Complement C5b-9-Mediated Arachidonic Acid Metabolism in Glomerular Epithelial Cells
2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65080-8
ISSN1525-2191
AutoresTomoko Takano, Andrey V. Cybulsky,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoIn the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG. E2 in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A2 was associated with a marked stimulation of PGE2 production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE2 production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG. E2 in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A2 was associated with a marked stimulation of PGE2 production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE2 production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury. Membranous nephropathy is a common cause of nephrotic syndrome in adults, and approximately one-third of affected patients will develop end stage renal failure, necessitating renal replacement therapy. In membranous nephropathy, injury of the glomerular capillary wall leads to impaired glomerular permselectivity and proteinuria. Passive Heymann nephritis (PHN)1Couser WG Pathogenesis of glomerulonephritis.Kidney Int. 1993; 44: S19-S26Crossref PubMed Scopus (77) Google Scholar in the rat closely resembles human membranous nephropathy, and PHN has been used to study the pathogenesis of the human disorder. In PHN, the complement C5b-9 membrane attack complex induces nonlytic injury of glomerular visceral epithelial cells (GEC), in association with altered GEC morphology and proteinuria.1Couser WG Pathogenesis of glomerulonephritis.Kidney Int. 1993; 44: S19-S26Crossref PubMed Scopus (77) Google Scholar, 2Salant DJ Quigg RJ Cybulsky AV Heymann nephritis: mechanisms of renal injury.Kidney Int. 1989; 35: 976-984Crossref PubMed Scopus (100) Google Scholar A number of studies have demonstrated that metabolites of arachidonic acid (eicosanoids) play an important role in the pathogenesis of proteinuria in membranous nephropathy. Specifically, prostaglandin (PG) and thromboxane (TX. A2 production is enhanced in glomeruli isolated from rats with PHN, and inhibition of cyclooxygenase (COX) or TX synthase, or shifting production of dienoic prostanoids to inactive metabolites using fish oil diet, reduces proteinuria in certain models of membranous nephropathy.3Cybulsky AV Lieberthal W Quigg RJ Rennke HJ Salant DJ A role for thromboxane in complement-mediated glomerular injury.Am J Pathol. 1987; 128: 45-51PubMed Google Scholar, 4Nagao T Nagamatsu T Suzuki Y Effect of DP-1904, a thromboxane A2 synthase inhibitor, on passive Heymann nephritis in rats.Eur J Pharmacol. 1996; 316: 73-80Crossref PubMed Scopus (18) Google Scholar, 5Weise WJ Natori Y Levine JS O'Meara YM Minto AW Manning EC Goldstein DJ Abrahamson DR Salant DJ Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis.Kidney Int. 1993; 43: 359-368Crossref PubMed Scopus (38) Google Scholar, 6Zoja C Benigni A Verroust P Ronco P Bertani T Remuzzi G Indomethacin reduces proteinuria in passive Heymann nephritis in rats.Kidney Int. 1987; 31: 1335-1343Crossref PubMed Scopus (54) Google Scholar The effect of TXA2 on proteinuria may be through an increase in glomerular transcapillary pressure, since this parameter is elevated in rat membranous nephropathy and appears to be responsible for a portion of the enhanced urine protein excretion.7Yoshioka T Rennke HG Salant DJ Deen WM Ichikawa I Role of abnormally high transmural pressure in the permselectivity defect of glomerular capillary wall: a study in early passive Heymann nephritis.Circ Res. 1987; 61: 531-538Crossref PubMed Scopus (141) Google Scholar, 8Gabbai FB Gushwa LC Wilson CB Blantz RC An evaluation of the development of experimental membranous nephropathy.Kidney Int. 1987; 31: 1267-1278Crossref PubMed Scopus (33) Google Scholar We have previously used rat GEC in culture to characterize biochemical changes induced by sublytic C5b-9, including arachidonic acid metabolism. We have shown that sublytic C5b-9 activates cytosolic phospholipase A2 (cPLA2) in a calcium- and protein kinase C-dependent manner.9Cybulsky AV Papillon J McTavish AJ Complement activates phospholipases and protein kinases in glomerular epithelial cells.Kidney Int. 1998; 54: 360-372Crossref PubMed Scopus (48) Google Scholar, 10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar, 11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Free arachidonic acid released by cPLA2 is further converted to bioactive eicosanoids, including prostaglandins (PGs) and TXA2. The metabolism of arachidonic acid to PGs is catalyzed by COX.12Smith WL Garavito RM DeWitt DL Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and 2.J Biol Chem. 1996; 271: 33157-33160Crossref PubMed Scopus (1870) Google Scholar To date, two isoforms of COX, namely COX-1 and COX-2, have been characterized. Although both isoforms have similar structures, enzymatic properties, and intracellular distribution, their modes of regulation are distinct. In contrast to COX-1, which is constitutively expressed in most mammalian cells, COX-2 protein is not expressed in most tissues under normal physiological conditions, but is induced in certain cell types in response to growth factors, tumor promoters, hormones, bacterial endotoxin, and cytokines.12Smith WL Garavito RM DeWitt DL Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and 2.J Biol Chem. 1996; 271: 33157-33160Crossref PubMed Scopus (1870) Google Scholar Thus, it has been proposed that COX-1 may be involved in PG synthesis for maintenance of normal physiology, whereas COX-2 may produce PGs for inflammatory processes or mitogenesis. Although there is considerable evidence to support a major pathogenetic role for eicosanoids in membranous nephropathy, little is known about the regulation and relative importance of the two COX isoforms in this disease. In the current study, we used cultured rat GEC to characterize the expression and regulation of COXs by C5b-9, and we defined the role of the two isoforms in complement-mediated arachidonic acid metabolism. We also extended the results in cultured GEC to in vivo C5b-9-dependent GEC injury by further characterizing COX-mediated arachidonic acid metabolism in the PHN model of membranous nephropathy. Tissue culture media, Trizol reagent, Random Primer DNA Labeling System, and RNase T1 were obtained from Gibco BRL (Burlington, ON). NuSerum was purchased from Collaborative Research (Bedford, MA). [3H]PGE2 (200 Ci/mmol), [α-32P]dCTP (3000 Ci/mmol), and [α-32P]CTP (3000 Ci/mmol) were purchased from New England Nuclear (Boston, MA). PGE2, anti-PGE2 antiserum, C8-deficient human serum (C8D), purified human C8, and RNase A were from Sigma Chemical Co. (St. Louis, MO). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting reagents were from BioRad Laboratories (Mississauga, ON). Goat anti-COX-1 antiserum was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-COX-2 antiserum was from Cayman Chemical (Ann Arbor, MI). β-actin cDNA probe was purchased from Ambion, Inc. (Austin, TX). Plasmids containing coding regions of rat COX-1 and -2 were kindly provided by Dr. Brian Kennedy (Merck-Frosst, Point Claire-Dorval, QC),13Kennedy BP Chan C-C Culp SA Cromlish WA Cloning and expression of rat prostaglandin endoperoxide synthase (cyclooxygenase)-2 cDNA.Biochem Biophys Res Comm. 1993; 197: 494-500Crossref PubMed Scopus (120) Google Scholar and each coding region was subcloned into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Primary cultures of rat GEC were established from explants of rat glomeruli.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar, 11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Characterization of GEC was published previously.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar, 11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar According to established criteria, the cells demonstrated polygonal shape and cobblestone appearance at confluency, cytotoxic susceptibility to low doses of aminonucleoside of puromycin, positive immunofluorescence staining for cytokeratin, and presence of junctional complexes and apical microvilli by electron microscopy. The standard medium used to maintain GEC cultures, K1, consisted of Dulbecco's modified Eagle's medium/Ham F-10 (1:1. containing 5.0% NuSerum and hormone supplements. A subclone of GEC that stably overexpresses cPLA2 (fivefold above the endogenous level), or neo-GEC (control) were used in this study. These clones were produced and characterized previously.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar, 11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Studies were done with cells between passages 4 and 70. For total RNA preparation, GEC were cultured in serum-poor medium (Dulbecco's modified Eagle's medium/Ham F-10 (1:1. with 0.5% fetal calf serum) for 16 hours before experiments. Rabbit antiserum to GEC10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar was used to activate complement on GEC membranes. Briefly, GEC were incubated with antiserum (5% v/v) in serum-poor medium for 40 minutes at 22°C. GEC were then incubated with normal human serum (2.5–3.0% v/v in serum-poor medium. or with heat-inactivated (decomplemented) human serum (56°C, 30 minutes) in controls, for the indicated times at 37°C. In some experiments, antibody-sensitized GEC were incubated with C8D (2.5–5.0% v/v) reconstituted with or without purified human C8 (80 μg/ml undiluted serum). We have generally used heterologous complement to facilitate studies with complement-deficient sera and to minimize possible signaling via complement-regulatory proteins. however, in previous studies, results of several experiments involving arachidonic acid metabolism were confirmed with homologous (rat. complement.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar Sublytic concentrations of complement (≤5. normal human serum) were established previously.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar Previous studies have shown that in GEC, complement is not activated in the absence of antibody. Northern blot hybridization was performed as described previously.14Takano T Fiore S Maddox JF Brady HR Petasis NA Serhan CN Aspirin-triggered 15-epi-Lipoxin A4 (LXA4) and LXA4 stable analogs are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.J Exp Med. 1997; 185: 1693-1704Crossref PubMed Scopus (393) Google Scholar Total RNA was extracted from GEC using the Trizol reagent according to manufacturer's protocol. RNA (15 μg) was separated by gel electrophoresis on 1% agarose gels containing 1.9. formaldehyde and transferred to a nylon membrane. Coding regions of rat COX-1 and -2 cDNAs were radiolabeled with [α-32P]dCTP using the Random Primer DNA Labeling System. Membranes were hybridized in hybridization buffer (1. bovine serum albumin, 7% SDS, 0.5 Mol/L phosphate buffer, pH 6.8, 1 mmol/L EDTA), containing 1–2 × 106 cpm/ml of radiolabeled probe for 16 hours at 42°C, followed by washing in buffer A (0.5% bovine serum albumin, 5% SDS, 40 mmol/L phosphate buffer, pH 6.8, 1 mmol/L EDTA) twice for 20 minutes at 65°C, and then in buffer B (1% SDS, 40 mmol/L phosphate buffer, pH 6.8, 1 mmol/L EDTA) 4 times for 20 minutes at 65°C. Membranes were exposed to X-ray film with an intensifying screen at −70°C for 48 to 72 hours. The amount of mRNA was quantitated using scanning densitometry (NIH Image software). RNase protection assay was performed using the methods described by Feng et al, except that pcDNA3 was used to construct probes for COX-1 and -2.15Feng L Sun W Xia Y Tang WW Chanmugam P Soyoola E Wilson CB Hwang D Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.Arch Biochem Biophys. 1993; 307: 361-368Crossref PubMed Scopus (466) Google Scholar A 341-bp fragment of rat COX-1 cDNA produced by BstXI (from bp 1297 to bp 1637 in the coding region) was blunted and subcloned into the EcoRV site in pcDNA3. A 241-bp fragment of rat COX-2 cDNA produced by BamHI and EcoRI (from bp 291 to bp 531 in the coding region) was subcloned in pcDNA3. XhoI was used to linearize both COX-1 and COX-2 templates for labeling. 32P-labeled antisense cRNA probes were synthesized by in vitro transcription, using linearized COX cDNA templates, or β-actin template, T7 RNA polymerase and [α-32P]CTP. Total RNA (5–8 μg) was hybridized with 1 × 105 cpm of each labeled antisense cRNA probe for 16 hours at 55°C. Unhybridized probes were digested with RNaseA (0.3 μg/ml) and RNase T1 (90 U/ml) for 1 hour at 30°C. Then, the RNases were digested with proteinase K (0.5 mg/ml. for 30 minutes at 37°C. After phenol/chloroform extraction and ethanol precipitation, the hybrids were denatured at 85°C for 3 minutes and electrophoresed on 6% polyacrylamide gels. After drying, gels were exposed to X-ray film at −70°C for 24 to 48 hours. GEC or rat glomeruli were lysed in Laemmli buffer (62.5 mmol/L Tris, 2% SDS, 10% glycerol, 0.01% bromphenol blue, pH 6.8) and boiled for 5 minutes. After centrifugation at 14,000 × g, supernatants were collected, and protein content was quantified by a modified Lowry method (Protein DC-assay, BioRad). Equal amounts of protein were separated by 8% SDS-PAGE under reducing conditions. Proteins were then electrophoretically transferred to nitrocellulose membrane, blocked with 5% dry milk, and incubated with goat anti-COX-1 antiserum for 2 hours at 22°C, or with rabbit anti-COX-2 antiserum for 16 hours at 4°C. After 3 washes, membranes were incubated with respective secondary antibodies conjugated with alkaline phosphatase, and alkaline phosphatase activity was detected by the addition of bromochloroindolyl phosphate with nitro blue tetrazolium. Protein content was quantitated using scanning densitometry (NIH Image software). For measurement of PGE2 production in the presence of exogenous arachidonic acid, GEC were incubated with antibody and complement, and then with arachidonic acid (15 μmol/L. in measurement buffer (145 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L MgSO4, 0.5 mmol/L CaCl2, 1 mmol/L Na2HPO4, 5 mmol/L glucose, 20 mmol/L Hepes, pH 7.4) for 20 minutes at 37°C. For measurement of PGE2 generation resulting from release of endogenous arachidonic acid, culture medium was supplemented with arachidonic acid (7.5–10 μmol/L) for 24 to 48 hours before experiments. GEC were incubated with antibody and complement in serum-poor medium. Supernatants were collected in 100 μmol/L indomethacin to prevent further metabolism, and were used for PGE2 measurement. In some experiments, lipids were extracted from cells and supernatants for PGE2 measurements, as described previously.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar The amount of PGE2 released into supernatants was equivalent to that from cells plus supernatants, indicating that most of the PGE2 was released from cells into supernatants. PGE2 was measured by radioimmunoassay using anti-PGE2 antibody and [3H]PGE2, according to the manufacturer's protocol. The range of the standard curve in the assay was 15–500 pg/0.1 ml of PGE2. Samples were incubated with [3H]PGE2 and anti-PGE2 antibody for 1 hour at 4°C, after which time unbound PGE2 was removed by the addition of activated charcoal. The radioactivity of the supernatant was counted in a β-scintillation counter, and PGE2 concentration was calculated from standard formulas. In some experiments, cells were lysed with 1% Triton X-100, and protein content was measured to normalize PGE2 concentration according to protein content. For measurement of TXA2, cells were stimulated as above, and the concentration of TXB2 (the stable metabolite of TXA2) in supernatants was determined by enzyme immunoassay kit (Cayman Chemical). The range of the standard curve in the assay was 7.8 to 1000 pg/ml of TXB2. Culture of Cos-1 cells was described previously.11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Transient transfection of Cos-1 cells was performed by the diethylaminoethyl-dextran method.11Panesar M Papillon J McTavish AJ Cybulsky AV Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.J Immunol. 1997; 159: 3584-3594PubMed Google Scholar Briefly, cells in 35-mm culture dish were transfected with 0.5 μg of plasmid DNA encoding rat COX-1 or COX-2. Three days after the transfection, cells were incubated with measurement buffer containing arachidonic acid (15 μmol/L) and NS-398 for 30 minutes at 37°C. PGE2 in supernatants was measured by radioimmunoassay. Anti-Fx1A was prepared as described previously.16Salant DJ Cybulsky AV Experimental glomerulonephritis.in: DiSabato G Methods in Enzymology. Academic Press, New York1988: 421-461Google Scholar Male Sprague-Dawley rats (150–175 g; Charles River, St. Constant, QC, Canada) were injected with 450 μl of sheep anti-Fx1A antiserum. This batch of antiserum caused minimal proteinuria in the heterologous phase (day 5) but induced significant proteinuria in the autologous phase (day 14). On day 14, rats were sacrificed and glomeruli were isolated by differential sieving,16Salant DJ Cybulsky AV Experimental glomerulonephritis.in: DiSabato G Methods in Enzymology. Academic Press, New York1988: 421-461Google Scholar yielding a glomerular preparation that was >95% pure. For Western blotting, glomeruli were lysed in Laemmli buffer as described above (Immunoblotting). For the RNase protection assay, glomerular RNA was prepared using the Trizol reagent. To measure PGE2 generation, glomeruli from each rat were resuspended in 2 ml of measurement buffer and divided into two aliquots. One aliquot was incubated with NS-398 for 15 minutes at 37°C and then centrifuged for 2 minutes at 1,000 × g at 22°C. The second aliquot was treated in an identical manner, except that dimethylsulfoxide was used instead of NS-398 (control). Supernatants were transferred to another set of tubes, which contained indomethacin (final concentration, 100 μmol/L) to terminate the reaction, and 100 μl of each supernatant was used for the measurement of PGE2 by radioimmunoassay. Data are presented as mean ± SEM. The t statistic was used to determine significant differences between two groups. One-way analysis of variance (ANOVA) was used to determine significant differences among groups. Two-way ANOVA was used to determine significant differences in multiple measurements among groups. Where significant differences were found, individual comparisons were made between groups using the t statistic, and adjusting the critical value according to the Bonferroni method. The first series of experiments were designed to determine whether sublytic C5b-9 regulates expression of COX mRNA. Unstimulated neo-GEC expressed COX-1 mRNA at a level readily detectable by Northern hybridization (using 15 μg of total RNA), whereas COX-2 mRNA was not detectable under the same conditions (Figure 1A). When neo-GEC were stimulated with a sublytic concentration of complement by serial exposure to anti-GEC antibody and normal serum, COX-2 mRNA was up-regulated significantly, peaking approximately 2 hours after the initiation of C5b-9 assembly and continuing for at least for 4 hours. When heat-inactivated (decomplemented) serum was used instead of normal serum (control incubations), COX-2 mRNA up-regulation was trivial, indicating that COX-2 up-regulation is most likely mediated by complement activation. The small up-regulation of COX-2 induced by heat-inactivated serum (at 40 minutes) may be attributed to some minor stimulatory component(s) in the serum, such as growth factors. At the same time points, there were no consistent complement-dependent changes in COX-1 mRNA levels. When the amounts of mRNA were quantified by densitometry at 100 minutes, cells treated with complement had COX-1 mRNA levels similar to control, whereas complement-treated cells had ∼4 times more COX-2 mRNA than control (Figure 1A). In addition to the well-described COX-2 transcript of approximately 4.2 kb, a larger transcript (∼5.0 kb) was also observed in Northern blots. The origin of the 5.0-kb transcript has not been clearly established, but posttranscriptional regulation, such as alternate polyadenylation described in other cell types,17Newton R Seybold J Liu SF Barnes PJ Alternate COX-2 transcripts are differentially regulated: implications for post-transcriptional control.Biochem Biophys Res Comm. 1997; 234: 85-89Crossref PubMed Scopus (51) Google Scholar may account for this phenomenon. These results suggest that expression of the two COX isozymes in GEC is differentially regulated by complement (Figure 1A). To demonstrate that C5b-9 assembly was actually required for COX-2 mRNA up-regulation, we incubated antibody-treated neo-GEC with C8D, with or without reconstitution with purified C8. C8D without C8 forms C5b-7, which was previously shown to be biologically inactive in GEC.10Cybulsky AV Monge JC Papillon J McTavish AJ Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.Am J Physiol. 1995; 269: F739-F749PubMed Google Scholar When evaluated by RNase protection assay, GEC incubated with C8D did not show significant COX-2 mRNA up-regulation, as compared with heat-inactivated serum. However, when C8D was reconstituted with purified C8, up-regulation of COX-2 mRNA was evident, indicating that formation of C5b-9 is required for COX-2 up-regulation (Figure 1B). Quantification of COX-2 mRNA by densitometry showed that the relative amounts of COX-2 mRNA were heat-inactivated serum (HIS), 1.0; normal serum (NS), 2.2; C8D, 1.1; C8D+C8, 1.7 (average of 2 experiments). It was noted that the effects of C8D reconstituted with C8 were less potent, as compared with normal serum, probably because there appears to be some general loss of complement activity during the immunodepletion of C8 (unpublished observation). We next examined if the complement-mediated COX mRNA regulation is reflected in protein expression. When neo-GEC were incubated with antibody and complement, COX-2 protein expression was up-regulated, as compared with GEC incubated with heat-inactivated serum (control). This up-regulation was transient, typically peaking at ∼3 hours of stimulation (Figure 1C). COX-1 protein expression was not affected by complement for up to 24 hours of stimulation (Figure 1C). When the amounts of protein were quantified by densitometry at 3 hours, cells treated with complement had similar amounts of COX-1 protein, as compared with control, whereas complement-treated cells had ∼1.6 times more COX-2 protein, as compared with control (Figure 1C). The results of mRNA analysis and immunoblotting suggested that COX-1 was the dominant COX isozyme in resting rat GEC in culture and in GEC incubated with heat-inactivated serum, but that COX-2 expression was up-regulated by sublytic C5b-9. To understand the role of the two COX isozymes in arachidonic acid metabolism in GEC, we next addressed the enzymatic activities of COX isozymes. In these experiments, exogenous arachidonic acid (15 μmol/L) was provided as substrate for COX. After stimulation of neo-GEC with sublytic concentrations of complement for 3 hours, PGE2 generation in the presence of exogenous arachidonic acid was increased by 32%, as compared with GEC that were incubated with heat-inactivated serum (control; Figure 2A). A similar increase in PGE2 was observed after 24 hours of stimulation with complement, but the change was not statistically significant (not shown). These results indicate that GEC treated with complement have an increase in total COX enzyme activity. To determine which isozyme was responsible for the complement-mediated increase in COX activity, we examined the effect of the COX-2 selective inhibitor, NS-398. When exogenous arachidonic acid was added together with NS-398 (10−6 M), the complement-mediated increase in PGE2 generation was abolished, indicating that increased COX activity was due to the up-regulation of COX-2 (Figure 2A), consistent with an increase in COX-2 protein (Figure 1C). Nevertheless, in the presence of exogenous arachidonic acid, ∼75% of total PGE2 production in complement-treated cells was due to constitutive COX-1 activity, and COX-2 contributed ∼25% (Figure 2A, NS). A downward trend in PGE2 production was induced by NS-398 in GEC incubated with HIS (Figure 2A). This result suggests that these GEC may contain a small amount of COX-2 activity and/or that NS-398 cross-reacted with COX-1 (see below). NS-398 (10−6 M) was shown previously not to affect human or ovine COX-1 activity.18Johnson JL Wimsatt J Buckel SD Dyer RD Maddipati KR Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons.Arch Biochem Biophys. 1995; 324: 26-34Crossref PubMed Scopus (110) Google Scholar, 19Barnett J Chow J Ives D Chiou M Mackenzie R Osen E Nguyen B Tsing S Bach C Freire J Chan H Sigal E Ramesha C Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system.Biochim Biophys Acta. 1994; 1209: 130-139Crossref PubMed Scopus (314) Google Scholar, 20Futaki N Takahashi T Yokoyama M Arai I Higuchi S Otomo S NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro.Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (810) Google Scholar We verified the selectivity of NS-398 using Cos-1 cells transfected with rat COX-1 or COX-2 cDNA. When untransfected Cos-1 cells were incubated with exogenous arachidonic acid, the amount of PGE2 released to the supernatant was trivial (∼12 pg/0.1 ml), indicating that untransfected Cos-1 cells do not have significant endogenous COX activity. In contrast, Cos-1 cells transfected with COX-1 or COX-2 cDNA released significant amounts of PGE2 into supernatants when incubated with exogenous arachidonic acid (823 ± 166 pg/0.1 ml, n = 6, and 585 ± 85 pg/0.1 ml, n = 6, respectively). At 10−7 or 10−6 M NS-398, COX-1 activity tended to
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