Characterization of the Promoter of Human Prostaglandin H Synthase-1 Gene

Edited by Paul E. FraserBurgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-D-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-D-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.The first committed reaction in the metabolism of arachidonic acid to prostaglandins and thromboxanes is catalyzed by two related heme-containing bis-oxygenases, cyclooxygenase (COX) 1 and 2. These enzymes share 90% similarity in amino acid sequence and exhibit nearly identical enzyme kinetics (1, 2). Both catalyze two separate reactions, the first metabolizing arachidonic acid to PGG 2 3 (cyclooxygenase reaction), an intermediate that is subsequently reduced in the second reaction to the product, PGH 2 (peroxidase reaction). PGH 2 is the substrate for various synthases that generate individual biologically active prostaglandins and thromboxanes, often in a cell typespecific manner (3). Although both metabolize arachidonic acid to PGH 2 , the transcriptional regulation of each isoform differs. The PTGS1 gene encoding COX-1 lacks a TATA box motif in its 5Ј promoter region and is generally constitutively active in cells (4, 5). In contrast, the promoter regulatory region of the PTGS2 gene encoding COX-2 is not typically active but can be strongly and rapidly induced under specific...

mentioning

confidence: 99%

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1)

Hewett

Shi

Gong

et al. 2016

“…A constitutive and inducible form of COX has been identified, and x-ray crystallographic analyses suggest strongly that they are monotopic, endoplasmic reticulum-associated homodimeric enzymes that possess heme-dependent peroxidase and cyclooxygenase activity (7). The constitutive COX-1 gene has been ascribed a homeostatic function and indeed has a GC-rich housekeeping promoter (8). In contrast, the COX-2 gene (mRNAs 4.6 and 2.8 kb) is rapidly induced by tumor promoters, growth factors, cytokines, and mitogens in many cell model systems (9 -11).…”

mentioning

confidence: 99%

Prostaglandin E2 Regulates the Level and Stability of Cyclooxygenase-2 mRNA through Activation of p38 Mitogen-activated Protein Kinase in Interleukin-1β-treated Human Synovial Fibroblasts

Faour

et al. 2001

187

146

The p38 MAPK mediates transcriptional and posttranscriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E 2 (PGE 2 )-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1␤-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE 2 release following a recombinant human (rh) IL-1␤ signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE 2 completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1␤-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1␤ stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1␤ for 3-4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE 2 , unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE 2 monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE 2 . Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3-untranslated region reporter constructs revealed that IL-1␤ increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE 2 /p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE 2 release by rhIL-1␤ is primarily the result of PGE 2 -dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase.

“…There are two isoforms of COX, cox-1 and cox-2, produced from differently regulated genes. cox-1 is constitutively expressed in most cell types and is thought to be involved in the maintenance of physiological functions (33,34). In contrast, cox-2 is undetectable in normal tissues or resting immune cells but is rapidly induced by cytokines, growth factors, tumor promoters, and bacterial products such as LPS and CpG DNA (26,27,(35)(36)(37)(38).…”

mentioning

confidence: 99%

Myeloid Differentiation Factor 88-dependent Post-transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA

Yeo

Yoon

2003

The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2-3-UTR-luciferase) was generated by inserting sequences within the 3-untranslated region (UTR) of cox-2 to the 3 end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2-3-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2-3-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2-3-UTR-luciferase activity induced by CpG DNA.