Cyclooxygenase-2 gene expression and regulation in human retinal pigment epithelial cells.
OBJECTIVE
Cyclooxygenases (COX) orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. The retinal pigment epithelium (RPE) cell performs a variety of regulatory functions within the retina. The conditions under which COX-1 and COX-2 are expressed and upregulated in human RPE (HRPE) cells were determined.


METHODS
COX gene expression was examined using RT-PCR analysis of untreated HRPE cultures or cultures exposed to bacterial lipopolysaccharide or various cytokines. COX proteins were detected by immunohistochemistry and Western blot analysis. Prostaglandin (PG) production was analyzed by EIA.


RESULTS
Examination of untreated RPE cells revealed the presence of COX-2 mRNA and the absence of COX-1 mRNA. Moreover, cytokine stimulation more readily enhanced COX-2 gene expression than COX-1 gene expression. IL-1 beta, the most potent inducer of COX-2, also resulted in detection of COX-2 protein by immunocytochemical staining and Western blot analysis. There was a direct relationship between both the appearance and amount of COX-2 mRNA and protein synthesis and the degree of PG synthesis by RPE cells. Furthermore, COX inhibitors significantly decreased PG production. Pretreatment of RPE cells with a NF-kappa B inhibitor, PDTC, resulted in dose-dependent decrease in IL-1 beta-induced COX-2 gene expression and PG production.


CONCLUSIONS
COX-2 was the predominant isoform of cyclooxygenase in untreated HRPE cells. When HRPE cells were treated with proinflammatory cytokines, COX-2 gene expression and synthesis of PGs were enhanced. NF-kappa B mediated the induction of COX-2 gene expression in HRPE cells. These studies indicate that RPE cells may participate in normal and pathologic retinal conditions through the induction of COX-2.
Chin
M S
MS
Virology and Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nagineni
C N
CN
Hooper
L C
LC
Detrick
B
B
Hooks
J J
JJ
D015153:Blotting, Western; D002478:Cells, Cultured; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D011453:Prostaglandins / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012485:Salmonella typhi; D013859:Thiocarbamates / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D011453:Prostaglandins; D012333:RNA, Messenger; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D011392:Proline; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases