Interleukin 1beta induces functional prostaglandin E synthase in cultured human umbilical vein endothelial cells.
Prostaglandin endoperoxide H2 (PGH2) is generated from arachidonic acid by either constitutive (COX-1) or inducible (COX-2) cyclooxygenases. In arterial wall PGH2 is converted by PGI2 synthase (PGI-S) to prostacyclin (PGI2), and in platelets by thromboxane synthase (TX-S) to thromboxane (TXA2). Other prostanoids as PGD2, PGF2alpha, or PGE2 were believed to arise non-enzymatically from PGH2. Only recently, human prostaglandin E synthase (PGE-S) has been identified and cloned as a membrane bound, microsomal, glutathione-dependent inducible enzyme. Here we demonstrated that interleukin 1beta (IL-1beta) is an inducer of COX-2 and PGE-S in human umbilical vein endothelial cells (HUVEC). Functional expression of PGE-S was measured at the level of specific mRNA by semi-quantitative RT-PCR, PGE-S protein was detected by Western blot in HUVEC, while PGE2 was measured by immunoassay in the supernatant. Actinomycin D, a classical transcription inhibitor, was used to prove that indeed IL-1beta induced the functional PGE-S enzyme. PGE2 generation in HUVEC was inhibited by indomethacin, acetaminophen and dexamethasone. In conclusion, we found that in cultured endothelial cells IL-1beta induced as evidenced by the appearance of its transcript and its functional enzyme. The induction of endothelial PGE-S and COX-2 appeared to be and their transcripts were induced as fast as one might expect from immediate early genes. It means that IL-1beta-triggered-PGE2 biosynthesis in endothelial cells is probably regulated by induction of both COX-2 and PGE-S. This is way we hypothesise the existence of at least two distinct pools of COX-2: the first selectively coupled to PGE-S and the second one that is coupled to PGI-S yielding the main endothelial product--PGI2.
Uracz
W
W
Molecular Pharmacology Laboratory, Chair of Pharmacology, Medical College of Jagiellonian University, Grzegórzecka 16 31-531 Krakow, Poland. miuracz@kinga.cyf-kr.edu.pl
Uracz
D
D
Olszanecki
R
R
Gryglewski
R J
RJ
D015153:Blotting, Western; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D009994:Osmolar Concentration; D000071876:Prostaglandin-E Synthases; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000201:enzymology*
D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases