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

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The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (؊1250 to ؉77) showed that an AP-2 element at ؊109 was required for activation and an E-box at ؉57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1␤ to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-B elements at ؊1106 and ؊291 were essential for this up-regulation. Binding of two NF-B subunits, p65 and c-Rel, to these two NF-B elements occurred after the interleukin-1␤ treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-B system. Lipocalin-type prostaglandin (PG)1 D synthase (L-PGDS) was first identified in the rat brain as an enzyme that catalyzes the conversion to PGD 2 from PGH 2 , the latter being a common precursor of all prostanoids (1). Later, L-PGDS was found to be identical to ␤-trace (2, 3), which had been identified earlier (4) as the major protein in human cerebrospinal fluid (CSF). PGD 2 is a major prostanoid in the brain, known as the most potent endogenous somnogenic substance thus far identified (5), and is involved in various physiological events such as regulation of sleep and pain responses (5-8). Because human L-PGDS-overexpressing transgenic mice (9) and L-PGDS-gene knock-out mice (10, 11) showed abnormality in the regulation of non-rapid eye movement (NREM) sleep and pain responses, PGD 2 produced by L-PGDS is thought to play important roles in the regulation of NREM sleep and pain sensation in the central nervous system. Alternatively L-PGDS binds small lipophilic molecules such as retinal and retinoic acid (K d ϭ 70 -80 nM) (12) and biliverdin and bilirubin (K d ϭ 33-37 nM) (13) with affinities higher than those of other members of the lipocalin family. Therefore, L-PGDS is a unique bifunctional protein acting as a PGD 2 -producing enzyme and as a lipophilic molecule-binding protein (6 -8).L-PGDS is expressed abundantly in the human heart (14), the male genital organs (15-17), and the central nervous system (18 -20) of various mammals. In the brain, this enzyme is expressed dominantly in the leptomeninges, chroid plexus, and...

Section: Regulation Of Rat L-pgds Gene Promotermentioning

confidence: 76%

“…Protein concentrations were determined according to the method of Bradford (47). EMSA was performed as described previously (48).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Lipocalin-type Prostaglandin D Synthase Gene Expression by Hes-1 through E-box and Interleukin-1β via Two NF-κB Elements in Rat Leptomeningeal Cells

Fujimori¹,

Fujitani²,

Kadoyama³

et al. 2003

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“…PCR amplification was conducted by using ExTaq DNA polymerase (Takara Shuzo, Kyoto, Japan) under the following conditions: initial denaturation at 95°C for 3 min, followed by 28 -35 cycles of 94°C for 20 s, 55°C for 20 s, and 74°C for 30 -90 s. Primers used in this study are described (35) and listed in Table I. The resultant PCR products were analyzed by electrophoresis in an agarose gel (1.5%).…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase C Activates Human Lipocalin-type Prostaglandin D Synthase Gene Expression through De-repression of Notch-HES Signaling and Enhancement of AP-2β Function in Brain-derived TE671 Cells

Fujimori

Kadoyama

Urade

2005

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When the AP-2 element at ؊98 of the promoter region was deleted or mutated, the promoter activity was drastically decreased to ϳ10% of normal. The AP-2 element was bound by AP-2␤ dominantly expressed in TE671 cells, according to the results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay. L-PGDS expression was induced by 12-O-tetradecanoylphorbol-13-acetate in TE671 cells, and this induction was inhibited by a protein kinase C inhibitor.

“…Plasmids, Transfection, and Luciferase Assay-The plasmid for mouse L-PGDS promoter-luciferase containing the promoter region from Ϫ1500 to ϩ76 was constructed by using the pGL4.10(luc2) vector (Promega, Madison, WI) as described previously (21). Site-directed mutagenesis was carried out by the use of a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instruction.…”

Section: Methodsmentioning

confidence: 99%

“…Electrophoretic Mobility Shift Assay and Chromatin Immunoprecipitation Assay-Preparation of nuclear extracts and EMSA were carried out by the method described previously (21). Oligonucleotides used in this experiment were 5Ј-TTTGC-CGGCAGGAGTGGGCAAGGTCTGAGCCAGTTCTGCC-3Ј for LRH-RE and 5Ј-CAGTTCTGCCTCTGGAGCTGGGGA-TGGGGGCAGGCAGA-3Ј for SRE, and modified at their 5Ј-end with Alexa680.…”

Section: Methodsmentioning

confidence: 99%

A Novel Pathway to Enhance Adipocyte Differentiation of 3T3-L1 Cells by Up-regulation of Lipocalin-type Prostaglandin D Synthase Mediated by Liver X Receptor-activated Sterol Regulatory Element-binding Protein-1c

Fujimori¹,

Aritake

Urade

2007

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Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) isexpressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at ؊233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at ؊194 to be cis- Adipocytes play a critical role in lipid homeostasis and energy balance. Their major role is storage of large amounts of lipid metabolites during periods of energy excess and utilization them during nutritional deprivation (1). Adipocytes are also known as endocrine cells that secrete various adipocytokines (2, 3). Disorders of lipid metabolism are associated with diseases such as obesity and diabetes (4). Adipocyte differentiation (adipogenesis) is a complex process involving coordinated changes in hormone sensitivity and gene expression.Mouse 3T3-L1 cells are well used in vitro model for adipogenesis (5). Previous studies demonstrated that lipocalin-type prostaglandin (PG) 3 D synthase (L-PGDS) gene was expressed in adipocytes (6, 7). L-PGDS is the member of the lipocalin gene family to be recognized as an enzyme that catalyzes the isomerization of PGH 2 , a common precursor of various prostanoids, to produce PGD 2 , a potent endogenous somnogen (8). Alternatively L-PGDS binds small lipophilic molecules such as retinal and retinoic acid (9), biliverdin and bilirubin (10), gangliosides (11), and amyloid ␤ peptides (12). L-PGDS knock-out mice did not show SeCl 4 -induced insomnia (13) and accelerated amyloid ␤-deposition (12). Ragolia et al. (14) demonstrated that L-PGDS knock-out mice become glucose-intolerant and insulin-resistant and that their adipocytes were significantly larger than those of wild-type mice. The L-PGDS expression level was shown to be high in the omental adipose tissue (15). A polymorphism in the 3Ј-untranslated region of the human L-PGDS gene appears to be associated with carotid atherosclerosis in Japanese individuals with hypertension (16). The balance between L-PGDS and PGE synthase levels was proposed to be a major determinant for stability/instability of atherosclerotic plaques (17). However, the role of L-PGDS and the regulation mechanism controlling its gene expression in adipocytes remain unclear.In this study, we showed that L-PGDS gene expression was enhanced during differentiation of mouse 3T3-L1 cells into adipocytes and that small interference RNA (siRNA)-mediated suppression of L-PGDS mRNA decreased the lipid accumulation in 3T3-L1 cells. Reporter analysis of the mouse L-PGDS promoter demonstr...