Abstract
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed 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-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.
Highlights
Tion them during nutritional deprivation [1]
PCR was conducted under the following conditions: Enhancement of lipocalin-type prostaglandin (PG)3 D synthase (L-PGDS) Gene Expression and Role of L-PGDS during Adipocyte Differentiation of Mouse 3T3-L1 Cells—To investigate the role of L-PGDS in adipocyte differentiation, we first examined the expression profile of the L-PGDS gene during the differentiation of 3T3-L1 cells into adipocytes. 3T3-L1 cells were treated with a differentiation medium containing IDX for 2 days and further cultured in the medium containing insulin
We examined whether LXRE and/or these two sterol regulatory elements (SREs) the nuclear extracts were omitted from this incubation mixture were involved in the activation of L-PGDS gene expression in (Fig. 3C, lane 1) and was decreased by the addition of an excess the 3T3-L1 cells by performing luciferase reporter assays amount (5- or 10-fold) of unlabeled oligonucleotide containing with the mutated inactive LXRE- or SREs-containing contreatment with T0901317 in adipocytic cells, indicating that induction of SREBP-1c precedes that of L-PGDS (Fig. 5B)
Summary
Cell Culture—Mouse 3T3-L1 cells were purchased from Health Science Research Resources Bank (Osaka, Japan) and cultured in Dulbecco’s modified Eagle’s medium supplemented with charcoal-treated fetal calf serum and antibiotics. 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]. The relative promoter activities were reported as the mean Ϯ S.D. Electrophoretic Mobility Shift Assay and Chromatin Immunoprecipitation Assay—Preparation of nuclear extracts and EMSA were carried out by the method described previously [21]. 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-TTTGCCGGCAGGAGTGGGCAAGGTCTGAGCCAGTTCTGCC-3Ј for LRH-RE and 5Ј-CAGTTCTGCCTCTGGAGCTGGGGATGGGGGCAGGCAGA-3Ј for SRE, and modified at their. Various deletion constructs of human L-PGDS promoter region were used to preadipocytic or adipocytic (3 days after start of differentiation) 3T3-L1 cells. The culture medium was collected and utilized for measurement of PGD2 by EIA
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