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Ohtani, K., Nakamura, M., Saito, S., Nagata, K., Sugamura, K. and Hinuma, Y. (1989) Electroporation: Application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type 1. Nucleic Acid Research, 17, 1589-1604. doi:10.1093/nar/17.4.1589

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• TITLE: Endogenous prostaglandin D2 synthesis inhibits e-selectin generation in human umbilical vein endothelial cells

  AUTHORS: Hideyuki Negoro, Hiroyuki Kobayashi, Yoshio Uehara

  KEYWORDS: Prostaglandin; E-selectin; PGDS; Endothelial Cell

  JOURNAL NAME: Health, Vol.3 No.5, May 19, 2011

  ABSTRACT: We examined the role of prostaglandin D2 (PGD2) in the formation of E-selectin following inter-leukin-1 (IL-1) stimulation in human umbilical vein endothelial cells (HUVEC) transfected with lipocaline-type PGD2 synthase (L-PGDS) genes. HUVEC were isolated from human umbilical vein and incubated with 20 U/mL IL-1 and various concentrations of authentic PGD2. The isolated HUVEC were also transfected with L-PGDS genes by electroporation. The L-PGDS-transfected HUVEC were used to investigate the role of endogenous PGD2 in IL-1-stimulated E-selectin biosynthesis. We also used an anti-PGD2 antibody to examine whether an intracrine mechanism was involved in E-selectin production. PGD2 and E-selectin levels were determined by radio-immunoassay and enzyme- immunoassay, respectively. E-selectin mRNA was assessed by real-time RT-PCR. IL-1-stimulated E-selectin production by HUVEC was dose-dependently inhibited by authentic PGD2 at concentrations greater than 10–6 mol/L. L-PGDS gene-transfected HUVEC produced more PGD2 than HUVEC transfected with the reporter gene alone. IL-1 induced increases in E-selectin production in HUVEC transfected with the reporter genes alone. However, this effect was significantly attenuated in the case of IL-1 stimulation of HUVEC trans-fected with L-PGDS genes, and accompanied by an apparent suppression of E-selectin mRNA expression. Neutralization of extracellular PGD2 by anti-PGD2- specific antibody influenced neither E-selectin mRNA expression nor E-selectin biosynthesis. HUVEC transfected with L-PGDS genes showed increased PGD2 synthesis. This increase was associated with attenuation of both E-selectin generation and E-selectin mRNA expression. The results suggest that endogenous PGD2 decreases E-selectin synthesis and E-selectin mRNA expression, probably through an intracrine mechanism.