Repression of Cyclooxygenase-2 and Prostaglandin E2Release by Dexamethasone Occurs by Transcriptional and Post-transcriptional Mechanisms Involving Loss of Polyadenylated mRNA

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1β (IL-1β) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25–40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1β-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1β treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3′-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3′-UTR for specificity. The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1β (IL-1β) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25–40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1β-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1β treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3′-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3′-UTR for specificity. prostaglandin prostaglandin E2 cyclooxygenase glucocorticoid receptor GR element interleukin untranslated region 4-morpholinepropanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase base pair(s) kilobase pair(s). Prostaglandins (PGs)1form a potent group of autocrine and paracrine lipid mediators (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). These compounds are implicated in many normal cellular processes as well as pathophysiological processes such as inflammation, edema, bronchoconstriction, platelet aggregation, fever, and hyperalgesia (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar, 3Portanova J.P. Zhang Y. Anderson G.D. Hauser S.D. Masferrer J.L. Seibert K. Gregory S.A. Isakson P.C. J. Exp. Med. 1996; 184: 883-891Crossref PubMed Scopus (379) Google Scholar).PG synthesis involves phospholipase catalyzed release of arachidonic acid from membrane phospholipids and its conversion by the two cyclooxygenase (COX) enzymes to PGH2. Subsequently, cell-specific isomerases and reductases result in production of biologically relevant PGs. The two COX isoforms are encoded by distinct genes of which COX-1 is a constitutively expressed housekeeping gene and COX-2 shows low basal expression that is rapidly induced by inflammatory and mitogenic stimuli (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). Pharmacologically, this pathway is important, since COX is the target for nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). The anti-inflammatory benefits of nonsteroidal anti-inflammatory drugs are thought to derive from inhibition of COX-2, while many of the undesirable side effects are due to COX-1 inhibition (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). This has now been confirmed by the use of selective COX-2 inhibitors (4Seibert K. Zhang Y. Leahy K. Hauser S. Masferrer J. Perkins W. Lee L. Isakson P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12013-12017Crossref PubMed Scopus (1455) Google Scholar, 5Masferrer J.L. Zweifel B.S. Manning P.T. Hauser S.D. Leahy K.M. Smith W.G. Isakson P.C. Seibert K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3228-3232Crossref PubMed Scopus (1284) Google Scholar).Despite the clinical usefulness of nonsteroidal anti-inflammatory drugs, the most effective drugs in the treatment of chronic inflammatory diseases, such as asthma, are synthetic glucocorticoids, which down-regulate inflammatory genes both in vitro and in vivo (6Barnes P.J. N. Engl. J. Med. 1995; 332: 868-875Crossref PubMed Scopus (635) Google Scholar). Synthetic glucocorticoids, such as dexamethasone, act by mimicking the natural steroid, cortisol, in binding the glucocorticoid receptor (GR). GR then disassociates from its cytoplasmic protein complex and translocates to the nucleus, where it can activate transcription (transactivation) of anti-inflammatory genes via cis-acting promoter elements known as glucocorticoid response elements (GREs) (7Beato M. Truss M. Chavez S. Ann. N. Y. Acad. Sci. 1996; 784: 93-123Crossref PubMed Scopus (114) Google Scholar). However, this fails to explain how glucocorticoids cause down-regulation of inflammatory genes such as COX-2. Other mechanisms for this effect may include transcriptional repression (transrepression) via negative GREs (8Drouin J. Sun Y.L. Chamberland M. Gauthier Y. De Lean A. Nemer M. Schmidt T.J. EMBO J. 1993; 12: 145-156Crossref PubMed Scopus (269) Google Scholar), repression of AP-1-dependent transactivation via direct AP-1/GR interactions (9Yang Yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.J. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar, 10Konig H. Ponta H. Rahmsdorf H.J. Herrlich P. EMBO J. 1992; 11: 2241-2246Crossref PubMed Scopus (233) Google Scholar), and repression of NF-κB-mediated transcription by up-regulation of the NF-κB inhibitor IκBα and/or direct NF-κB/GR interactions (11Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin Jr., A.S. Science. 1995; 270: 283-286Crossref PubMed Scopus (1586) Google Scholar, 12Auphan N. DiDonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Crossref PubMed Scopus (2143) Google Scholar, 13Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 14Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar).Epithelial cells are known to play an active role in inflammation by producing multiple mediators and therefore represent an important site for glucocorticoid action (15Devalia J.L. Davies R.J. Respir. Med. 1993; 87: 405-408Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Airway epithelial cells respond to proinflammatory cytokines, such as IL-1β, by induction of COX-2 and PGE2 release (16Mitchell J.A. Belvisi M.G. Akarasereenont P. Robbins R.A. Kwon O.J. Croxtall J. Barnes P.J. Vane J.R. Br. J. Pharmacol. 1994; 113: 1008-1014Crossref PubMed Scopus (287) Google Scholar). This response is also observed in human A549 cells, and in both cases the response is suppressed by dexamethasone (16Mitchell J.A. Belvisi M.G. Akarasereenont P. Robbins R.A. Kwon O.J. Croxtall J. Barnes P.J. Vane J.R. Br. J. Pharmacol. 1994; 113: 1008-1014Crossref PubMed Scopus (287) Google Scholar, 17Newton R. Kuitert L.M. Slater D.M. Adcock I.M. Barnes P.J. Life Sci. 1997; 60: 67-78Crossref PubMed Scopus (135) Google Scholar). We have therefore used these cells to examine the mechanisms of dexamethasone repression of IL-1β-dependent induction of COX-2 and relate these effects to functional changes in released PGE2.DISCUSSIONRepression of gene expression by activated GR accounts for two major functions of glucocorticoids, namely classical negative feedback of the hypothalamo-pituitary-adrenal axis and immunosuppression. Clinically, the immunosuppressive nature of glucocorticoids is important in the treatment of inflammatory disease by down-regulating expression of many proinflammatory genes (6Barnes P.J. N. Engl. J. Med. 1995; 332: 868-875Crossref PubMed Scopus (635) Google Scholar). One target of glucocorticoid repression is COX-2, whose expression is responsible for inflammatory prostaglandin synthesis and plays a major role in inflammation (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar, 3Portanova J.P. Zhang Y. Anderson G.D. Hauser S.D. Masferrer J.L. Seibert K. Gregory S.A. Isakson P.C. J. Exp. Med. 1996; 184: 883-891Crossref PubMed Scopus (379) Google Scholar, 4Seibert K. Zhang Y. Leahy K. Hauser S. Masferrer J. Perkins W. Lee L. Isakson P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12013-12017Crossref PubMed Scopus (1455) Google Scholar, 5Masferrer J.L. Zweifel B.S. Manning P.T. Hauser S.D. Leahy K.M. Smith W.G. Isakson P.C. Seibert K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3228-3232Crossref PubMed Scopus (1284) Google Scholar). Cytokine-induced COX-2 and PG synthesis is strongly repressed by dexamethasone in a number of experimental systems; however, the molecular basis for this effect is presently unclear (16Mitchell J.A. Belvisi M.G. Akarasereenont P. Robbins R.A. Kwon O.J. Croxtall J. Barnes P.J. Vane J.R. Br. J. Pharmacol. 1994; 113: 1008-1014Crossref PubMed Scopus (287) Google Scholar, 17Newton R. Kuitert L.M. Slater D.M. Adcock I.M. Barnes P.J. Life Sci. 1997; 60: 67-78Crossref PubMed Scopus (135) Google Scholar, 34Crofford L.J. Wilder R.L. Ristimaki A.P. Sano H. Remmers E.F. Epps H.R. Hla T. J. Clin. Invest. 1994; 93: 1095-1101Crossref PubMed Scopus (662) Google Scholar, 35O'Banion M.K. Winn V.D. Young D.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4888-4892Crossref PubMed Scopus (800) Google Scholar, 36Ristimaki A. Narko K. Hla T. Biochem. J. 1996; 318: 325-331Crossref PubMed Scopus (209) Google Scholar).Two mechanisms of glucocorticoid-mediated repression that have recently received much attention are those involving suppression of AP-1 and NF-κB-dependent transcription (9Yang Yen H.F. Chambard J.C. Sun Y.L. Smeal T. Schmidt T.J. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1314) Google Scholar, 10Konig H. Ponta H. Rahmsdorf H.J. Herrlich P. EMBO J. 1992; 11: 2241-2246Crossref PubMed Scopus (233) Google Scholar, 11Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin Jr., A.S. Science. 1995; 270: 283-286Crossref PubMed Scopus (1586) Google Scholar, 12Auphan N. DiDonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Crossref PubMed Scopus (2143) Google Scholar, 13Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 14Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar). Promoter analyses in nonhuman cells have previously identified the NF-κB, CCAAT/enhancer-binding protein, and cAMP response element sites as important in induction of COX-2 in response to phorbol esters, lipopolysaccharide, and tumor necrosis factor-α (37Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 38Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). Since AP-1 components, for example c-Jun, can activate transcription via the COX-2 cAMP response element (39Xie W. Herschman H.R. J. Biol. Chem. 1995; 270: 27622-27628Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), this site may provide sensitivity to dexamethasone. Furthermore, involvement of NF-κB in the transcriptional control of COX-2 is also suggested in human cells, including A549 cells where overexpression of NF-κB subunits activates the COX-2 promoter (22Newton R. Kuitert L.M. Bergmann M. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1997; 237: 28-32Crossref PubMed Scopus (358) Google Scholar, 40Roshak A.K. Jackson J.R. McGough K. Chabot-Fletcher M. Mochan E. Marshall L.A. J. Biol. Chem. 1996; 271: 31496-31501Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 41Schmedtje Jr., J.F. Ji Y.-S. Liu W.-L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Analysis of COX-2 transcription rate indicated dexamethasone-dependent repression of between 30 and 40%, suggesting a partial role for GR-mediated transrepression in this system. Interestingly, these data are consistent with the fact that NF-κB-dependent transcription from a κB-dependent reporter stably transfected in A549 cells also showed 30–40% repression by dexamethasone (42Newton R. Hart L.A. Stevens D.A. Bergmann M. Donnelly L.E. Adcock I.M. Barnes P.J. Eur. J. Biochem. 1998; 254: 81-89Crossref PubMed Scopus (98) Google Scholar). Since GR-dependent transrepression of AP-1 and NF-κB involves direct interactions between pre-existing proteins, there is no requirement for protein synthesis, so repression would be expected to occur even in the presence of cycloheximide (13Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (916) Google Scholar, 43Jonat C. Rahmsdorf H.J. Park K.K. Cato A.C. Gebel S. Ponta H. Herrlich P. Cell. 1990; 62: 1189-1204Abstract Full Text PDF PubMed Scopus (1363) Google Scholar). Thus, the 35–40% repression of COX-2 mRNA observed in the presence of cycloheximide further substantiates these transcriptional effects.The fact that COX-2 transcription rate was only reduced by a maximum of 40%, while repression of COX-2 protein and mRNA (at 6 h) was complete indicates that other, post-transcriptional, mechanisms of repression must also exist. This was confirmed by the addition of dexamethasone to cells that had been preinduced with IL-1β. In this case, dexamethasone resulted in total loss of preformed COX-2 mRNA within 6 h, presumably while COX-2 transcription was still occurring at up to 60% of the IL-1β-induced level. This contrasted with total inhibition of transcription by actinomycin D, which had little effect on preformed COX-2 mRNA levels. Furthermore, the dexamethasone-dependent repression took place after a lag of almost 2 h, suggesting the need for new transcription and translation. This was confirmed by the ability of actinomycin D and cycloheximide to block the dexamethasone effect.In addition, the steroid antagonist, RU38486, also inhibited the dexamethasone-dependent repression, yet showed little repressive effect by itself. This is explained by RU38486 being an effective antagonist and only a weak agonist of GR-dependent transactivation (26Vayssiere B.M. Dupont S. Choquart A. Petit F. Garcia T. Marchandeau C. Gronemeyer H. Resche-Rigon M. Mol. Endocrinol. 1997; 11: 1245-1255Crossref PubMed Scopus (298) Google Scholar, 28Liden J. Delaunay F. Rafter I. Gustafsson J.-A. Okret S. J. Biol. Chem. 1997; 272: 21467-21472Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 44Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar). Yet in AP-1 transrepression assays, RU38486 showed much of the activity of dexamethasone (26Vayssiere B.M. Dupont S. Choquart A. Petit F. Garcia T. Marchandeau C. Gronemeyer H. Resche-Rigon M. Mol. Endocrinol. 1997; 11: 1245-1255Crossref PubMed Scopus (298) Google Scholar, 44Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar), while in NF-κB transrepression assays significant transrepression by RU38486 was also observed (14Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 28Liden J. Delaunay F. Rafter I. Gustafsson J.-A. Okret S. J. Biol. Chem. 1997; 272: 21467-21472Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Taken together, this information, along with the actinomycin D, cycloheximide, and RU38486 data reported here, indicates a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 mRNA. This mechanism (or mechanisms) requires GR-mediated transactivation, which leads to de novo transcription and translation of genes that either directly or indirectly suppress COX-2 mRNA levels.To evaluate the functional significance of these effects, we examined the ability of dexamethasone and actinomycin D to inhibit COX-2 protein, COX activity, and PGE2 release when added with or after IL-1β. To our surprise, actinomycin D was only effective in suppressing these indicators of COX induction when added with IL-1β or within the first hour of IL-1β treatment, whereas dexamethasone effectively suppressed induction of COX-2 when added up to 10–14 h after the IL-1β. Since total suppression of transcription is not observed with dexamethasone, these data point to an important functional role for post-transcriptional, possibly including translational and post-translational, mechanisms in the dexamethasone inhibition of COX-2. Again, the dexamethasone effect was blocked by actinomycin D, indicating the requirement for de novo gene synthesis.The role of mRNA stability as a major determinant in the control of gene expression is now well established (29Jacobson A. Peltz S.W. Annu. Rev. Biochem. 1996; 65: 693-739Crossref PubMed Scopus (576) Google Scholar). However, the actual mechanisms for these processes are only now being elucidated. One prevailing theme is that shortening and/or loss of the poly(A) tail precedes degradation of the main body of many mRNA species. Consequently, we examined the effect of dexamethasone on COX-2 poly(A) length variation. Consistent with the above hypothesis, we observed a shortening of the average length of COX-2 poly(A) tails following dexamethasone treatment via what appeared to be a processive mechanism. This involves rapid shortening of the poly(A) tail and accumulation of deadenylated intermediates prior to degradation and loss of the main body of the RNA (31Chen C.Y. Xu N. Shyu A.B. Mol. Cell. Biol. 1995; 15: 5777-5788Crossref PubMed Scopus (249) Google Scholar).Many cytokine genes and other acute phase genes are regulated by post-transcriptional mRNA stability as well as transcriptionally (45Yang L. Yang Y.C. J. Biol. Chem. 1994; 269: 32732-32739Abstract Full Text PDF PubMed Google Scholar, 46Razanajaona D. Maroc C. Lopez M. Mannoni P. Gabert J. Cell Growth Differ. 1992; 3: 299-305PubMed Google Scholar). Indeed, COX-2 mRNA is stabilized by IL-1, and some evidence indicates a possible role for the 3′-UTR in this effect (20Newton R. Seybold S. Liu S.F. Barnes P.J. Biochem. Biophys. Res. Commun. 1997; 234: 85-89Crossref PubMed Scopus (51) Google Scholar,24Ristimaki A. Garfinkel S. Wessendorf J. Maciag T. Hla T. J. Biol. Chem. 1994; 269: 11769-11775Abstract Full Text PDF PubMed Google Scholar). Furthermore, the human COX-2 3′-UTR contains 22 repeats of the sequence AUUUA, which is common in many cytokine genes and was shown to confer instability to granulocyte-macrophage colony-stimulating factor mRNA (33Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar, 47Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar). The addition of the COX-2 3′-UTR to a heterologous reporter failed to confer dexamethasone sensitivity, indicating that the region is not in itself sufficient to confer dexamethasone sensitivity. However, the actual mechanism of degradation appears to involve processive loss of the poly(A) tail. This aspect of mRNA degradation can be conferred by AU elements from the granulocyte-macrophage colony-stimulating factor 3′-UTR, and similar elements in the COX-2 3′-UTR may be expected to show a similar function (31Chen C.Y. Xu N. Shyu A.B. Mol. Cell. Biol. 1995; 15: 5777-5788Crossref PubMed Scopus (249) Google Scholar). Consequently, while the actual mechanism of degradation of COX-2 mRNA may be dictated by sequences in the 3′-UTR, this region is not sufficient to confer dexamethasone responsiveness.Thus, we have established that repression of IL-1β-induced COX-2 by dexamethasone involves some degree (<40%) of transcriptional repression in A549 cells. However, this is not sufficient to account for the observed degree of repression at the product level. In contrast, mouse 3T3 cells showed substantial transcriptional repression of COX-2 by dexamethasone (48DeWitt D.L. Meade E.A. Arch. Biochem. Biophys. 1993; 306: 94-102Crossref PubMed Scopus (230) Google Scholar, 49Herschman H.R. Kujubu D.A. Fletcher B.S. Ma Q. Varnum B.C. Gilbert R.S. Reddy S.T. Prog. Nucleic Acids Res. Mol. Biol. 1994; 47: 113-148Crossref PubMed Scopus (27) Google Scholar). However, in common with our data, the degree of transcriptional repression was again insufficient to account for the total dexamethasone-dependent inhibition of COX-2 protein, indicative of further repressive mechanisms (48DeWitt D.L. Meade E.A. Arch. Biochem. Biophys. 1993; 306: 94-102Crossref PubMed Scopus (230) Google Scholar). Our data point to the existence of significant post-transcriptional mechanisms that require the GR transactivation function for de novo transcription and translation of a gene or genes that mediate dexamethasone-dependent repression. Furthermore, we demonstrate a functional relevance for this mechanism and indicate that loss of COX-2 mRNA as a result of dexamethasone treatment involves prior loss of the COX-2 poly(A) tail. However, the sequences involved in specificity and the events needed to initiate the response remain to be elucidated, and more detailed models are required to address these outstanding issues. Consequently, dexamethasone-dependent repression of COX-2 involves transcriptional as well as additional post-transcriptional or possibly translational mechanisms. It therefore seems that the exact contribution of each repressive mechanism to the overall repressive effect may depend on the cell type and the actual stimulation as well as the time of dexamethasone addition in relation to the stimulus.Recently, the transactivation function of GR, in driving GRE or mouse mammary tumor virus (MMTV)-dependent transcription or expression of the GR-dependent gene tyrosine aminotransferase, was shown to be defective in transgenic mice carrying a point mutation in GR that prevents dimerization (50Reichardt H.M. Kaestner K.H. Tuckermann J. Kretz O. Wessely O. Bock R. Gass P. Schmid W. Herrlich P. Angel P. Schutz G. Cell. 1998; 93: 531-541Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). In these mice, the repressive effect of dexamethasone on collagenase-3 and gelatinase B gene expression was essentially unaltered, whereas expression of the proopiomelanocortin and prolactin genes, which are thought to be negatively regulated via negative GRE sites, was markedly increased. These observations have been interpreted as showing that GR dimerization is required for GR-dependent transactivation via positive GREs and repression via negative GREs, while the transcriptional interference or transrepression functions of GR are unaffected (51Karin M. Cell. 1998; 93: 487-490Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). While this is likely to be correct, one further activation function of GR, namely the positive synergistic activation of transcription via interactions with members, for example, of the STAT transcription factor family has been overlooked (52Zhang Z. Jones S. Hagood J.S. Fuentes N.L. Fuller G.M. J. Biol. Chem. 1997; 272: 30607-30610Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 53Stocklin E. Wissler M. Gouilleux F. Groner B. Nature. 1997; 383: 726-728Crossref Scopus (570) Google Scholar). Cooperation with STAT proteins does not require GR dimerization and may therefore be unaffected in the GR dimerization-defective mice. We therefore speculate that positive cooperative functions of GR leading to post-transcriptional repressive mechanisms may, in addition to transrepression with factors such as AP-1 and NF-κB, account for GR-dependent repression of inflammatory genes such as COX-2. Prostaglandins (PGs)1form a potent group of autocrine and paracrine lipid mediators (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). These compounds are implicated in many normal cellular processes as well as pathophysiological processes such as inflammation, edema, bronchoconstriction, platelet aggregation, fever, and hyperalgesia (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar, 3Portanova J.P. Zhang Y. Anderson G.D. Hauser S.D. Masferrer J.L. Seibert K. Gregory S.A. Isakson P.C. J. Exp. Med. 1996; 184: 883-891Crossref PubMed Scopus (379) Google Scholar). PG synthesis involves phospholipase catalyzed release of arachidonic acid from membrane phospholipids and its conversion by the two cyclooxygenase (COX) enzymes to PGH2. Subsequently, cell-specific isomerases and reductases result in production of biologically relevant PGs. The two COX isoforms are encoded by distinct genes of which COX-1 is a constitutively expressed housekeeping gene and COX-2 shows low basal expression that is rapidly induced by inflammatory and mitogenic stimuli (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). Pharmacologically, this pathway is important, since COX is the target for nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol. 1995; 50: 1535-1542Crossref PubMed Scopus (327) Google Scholar). The anti-inflammatory benefits of nonsteroidal anti-inflammatory drugs are thought to derive from inhibition of COX-2, while many of the undesirable side effects are due to COX-1 inhibition (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar, 2Mitchell J.A. Larkin S. Williams T.J. Biochem. Pharmacol