Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2) in human osteosarcoma cells.
The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 beta and TNF alpha in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were preincubated with inhibitors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1-30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2-0.4 microM). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released more PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.
Wong
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E
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada.
DeLuca
C
C
Boily
C
C
Charleson
S
S
Cromlish
W
W
Denis
D
D
Kargman
S
S
Kennedy
B P
BP
Ouellet
M
M
Skorey
K
K
O'Neill
G P
GP
Vickers
P J
PJ
Riendeau
D
D
D016718:Arachidonic Acid / Q000494:pharmacology / Q000502:physiology; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D016207:Cytokines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins; D012516:Osteosarcoma / Q000201:enzymology* / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D016207:Cytokines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D016718:Arachidonic Acid; D003907:Dexamethasone; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases