Multiple factors regulating the expression of human thromboxane synthase gene

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5-flanking region and the untranslated exon 1 (؊1044 to ؉290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5-flanking region decreased the promoter activity by ϳ30% compared with that of the entire region from ؊1044 to ؉290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (؉1 to ؉290) showed ϳ60% of the promoter activity of the entire region from ؊1044 to ؉290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2␣. These findings indicate that the Oct-1 element in the 5-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells. Prostaglandin (PG)1 D 2 is actively produced in a variety of tissues by prostaglandin D synthase (EC 5.3.99.2) from PGH 2 , which is a common precursor for various PGs and thromboxane of the two series synthesized from arachidonic acid by cyclooxygenase. PGD 2 inhibits platelet aggregation, induces vasodilatation and bronchoconstriction (1, 2), and acts as an allergic mediator (3). Furthermore, PGD 2 is converted to 9␣,11␤-PGF 2 or dehydrated to yield the J series of PGs such as ⌬ 12

Section: Discussionsupporting

confidence: 76%

Transcriptional Activation of the Human Hematopoietic Prostaglandin D Synthase Gene in Megakaryoblastic Cells

Fujimori¹,

Kanaoka²,

Sakaguchi³

et al. 2000

“…A point mutation at the NF-E2 binding site (TGATTCA to GGATCCA) in pMCϪ280/ϩ137, called pMCϪ280/ϩ137-mNF-E2, was generated by PCR. NF-E2 cDNA was obtained by reverse transcription-PCR from total MEG-01 cellular RNA (17) and first subcloned into TA vector and then into the pcDNA3.1 eukaryotic expression vector (Invitrogen) at the BamHI/HindIII site. All constructs generated by PCR were confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Role of NF-E2 Site in TXAS Gene Transcription in a Native Chromatin Context-In transient transfection experiments, the Ϫ300/Ϫ40 TXAS upstream promoter sequence, particularly at the NF-E2 site between Ϫ83 and Ϫ77, is crucial for transcriptional activity in several types of cells (15,17,18,20). To test whether NF-E2 activates the TXAS gene in a natural chromatin context, we generated a mutation at the NF-E2 site in the pMCϪ280/ϩ137 minichromosome (pMCϪ280/ϩ137-mNF-E2) and stably transfected it into HEL cells.…”

Section: Mnase-coupled Southern Blot Analyses Of Txas Promoter/ Reportermentioning

confidence: 99%

“…An upstream sequence between nucleotides Ϫ90 and Ϫ56 relative to the TXAS transcriptional start site was shown to be important for TXAS promoter activity in a megakaryotic cell line. Further detailed analysis identified transcription factor NF-E2 binding at the nucleotide region Ϫ83/Ϫ77 as being responsible for TXAS transactivation (17,18). The NF-E2 site also plays an important role in activation of the mouse and rat TXAS promoters (19,20).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Transcriptional Control of the Human Thromboxane Synthase Genein Vivo and in Vitro

Yaekashiwa

Wang

2002

Thromboxane A 2 , a potent mediator of vasoconstriction and platelet aggregation, is synthesized from prostaglandin H 2 by thromboxane synthase (TXAS). We report here on promoter analyses of human TXAS using in vitro transcription and in vivo transfection methods. The 39-bp core promoter, containing both TATA and initiator elements, accurately initiates transcription in an orientation-dependent manner in a cell-free transcription system. Mutation of either TATA or initiator abolished transcriptional activity, but the upstream sequence had no effect on TXAS promoter activities in vitro, suggesting that the core promoter is sufficient for transcriptional activity from a naked DNA template. In contrast, mutation of both elements drastically decreased the promoter activity in vivo. Furthermore, TXAS proximal promoter containing the nucleotides ؊90 to ؊56 relative to the transcriptional start site was necessary for promoter transactivation in vivo. Transcriptional activities from 5-deletion mutants indicated that the effects of the nucleotides ؊90/؊56 were more pronounced in stably transfected cells (a 150-fold difference) than in the transiently transfected cells (an 8-fold difference), reflecting the effects of TXAS transcriptional activation from replicating and nonreplicating DNA templates. Partial micrococcal nuclease digestion indicated that the sequence ؊90/؊56, where the NF-E2 site is located, is associated with alterations of nucleosomal structure at the regions of promoter and reporter gene but not the region further downstream. Mutagenesis and forced expression studies demonstrated a critical role of p45 NF-E2 in controlling TXAS expression under native chromatin conditions. Band shifting and chromatin immunoprecipitation assays indicated that p45 NF-E2 was bound to the TXAS promoter in vitro and in vivo. Indirect end labeling and ligation-mediated PCR analyses further demonstrated that the occupation of TXAS promoter NF-E2 site was associated with disruption of nucleosomal structure. Collectively, these results indicate that binding of NF-E2 is critical both for alteration of the nucleosomal structure and for activation of the TXAS promoter, whereas the TATA and initiator elements are essential for transcription.

“…We also have observed that TXS mRNA is induced in an inflammatory model of hydronephrosis characterized by infiltration of macrophages in the renal interstitium (8). Although the transcriptional regulatory regions of the human (9) and mouse TXS (10) genes have been cloned, analysis of their gene transcription has not yet been extensively performed, and any role of the peroxisome proliferatoractivated receptor (PPAR) in TXS gene transcription has never been examined.…”

mentioning

confidence: 99%

Suppression of Rat Thromboxane Synthase Gene Transcription by Peroxisome Proliferator-activated Receptor γ in Macrophages via an Interaction with NRF2

Ikeda¹,

Sugawara²,

Taniyama³

et al. 2000

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor ␥ (PPAR␥) in macrophages. The transcription activity of a cloned 5-flanking region (1.6 kilobases) of the rat TXS gene (5FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5FL-TXS were inhibited by PPAR␥ ligands, 15-deoxy-⌬ 12,14 -prostaglandin J 2 (PGJ 2 ), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPAR␥ also significantly suppressed transcription, and further addition of PGJ 2 or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPAR␥ effect is located in a region containing the nuclear factor E2 (NF-E2)/ AP-1 site (؊98/؊88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ 2 , TRO, or in vitro translated PPAR␥. The transcription suppression by PPAR␥ was confirmed using this truncated NRF2-binding element (؊98/؊88) by the reporter gene assay. Finally, a direct interaction between PPAR␥ and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (؊98/؊88) is the major promoter of 5FL-TXS which can be suppressed by activated PPAR␥ via a protein-protein interaction with NRF2 in macrophages.