Regular ArticleSerum and Glucocorticoid Regulation of Gene Transcription and Expression of the Prostaglandin H Synthase-1 and Prostaglandin H Synthase-2 Isozymes
Abstract
Mitogenic stimulation has been shown to increase both prostaglandin H (PGH) synthase-1 (PGHS-I) and PGH synthase-2 (PGHS-2) mRNA levels, although the time course and magnitude of induction are different for the two genes. To investigate the mechanism for mRNA induction, we conducted nuclear run-off assays of these two genes in 3T3 cells and correlated mitogen-induced changes in PGHS gene transcription with changes in PGHS mRNA and PGHS isozyme expression. We also examined the mechanism for glucocorticoid inhibition of PGHS mRNA expression and the effects of glucocorticoids on PGHS isozyme expression. Serum stimulation of quiescent 3T3 cells led to a sequential increase in PGHS-2 gene transcription, PGHS-2 mRNA. and PGHS-2 enzyme levels. PGHS-2 gene transcription increased over 25-fold within 30 min of serum addition resulting in an over 70-fold increase in PGHS-2 mRNA by 1 h, and maximal PGHS-2 enzyme expression by 2 h. Increased PGHS-2 isozyme expression thus appears to depend on transcriptional activation of the gene. Transcription of the PGHS-2 gene declined after 30 min, and PGHS-2 mRNA levels declined similarly after 1 h, leading to a return of PGHS-2 levels to near basal levels by 6 h. Glucocorticoids, which previously have been shown to inhibit mitogen-stimulated increases in PGHS-2 levels, were found to inhibit serum-stimulated increases in PGHS-2 gene transcription by 70%, resulting in a 70% reduction in peak serum-stimulated PGHS-2 mRNA levels also. Western blotting with PGHS-2 specific antisera demonstrated that while dexamethasone simply reduced PGHS-2 mRNA levels, it completely suppressed expression of PGHS-2 protein. The coincidental reduction in PGHS-2 transcription, PGHS-2 mRNA, and enzyme levels by dexamethasone, provides further support for the hypothesis that control of transcription is one primary control mechanism for regulating PGHS-2 expression. That complete suppression of PGHS-2 enzyme expression occurs following partial suppression of PGHS-2 mRNA, however, suggests that other mechanisms may also contribute to the glucocorticoid effect. A small, but reproducible, increase in transcription of the PGHS-1 gene occurred 3 h following serum stimulation, coincident with a three- to fourfold increase in PGHS-1 mRNA; PGHS-1 mRNA remained elevated for at least 3 h. Dexamethasone reduced, but did not completely inhibit, the serum-stimulated increases in PGHS-1. However, changes in PGHS-1 mRNA were not accompanied by detectable changes in PGHS-1 protein in the presence or absence of dexamethasone.
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Prostaglandins and bone metabolism
2019, Principles of Bone BiologyProstaglandins (PGs) are autocrine–paracrine fatty acids produced by most cells. They are not stored but synthesized and released as needed and rapidly metabolized. PGs are produced by the metabolism of arachidonic acid, which is released from membranes by phospholipases, by two cyclooxygenase (COX) enzymes. PGE2 is produced abundantly by both osteoblastic and osteoclastic lineage cells, as well as other cells in the bone environment. Much of the PGE2 in bone is produced by COX-2, which can be induced by multiple systemic hormones and local factors important for bone metabolism. PGE2 can stimulate both bone resorption and bone formation and mediate some actions of the COX-2 agonists. PGE2 acts via four receptors to engage an extensive G-protein signaling network that can integrate, amplify, or, as we have shown for parathyroid hormone, block responses to COX-2 agonists at the cellular level.
Targeted exchange of an expression cassette encoding cyclooxygenase-2 at the Ptgs1 locus
2012, Prostaglandins and Other Lipid MediatorsDefining the multi-faceted roles of prostaglandins has been facilitated by studying mice with manipulated expression of the two enzymes encoding cyclooxygenase (COX) via gene targeting, with either knocked down expression of COX-1 or COX-2, a knocked-in COX-2 active site mutation and exchange of COX isoforms by insertion of a cassette encoding COX-1 into the COX-2 (Ptgs2) gene to create COX-1 > COX-2 mice. Here, we sought to extend these studies by creating a new induced mutant strain with manipulated COX expression. We carried out gene targeting at the Ptgs1 locus to knock-in an expression cassette encoding COX-2 under Ptgs1 regulatory elements in a manner analogous used in COX-1 > COX-2 targeting. While successful gene targeting at the Ptgs1 locus was achieved, the strategy did not yield a “basal” increase of COX-2 under Ptgs1 gene regulatory control in various cells and tissues from COX-2 > COX-1 mice but rather resulted in a Ptgs1 null allele. Possible explanations as to why this strategy was unsuccessful include non-functionality of the hybrid signal peptide and aberrant transcript processing. Since a similar strategy had previously worked (i.e. COX-1 cDNA knocked-in to the Ptgs2 locus; COX-1 > COX-2 mice) interpretations of our findings on murine COX biology and gene targeting are discussed.
Dynamics of different arachidonic acid orientations bound to prostaglandin endoperoxide synthases
2011, European Journal of Medicinal ChemistryProstaglandin endoperoxide synthases (PGHSs) catalyze the conversion of arachidonic acid (AA) into prostaglandin endoperoxide H2. This reaction requires a specific orientation of AA within the active site, but an alternative crystallographic binding orientation for AA also exists. Since the origin of this alternative complex, and its potential relevance, have been neglected so far, we have characterized the dynamics of both orientations of AA, bound to PGHS-1 and -2, in order to obtain new insights for designing PGHSs inhibitors. Our results indicate that AA in the alternative orientation seems to be less stable, moving toward Arg120. Such potentially minor orientation of AA can be related to crystallographic complexes of anti-inflammatory agents, pointing to an alternate SAR on PGHSs inhibitors.
To use an in vitro model of the ovine placenta to determine effects of n-6 polyunsaturated fatty acid (PUFA) supplementation on prostaglandin (PG) production. PGs are key regulators of fetal maturation and parturition.
Fetal allantochorion tissue (FC) was collected in late pregnancy (day 135). FC cells were isolated and cultured with 0–100 μM of linoleic acid (LA), γ-linolenic acid (GLA) or arachidonic acid (AA) in serum free medium and challenged with control medium, lipopolysaccharide (LPS, 0.1 μg/ml), dexamethasone (DEX, 5 μM) or a combination of LPS (0.1 μg/ml) with DEX (5 μM). Spent medium was harvested at 2 h and 24 h post challenge for measuring PGs.
To assess the effects of treatment on placental 1- and 2-series PGE production.
LA supplementation inhibited both PGE1 and PGE2 production. GLA predominantly stimulated PGE1 generation, although it also increased PGE2 production. AA supplementation predominantly increased PGE2 production, but also stimulated PGE1. DEX treatment with or without LPS inhibited PG production. Supplementation with n-6 PUFAs attenuated or neutralised the stimulatory effect of LPS challenge on FC cells for both PGE1 and PGE2 production.
These data show that supplementation with n-6 PUFAs alters placental PG production, but their precise effects depend on their position in the biosynthetic pathway for PG synthesis. This study supports the possibility that GLA containing oils, widely promoted as dietary supplements, might reduce the risk of pre-term labour by inhibiting the responsiveness of PGE2 production to LPS challenge in the placenta.
Two distinct pathways for cyclooxygenase-2 protein degradation
2008, Journal of Biological ChemistryCyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.
Prostaglandins and Bone Metabolism
2008, Principles of Bone Biology: Volume 1-2, Third EditionProstaglandins PGs) and other eicosanoids have important physiologic and pathologic roles in skeletal metabolism. This chapter summarizes the current knowledge on the regulation of PG production in bone and the effects of PGs and other eicosanoids on bone resorption and formation. PGs are not stored but are synthesized and released as needed and rapidly metabolized in their passage through the lung. PG production is regulated by many systemic hormones and other local factors involved in bone metabolism, and PGs may function to integrate or amplify responses to these agents at the cellular level. In vitro, PGE2 generally stimulates the differentiation of both osteoblasts and osteoclasts, but inhibitory effects on bone formation and resorption are also seen. In vivo, the predominant effects of exogenous PGE2 are to stimulate both bone formation and bone resorption, but the relative magnitude of theses can vary so that in some models there is net bone loss whereas in others there is a gain in bone mass. Many studies have now implicated COX-2 and PGE2 in the promotion of tumorigenesis, suggesting that PGE2 functions as a general regulator of cell growth and apoptosis. Studies of the role of COX-2 and PGE2 in cancer have highlighted new and complex signaling pathways for the EP2 and EP4 receptors that may also be important for PGE2 effects in bone cells.