Up-regulation of Prostaglandin E2 Synthesis by Interleukin-1β in Human Orbital Fibroblasts Involves Coordinate Induction of Prostaglandin-Endoperoxide H Synthase-2 and Glutathione-dependent Prostaglandin E2 Synthase Expression
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m111246200
ISSN1083-351X
AutoresRui Han, Shanli Tsui, Terry J. Smith,
Tópico(s)Glutathione Transferases and Polymorphisms
ResumoProstaglandin E2(PGE2) production involves the activity of a multistep biosynthetic pathway. The terminal components of this cascade, two PGE2 synthases (PGES), have very recently been identified as glutathione-dependent proteins. cPGES is cytoplasmic, apparently identical to the hsp90 chaperone, p23, and associates functionally with prostaglandin-endoperoxide H synthase-1 (PGHS-1), the constitutive cyclooxygenase. A second synthase, designated mPGES, is microsomal and can be regulated. Here we demonstrate that mPGES and PGHS-2 are expressed at very low levels in untreated human orbital fibroblasts. Interleukin (IL)-1β treatment elicits high levels of PGHS-2 and mPGES expression. The induction of both enzymes occurs at the pretranslational level, is the consequence of enhanced gene promoter activities, and can be blocked by dexamethasone (10 nm). SC58125, a PGHS-2-selective inhibitor, could attenuate the induction of mPGES, suggesting a dependence of this enzyme on PGHS-2 activity. IL-1β treatment activates p38 and ERK mitogen-activated protein kinases. Induction of both mPGES and PGHS-2 was susceptible to either chemical inhibition or molecular interruption of these pathways with dominant negative constructs. These results indicate that the induction of PGHS-2 and mPGES by IL-1β underlies robust PGE2 production in orbital fibroblasts. Prostaglandin E2(PGE2) production involves the activity of a multistep biosynthetic pathway. The terminal components of this cascade, two PGE2 synthases (PGES), have very recently been identified as glutathione-dependent proteins. cPGES is cytoplasmic, apparently identical to the hsp90 chaperone, p23, and associates functionally with prostaglandin-endoperoxide H synthase-1 (PGHS-1), the constitutive cyclooxygenase. A second synthase, designated mPGES, is microsomal and can be regulated. Here we demonstrate that mPGES and PGHS-2 are expressed at very low levels in untreated human orbital fibroblasts. Interleukin (IL)-1β treatment elicits high levels of PGHS-2 and mPGES expression. The induction of both enzymes occurs at the pretranslational level, is the consequence of enhanced gene promoter activities, and can be blocked by dexamethasone (10 nm). SC58125, a PGHS-2-selective inhibitor, could attenuate the induction of mPGES, suggesting a dependence of this enzyme on PGHS-2 activity. IL-1β treatment activates p38 and ERK mitogen-activated protein kinases. Induction of both mPGES and PGHS-2 was susceptible to either chemical inhibition or molecular interruption of these pathways with dominant negative constructs. These results indicate that the induction of PGHS-2 and mPGES by IL-1β underlies robust PGE2 production in orbital fibroblasts. The past decade has witnessed dramatic advances in our understanding of the prostanoid biosynthetic pathways in mammalian cells. Of particular importance was the discovery and molecular characterization of two cyclooxygenase isoforms and the realization that each protein might possess distinct functions and patterns of expression and regulation (1.Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1868) Google Scholar, 2.Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2521) Google Scholar). Prostaglandin-endoperoxide H synthase-1 and -2 (EC 1.14.99.1, PGHS) 1The abbreviations used are: PGHSprostaglandin-endoperoxide H synthaseDNdominant negativeDRB5,6-dichlorobenzimidazoleERKextracellular signal-regulated kinaseFBSfetal bovine serumGAPDHglyceraldehyde-3-phosphate dehydrogenaseILinterleukinMAP kinasemitogen-activated protein kinasePBSphosphate-buffered salinePGE2prostaglandin E2PGESprostaglandin synthasecPGEScytoplasmic PGESmPGESmicrosomal PGESTAOthyroid-associated ophthalmopathyTNFtumor necrosis factorTGFtransforming growth factor are membrane-associated and contain a heme prosthetic group. The PGHS isoforms have received substantial attention, in large part because they are the targets of aspirin and a large series of nonsteroidal anti-inflammatory compounds (3.Smith T.J. Rheum. Dis. Clin. North Am. 1998; 24: 501-523Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). PGHS-1 is constitutively expressed in most tissues and is thought to generate prostaglandins involved in housekeeping activities (1.Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1868) Google Scholar, 2.Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2521) Google Scholar, 4.Yokoyama C. Tanabe T. Biochem. Biophys. Res. Commun. 1989; 165: 888-894Crossref PubMed Scopus (301) Google Scholar, 5.Funk C.D. Funk L.B. Kennedy M.E. Pong A.S. Fitzgerald G.A. FASEB J. 1991; 5: 2304-2312Crossref PubMed Scopus (541) Google Scholar). In contrast, PGHS-2 is expressed in most tissues only following cell activation by cytokines, growth factors, and mitogens (6.O'Banion M.K. Sadowski H.B. Winn V. Young D.A. J. Biol. Chem. 1991; 266: 23261-23267Abstract Full Text PDF PubMed Google Scholar, 7.Xie W. Chipman J.G. Robertson D.L. Erikson R.L. Simmons D.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2692-2696Crossref PubMed Scopus (1715) Google Scholar, 8.Kujubu D.A. Herschman H.R. J. Biol. Chem. 1992; 267: 7991-7994Abstract Full Text PDF PubMed Google Scholar, 9.Ristimäki A. Garfinkel S. Wessendorf J. Maciag T. Hla T. J. Biol. Chem. 1994; 269: 11769-11775Abstract Full Text PDF PubMed Google Scholar, 10.Hla T. Neilson K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7384-7388Crossref PubMed Scopus (1497) Google Scholar). Prostanoids generated through the activities of PGHS-2 are thought to represent those produced under circumstances of inflammation and tissue disruption. prostaglandin-endoperoxide H synthase dominant negative 5,6-dichlorobenzimidazole extracellular signal-regulated kinase fetal bovine serum glyceraldehyde-3-phosphate dehydrogenase interleukin mitogen-activated protein kinase phosphate-buffered saline prostaglandin E2 prostaglandin synthase cytoplasmic PGES microsomal PGES thyroid-associated ophthalmopathy tumor necrosis factor transforming growth factor A very recent and significant advance has resulted from the identification of two prostaglandin E2 (PGE2) synthase enzyme isoforms (EC 5.3.99.3) (11.Jakobsson P.-J. Thorén S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 12.Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 13.Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Both are glutathione-dependent and catalyze the terminal conversion reaction of PGH2 to PGE2. One enzyme is a constitutively expressed cytosolic protein, designated cPGES (12.Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Evidence has been advanced indicating that cPGES is identical to p23, a putative chaperone of hsp90 that stabilizes steroid hormone receptor-hsp90 complexes (14.Freeman B.C. Felts S.J. Toft D.O. Yamamoto K.R. Genes Dev. 2000; 14: 422-434PubMed Google Scholar). It exhibits substantial constitutive expression in many tissues. Moreover, this expression was reported to be invariant in several cell lines in vitro with regard to treatment with IL-1β or TNF-α (12.Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). cPGES protein was found to be induced modestly in rat brain 48 h after injection of lipopolysaccharide (12.Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). The other isoform, mPGES, is a 16-kDa microsomal protein that can be regulated (11.Jakobsson P.-J. Thorén S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 13.Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). This enzyme may be expressed and regulated in a cell type-specific manner. mPGES is induced by lipopolysaccharide in rat macrophages, and this up-regulation can be blocked by the glucocorticoid, dexamethasone (13.Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Neither treatment altered the levels of cPGES in macrophages. Thus mPGES represents a regulated enzyme and a potentially important drug target. Very recent studies imply a preferential, functional association of each PGES isoform with a particular PGHS enzyme (12.Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar,13.Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). The studies demonstrated that cPGES is linked to PGHS-1 and that mPGES utilizes PGH2 generated by PGHS-2. Further studies have extended the notion that functional, stimuli-dependent linkage exists between specific PGHS isoforms and cell type-specific downstream enzymes (15.Ueno N. Murakami M. Tanioka T. Fujimori K. Tanabe T. Urade Y. Kudo I. J. Biol. Chem. 2001; 276: 34918-34927Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). These observations were made in transfected cells where one or more of the relevant enzymes had been over-expressed. Importantly, nothing is known currently about whether the expression of endogenous PGHS isoforms might in some way exhibit physiological coordination with that of the PGES enzymes. Such a coupling would be consistent with a model of PGE2production that exhibited functional cellular compartmentalization. Fibroblasts have been shown to express components of the prostanoid biosynthetic pathways, and cultures derived from particular anatomic regions and tissues can generate large amounts of PGE2 when provoked (16.Smith T.J. Exp. Clin. Endocrinol. Diabetes. 1999; 107 (Suppl.): S160-S163Google Scholar). These sentinel cells are critical to the orchestration of inflammatory responses, tissue remodeling, and wound healing and are key participants in the evolution of fibrosis (17.Smith R.S. Smith T.J. Blieden T.M. Phipps R.P. Am. J. Pathol. 1997; 151: 317-322PubMed Google Scholar). Diversity among human fibroblasts has only recently been appreciated as being potentially important to normal tissue function and disease manifestation. It is now clear that fibroblast subsets represent highly specialized cells that participate in reactive processes in the context of both normal function and pathology (18.Smith T.J. Sempowski G.D. Wang H.-S. Del Vecchio P.J. Lippe S.D. Phipps R.P. J. Clin. Endocrinol. Metab. 1995; 80: 2620-2625Crossref PubMed Google Scholar, 19.Young D.A. Evans C.H. Smith T.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8904-8909Crossref PubMed Scopus (58) Google Scholar). Fibroblasts function in various aspects of immunity, the recruitment of bone marrow-derived cells, tissue repair, and remodeling and provide the molecular and structural infrastructure supporting cellular cross-talk (17.Smith R.S. Smith T.J. Blieden T.M. Phipps R.P. Am. J. Pathol. 1997; 151: 317-322PubMed Google Scholar, 18.Smith T.J. Sempowski G.D. Wang H.-S. Del Vecchio P.J. Lippe S.D. Phipps R.P. J. Clin. Endocrinol. Metab. 1995; 80: 2620-2625Crossref PubMed Google Scholar, 20.Sciaky D. Brazer W. Center D.M. Cruikshank W.W. Smith T.J. J. Immunol. 2000; 164: 3806-3814Crossref PubMed Scopus (99) Google Scholar). Human orbital fibroblasts exhibit a phenotype that sets them apart from fibroblasts derived from other anatomic regions. They are composed of discrete subsets (18.Smith T.J. Sempowski G.D. Wang H.-S. Del Vecchio P.J. Lippe S.D. Phipps R.P. J. Clin. Endocrinol. Metab. 1995; 80: 2620-2625Crossref PubMed Google Scholar), one of which represents pre-adipocytes (21.Sorisky A. Pardasani D. Gagnon A. Smith T.J. J. Clin. Endocrinol. Metab. 1996; 81: 3428-3431Crossref PubMed Scopus (151) Google Scholar); possess a characteristic morphology (22.Smith T.J. Bahn R.S. Gorman C.A. J. Clin. Endocrinol. Metab. 1989; 69: 1019-1023Crossref PubMed Scopus (98) Google Scholar); and display a distinct pattern of gangliosides (23.Berenson C.S. Smith T.J. J. Clin. Endocrinol. Metab. 1995; 80: 2668-2674PubMed Google Scholar) and surface receptors (24.Smith T.J. Kottke R.J. Lum H. Andersen T.T. Am. J. Physiol. 1993; 265: C138-C142Crossref PubMed Google Scholar). Of particular relevance to their proposed participation in orbital inflammation are the robust responses to a variety of disease mediators such as cytokines, growth factors, and bioactive lipids (25.Wang H.-S. Cao H.J. Winn V.D. Rezanka L.J. Frobert Y. Evans C.H. Sciaky D. Young D.A. Smith T.J. J. Biol. Chem. 1996; 271: 22718-22728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 26.Cao H.J. Smith T.J. Am. J. Physiol. 1999; 277: C1075-C1085Crossref PubMed Google Scholar, 27.Smith T.J. Ahmed A. Hogg M.G. Higgins P.J. Am. J. Physiol. 1992; 263: C24-C29Crossref PubMed Google Scholar, 28.Smith T.J. Wang H.-S. Hogg M.G. Henrikson R.C. Keese C.R. Giaever I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5094-5098Crossref PubMed Scopus (64) Google Scholar, 29.Wang H.-S. Keese C.R. Giaever I. Smith T.J. J. Clin. Endocrinol. Metab. 1995; 80: 3553-3560Crossref PubMed Google Scholar). These fibroblasts have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune process. Two hallmarks of the tissue remodeling observed in TAO are the accumulation of hyaluronan and an often intense inflammatory reaction (30.Hufnagel T.J. Hickey W.F. Cobbs W.H. Jakobiec F.A. Iwamoto T. Eagle R.C. Ophthalmology. 1984; 91: 1411-1419Abstract Full Text PDF PubMed Scopus (184) Google Scholar). We hypothesize that it is this set of attributes that renders connective tissues in the human orbit susceptible to the remodeling associated with TAO. Among the characteristics that set apart orbital fibroblasts is the dramatic up-regulation of PGE2 synthesis observed following exposure of these cells in culture to proinflammatory cytokines (25.Wang H.-S. Cao H.J. Winn V.D. Rezanka L.J. Frobert Y. Evans C.H. Sciaky D. Young D.A. Smith T.J. J. Biol. Chem. 1996; 271: 22718-22728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,26.Cao H.J. Smith T.J. Am. J. Physiol. 1999; 277: C1075-C1085Crossref PubMed Google Scholar). These increases in PGE2 can be blocked by specific inhibitors such as SC58125 and NS-398, suggesting a dominant role for PGHS-2 in mediating the up-regulation (25.Wang H.-S. Cao H.J. Winn V.D. Rezanka L.J. Frobert Y. Evans C.H. Sciaky D. Young D.A. Smith T.J. J. Biol. Chem. 1996; 271: 22718-22728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In fact, orbital fibroblasts when provoked by IL-1β or leukoregulin or through engagement of surface-displayed CD40 by CD154, exhibit a particularly robust induction of PGHS-2 (25.Wang H.-S. Cao H.J. Winn V.D. Rezanka L.J. Frobert Y. Evans C.H. Sciaky D. Young D.A. Smith T.J. J. Biol. Chem. 1996; 271: 22718-22728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 26.Cao H.J. Smith T.J. Am. J. Physiol. 1999; 277: C1075-C1085Crossref PubMed Google Scholar, 31.Cao H.J. Wang H.-S. Zhang Y. Lin H.-Y. Phipps R.P. Smith T.J. J. Biol. Chem. 1998; 273: 29615-29625Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Here, we report that the treatment of orbital fibroblasts with IL-1β results in dramatic increases in PGE2 production and is associated with coordinate induction of both PGHS-2 and mPGES expression. These responses are mediated through elevations in the steady-state levels of their respective mRNAs and involve the use of overlapping intracellular signaling pathways, including the p38 and ERK mitogen-activated protein (MAP) kinases. Inhibiting PGHS-2 activity results in the blockade of mPGES induction by IL-1β, indicating some involvement of the products of the former enzyme in the expression of the latter. These latest findings provide insights into the complex interactions between PGHS-2 and mPGES in orbital fibroblasts that culminate in the generation of PGE2. SC58125 was obtained form Searle (Skokie, IL). Dexamethasone (1,4 pregnadien-9-fluoro-16α-methyl-11β,17α,21-triol-3,20-dione), 5,6-dichlorobenzimidazole (DRB), arachidonate, and cycloheximide were from Sigma. IL-1α, IL-1β, IL-4, TNF-α, and TGF-β were purchased from BIOSOURCE (Camarillo, CA). Interferon γ was obtained from Invitrogen. The cDNA encoding the human mPGES and antibodies directed against human mPGES protein were kindly provided by Dr. Per-Johan Jakobsson (Karolinska Institute, Stockholm, Sweden). An affinity-purified antibody against cPGES was kindly provided by Prof. I. Kudo (Showa University, Tokyo, Japan). PD98059 and SB203580 were obtained from Calbiochem (La Jolla, CA). p23 (cPGES) cDNA was a kind gift of Dr. David Toft (Mayo Clinic, Rochester, MN); full-length human PGHS-1 and PGHS-2 cDNAs were gifts from Dr. Kerry O'Banion (University of Rochester, Rochester, NY), and the plasmid designated −1800pGL2, containing a 1.8-kb fragment of the human PGHS-2 promoter, was generously supplied by Dr. Stephen M. Prescott (University of Utah, Salt Lake City). A dominant negative (DN) expression vector for p38 was generously provided by Dr. Roger Davis (University of Massachusetts, Worcester, MA), and the DN expression vector for ERK 1 was a gift from Dr. Melanie Cobb (Southwestern School of Medicine, Dallas, TX). Monoclonal antibodies directed against both human PGHS isoforms were purchased from Cayman (Ann Arbor, MI). Recombinant human CD154 was kindly supplied by Dr. R. P. Phipps (Rochester, NY) and prepared by the method of Kehry (32.Kehry M.R. Castle B.E. Semin. Immunol. 1994; 6: 287-294Crossref PubMed Scopus (79) Google Scholar). PGE2 radioimmunoassay kits were obtained from Amersham Biosciences. Antibodies against p38 and ERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Orbital fibroblast cultures were initiated from tissue explants obtained as surgical waste during decompression surgery from severe TAO or were from normal appearing orbital tissues in patients undergoing surgery for non-inflammatory conditions. These activities have been approved by the Institutional Review Boards of Albany Medical College and Harbor-UCLA Medical Center. Some of the fibroblast strains were kindly provided by Dr. Rebecca Bahn (Mayo Clinic, Rochester, MN). Tissue fragments were generated by mechanical disruption of explants, and fibroblasts were then allowed to adhere to plastic culture plates. They were covered with Eagle's medium to which 10% fetal bovine serum (FBS), glutamine (435 μg/ml), and penicillin/streptomycin were added as described previously (33.Smith T.J. J. Clin. Invest. 1987; 79: 1493-1497Crossref PubMed Scopus (33) Google Scholar). Medium was changed every 3–4 days, and monolayers were maintained in a 5% CO2, humidified incubator at 37 °C. Culture strains were utilized between the second and twelfth passage from initiation. All experimental manipulations were conducted after a state of confluence had been reached. We have already established the purity of these cultures and found them to be essentially free of contamination by endothelial and smooth muscle cells (18.Smith T.J. Sempowski G.D. Wang H.-S. Del Vecchio P.J. Lippe S.D. Phipps R.P. J. Clin. Endocrinol. Metab. 1995; 80: 2620-2625Crossref PubMed Google Scholar). Fibroblasts were cultivated in 100-mm-diameter plates to a confluent state and were then treated with the test agents specified in the figure legends. Cellular RNA was extracted from rinsed monolayers by the method of Chomczynski and Sacchi (34.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (64628) Google Scholar) with an RNA isolating system purchased from Biotecx (Houston, TX). The nucleic acid was subjected to electrophoresis through denaturing 1% agarose, formaldehyde gels. Integrity of the RNA was established by determining the 260/280 spectroscopic ratios and by staining the electrophoresed samples with ethidium bromide and inspecting them under UV light. The RNA was transferred to Zeta-probe membrane (Bio-Rad), and immobilized samples were hybridized with [32P]dCTP-labeled PGHS-1, PGHS-2, p23, and mPGES cDNA probes generated by the random primer method. Hybridization was conducted in a solution containing 5× SSC, 50% formamide, 5× Denhardt's solution, 50 mm phosphate buffer (pH 6.5), 1% SDS, and 0.25 mg/ml salmon sperm at 48 °C overnight. Membranes were washed under high stringency conditions, and then the RNA/DNA hybrids were visualized by autoradiography on X-Omat film (Kodak, Rochester, NY) following exposure at −70 °C. Bands resulting from radioactive hybrids were scanned by densitometry. Membranes were then stripped according to the instructions of the manufacturer and rehybridized with a GAPDH cDNA probe, and the band densities were normalized to this signal. For mPGES and PGHS-2 mRNA stability studies, cultures were treated with IL-1β for 3 h as a pretreatment. Cells were washed and incubated in growth medium for 4 h. At time 0, DRB (20 μg/ml), an inhibitor of gene transcription, was added to the medium of all plates without or with IL-1β (10 ng/ml) for the intervals indicated in Fig. 4. Abundance of mRNAs for the two enzymes was quantified by Northern blot hybridization. mPGES and PGHS-2 mRNA signals were normalized to their respective GAPDH levels. Cellular proteins were solubilized from rinsed fibroblast monolayers following the treatments indicated in the figure legends. The ice-cold harvest buffer contained 0.5% Nonidet P-40, 50 mm Tris-HCl (pH 8.0), and 10 μm phenylmethylsulfonyl fluoride. Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad). Primary monoclonal antibodies directed against PGHS-1 and PGHS-2 (10 μg/ml, Cayman) were incubated with the membranes for 2 h at room temperature. Following washes, membranes were reincubated with secondary peroxidase-labeled antibodies. In other experiments, primary antibodies directed against human mPGES and cPGES were utilized for the detection of these enzyme proteins. The ECL (Amersham Biosciences) chemiluminescence detection system was used to generate signals, and the resulting bands were analyzed with a densitometer. With regard to assessing the activation of p38 and ERK MAP kinases, cell lysates from untreated orbital fibroblast cultures and those treated with IL-1β (10 ng/ml) were subjected to SDS-PAGE; the proteins were transferred to membrane and then probed with phospho-specific antibodies against the two kinases. Fibroblasts were grown to confluence in 24-well plastic cluster plates in medium containing 10% FBS. Monolayers were shifted to serum-free medium for the final 24 h of incubation. IL-1β and the other test compounds were added at the times and concentrations indicated in the figure legends. Medium was removed from the cultures, and the monolayers were covered with phosphate-buffered saline (PBS) in the presence of the respective agents for the final 30 min of the treatment period. PBS was collected quantitatively, clarified by centrifugation, and subjected to PGE2 radioimmunoassay using a commercially available kit (Amersham Biosciences). For studies involving the transient transfection of human fibroblasts, cultures were allowed to proliferate to 80–90% confluence in medium containing 10% FBS. With regard to assessment of promoter activities, a 510-bp fragment spanning −538 to −28 of the putative mPGES promoter was cloned with the Human GenomeWalker kit (CLONTECH, Palo Alto, CA) according to the instructions of the supplier. Two reverse primers used for the PCR reactions included 5′-CGCAGCTCAACTGTGGGTGTGATC-3′ and 5′-GTGATCAGCTCGACAGAGGAGCAG-3′. The amplified fragment was sequenced and subcloned from pCR2.1-TOPO vector (Invitrogen) into a promoter-less pGL2-luciferase vector (Promega, Madison, WI). With regard to the human PGHS-2 promoter, a plasmid designated −1800pGL2, containing −1840 to +123 and thus located five base pairs upstream from the ATG of the human PGHS-2 promoter, was used. Promoter constructs were transiently transfected into fibroblasts using the LipofectAMINE PLUS system (Invitrogen). 0.75 μg of pGL2 promoter DNA and 0.1 μg of pRL-TK vector DNA (Promega), serving as a transfection efficiency control, were mixed with PLUS reagent for 15 min before being combined with LipofectAMINE PLUS for another 15 min. The DNA-lipid mixture was added to culture medium of 80% confluent cells for 3 h at 37 °C. Dulbecco's modified Eagle's medium containing 10% FBS replaced the transfection mixture overnight. Transfected cultures were then serum-starved, and some received either IL-1β (10 ng/ml) for 2 h or nothing (control) as indicated in the figure legends. Cellular material was harvested in buffer provided by the manufacturer (Promega) and stored at −80 °C until assayed. Luciferase activity was monitored with the Dual-Luciferase Reporter Assay System (Promega) in an FB12 tube luminometer (Zylux). Values were normalized to internal controls, and each experiment was performed at least three times. To interrupt the expression of potentially relevant signaling pathway components, DN constructs for p38 and ERK1 were ligated into pcDNA3.1 (Invitrogen). These were transiently transfected into cells as described above. Control cultures received a constant amount (2 μg) of empty vector DNA. The diminished levels of the kinases were documented by Western blotting an aliquot of the lysate with relevant antibodies. Confluent orbital fibroblast monolayers were shifted to serumless medium without or with IL-1β (10 ng/ml) for up to 48 h. As the data in Fig. 1 Asuggest, the cytokine elicits a dramatic increase in PGE2levels, which are 90-fold above control levels at 6 h, remain near peak values for 24 h, and then begin to decline at 48 h. Western blot analysis of cellular proteins from fibroblasts treated under identical conditions reveal a large, time-dependent induction of mPGES and PGHS-2. Neither is expressed at detectable levels under basal conditions. With regard to PGHS-2, the protein is detectable at 6 h. At 12 h, PGHS-2 levels are near maximal, at least 100-fold above control values, and remain elevated at 24 h. At the final time point in the experiment (48 h), PGHS-2 protein levels are again undetectable. The induction of mPGES lags behind that of PGHS-2 in that its levels are modestly elevated at 6 and 12 h and do not reach an apparent maximum until 24 h. Unlike PGHS-2, mPGES protein levels remain close to their peak at 48 h. In contrast, levels of cPGES and PGHS-1 are not altered as a consequence of IL-1β treatment. The effects of IL-1β on PGE2 synthesis, the expression of PGHS-2 and mPGES are dose-dependent, as the data in Fig. 1 B suggest. A near maximal effect on PGHS-2 expression and PGE2 production is achieved at a concentration of 1 ng/ml. That concentration yields a suboptimal induction of mPGES. IL-1β at 10 ng/ml, the highest concentration of the cytokine used, results in a considerably higher level of mPGES. The induction of both mPGES and PGHS-2 by IL-1β was susceptible to blockade by the synthetic glucocorticoid, dexamethasone (10 nm, Fig. 2). That concentration of dexamethasone is associated with a high fractional occupancy of the nuclear glucocorticoid receptor and near maximal effects on human fibroblast metabolism (35.Smith T.J. Metabolism. 1988; 37: 179-184Abstract Full Text PDF PubMed Scopus (33) Google Scholar). The steroid had no effect on basal enzyme expression. The blockade of IL-1β-dependent enzyme expression was accompanied by a substantial inhibition of cytokine-provoked PGE2production. Northern blot analysis was employed to determine the relationship in orbital fibroblasts between the induction of PGHS-2 and of mPGES mRNAs. Confluent cultures were shifted to medium without FBS overnight, and then IL-1β (10 ng/ml) was added at various times prior to monolayer harvest. As the Northern blot shown in Fig. 3 A indicates, the transcript encoding PGHS-2 is not detectable under basal culture conditions but was induced as a single, 4.8-kb mRNA with the addition of IL-1β within 6 h. At 12 h, the mRNA levels were at least 100-fold above control levels. At 24 h, PGHS-2 mRNA levels had dropped substantially and were again undetectable at 48 h. When the membrane was rehybridized with an mPGES cDNA probe, a 2-kb transcript was apparent under control conditions and was strongly up-regulated with IL-1β, an effect that reached a maximum at 12 h, when it was ∼7-fold above control levels. The induction was partially sustained for 48 h, the duration of the study, when it remained increased by 2.7-fold. Steady-state levels of cPGES and PGHS-1 mRNA were relatively constant following the addition of IL-1β to the culture medium (Fig. 3 A). The former is expressed on Northern blot analysis as a single band. PGHS-1 mRNA m
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