Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells.
OBJECTIVE
The peroxisome proliferator-activated receptor-gamma (PPARgamma), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the control of cyclooxygenase (COX) 2 expression in some tissue, although the exact mechanism(s) of this activity has not been elucidated. In this study we explored the possible mechanism(s) of control of COX-2 gene expression through PPARgamma signaling in human cervical cancer.


METHODS
Using primary human cervical tissues and the CaSki human cervical cancer cell line, we assayed for PPARgamma and COX-2 mRNA expression by reverse transcription-PCR. Nuclear protein binding activities to three response elements located in the COX-2 promoter [nuclear factor kappaB (NFkappaB), cyclic AMP response element, and activator protein (AP)-2] were measured by gel mobility shift assays. We used transient transfection assays with COX-2 promoter reporter gene constructs to determine the regulatory sites in this promoter, which mediates PPARgamma regulation of COX-2 activity.


RESULTS
We showed, for the first time, that primary human cervical cancer tissues express PPARgamma. Using CaSki cells, we demonstrated that COX-2 and PPARgamma mRNA levels were inversely regulated by PPARgamma ligands in that these compounds up-regulated PPARgamma but down-regulated COX-2. In contrast, epidermal growth factor (EGF), a potent activator of COX-2, decreased PPARgamma mRNA levels. This down-regulation of PPARgamma mRNA by EGF was blocked in the presence of NS-398, a selective COX-2 inhibitor. PPARgamma ligands suppressed the binding activities of AP-1 (binding to CRE) and NFkappaB but not AP-2. Transient transfection results indicated that EGF stimulated whereas PPARgamma ligands inhibited COX-2 promoter (-327/+59) activity. This effect by PPARgamma ligands on the COX-2 promoter was blocked when the CRE, but not the NFkappaB, binding site was mutagenized.


CONCLUSIONS
Cervical cancer cells express readily detectable levels of PPARgamma. There is reciprocal negative regulation between COX-2 and PPARgamma signaling in human cervical cancer cells. The ability of PPARgamma ligands to inhibit COX-2 appears to be mediated predominantly through inhibition of AP-1 protein binding to the CRE site in the COX-2 promoter.
Han
Shouwei
S
Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Inoue
Hiroyasu
H
Flowers
Lisa C
LC
Sidell
Neil
N
D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009578:Nitrobenzenes / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D013449:Sulfonamides / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D009578:Nitrobenzenes; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D013449:Sulfonamides; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D004815:Epidermal Growth Factor; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases