well as the functional enzyme. Three mechanistically distinct inhibitors of translation stimulated the expression of the Cox-2 mRNA and potentiated the effect of IL-la. Furthermore, IL-la induced rapid but transient activation of Cox-2 transcription and, in the absence of transcription, prolonged the half-life of the Cox-2 mRNA. Together, these data suggest that post-transcriptional mechanisms are important in the sustained induction of the Cox-2 mRNA and that IL-la may increase the stability of the Cox-2 transcript.
Prostaglandins and thromboxanes, collectively known as prostanoids (PGs), arc produced by biologi... more Prostaglandins and thromboxanes, collectively known as prostanoids (PGs), arc produced by biologic oxidation of arachidonic acid.'PGs are synthesized and sccretcd by a wide variety of cells only when stimulated by a multitude of ccll pcrturbations, ranging from ...
We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, seve... more We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, several cDNA clones that are induced by phorbol myristic acetate in human umbilical vein endothelial cells (HUVEC). One such clone, termed edg-2, was sequenced and was found to encode a human homologue of a Xenopus maternal transcript G10. The deduced amino acid sequence of edg-2 contains a putative nuclear translocation sequence, an N-terminal acidic domain and a cysteine-rich C-terminal domain containing a putative Zinc-finger structure. The structure of edg-2 polypeptide suggests that it may be a nuclear regulator of transcription. The edg-2 mRNA was expressed ubiquitously in cell lines of epithelial and mesenchymal lineages. In addition, the edg-2 polypeptide sequence is highly conserved in evolution and is expressed by lower organisms such as yeast and C. elegans, suggesting that it may be an important regulator of general nuclear function.
The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced du... more The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1.
Journal of Pharmacology and Experimental Therapeutics
We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the... more We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors bel... more The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR␥ has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR␥ ligand, 15-deoxy-⌬ 12,14 -PGJ 2 (15d-PGJ 2 ) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR␥ 1. The PPAR␥ expressed is of functional significance in that two ligands for PPAR␥, 15d-PGJ 2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC.
Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In th... more Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.
The bioactive lipid sphingosine-1-phosphate (SPP) is abundantly formed and released during the ac... more The bioactive lipid sphingosine-1-phosphate (SPP) is abundantly formed and released during the activation of platelets by thrombotic stimuli. Once exported, SPP interacts with the G-protein-coupled receptors (GPCR) of the EDG-1 family. SPP binds to EDG-1 with the dissociation constant of approximately 8 nM and induces signal transduction events such as mitogen-activated protein kinase (MAP kinase) activation, decrease of cAMP levels, remodeling of the actin cytoskeleton, among others. EDG-1 is a prototypical member of a large family of GPCRs that interact with glycero- and sphingolysolipid phosphates, namely, SPP and lysophosphatidic acid (LPA). Three other GPCRs, trivially termed EDG-3, EDG-5, and EDG-8, are also high-affinity receptors for SPP. The four SPP receptor subtypes regulate different intracellular signal transduction pathways. In vascular endothelial cells, cooperative signaling between EDG-1 and EDG-3 subtypes of SPP receptors results in adherens junction assembly, cell survival, morphogenesis into capillary-like networks, and angiogenesis. SPP acts distinctly, albeit cooperatively, with polypeptide angiogenic factors, resulting in the formation of mature neovessels. Thus SPP signaling as an extracellular mediator via the EDG-1 family of GPCRs may be a heretofore unrecognized mechanism for the regulation of angiogenesis and vascular endothelial cell function.
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Papers by timothy hla