Complement C5b-9 induces cyclooxygenase-2 gene transcription in glomerular epithelial cells

Tomoko Takano, Andrey V. Cybulsky, Xiaoxia Yang, and Lamine Aoudjit

Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada H3A 2B4


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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First published July 12, 2001; 10.1152/ajprenal.0048.2001.---In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. Rat GEC in culture express cyclooxygenase (COX)-1 constitutively, whereas COX-2 expression is induced by C5b-9. Both isoforms contribute to complement-induced prostaglandin generation. The present study addresses mechanisms of complement-induced COX-2 expression in GEC. Downregulation of protein kinase C (PKC) blunted complement-induced upregulation of COX-2 mRNA. Complement and phorbol 12-myristate 13-acetate (PMA) both stimulated COX-2 promoter activity. C5b-9 activated c-Jun NH2-terminal kinase (JNK), and inhibition of JNK activity by transfection of a kinase-inactive JNK1 partially inhibited complement-induced (but not PMA-induced) COX-2 promoter activation. Conversely, a constitutively active mitogen-activated protein or extracellular signal-regulated kinase kinase kinase (MEKK)-1, a kinase upstream of JNK, increased COX-2 promoter activity. MEKK-induced COX-2 promoter activation was not affected by downregulation of PKC and was augmented by PMA. Thus, in GEC, PKC and JNK pathways contribute independently to complement-induced COX-2 expression. Nuclear factor-kappa B was also activated by complement in GEC but did not contribute to complement-induced COX-2 upregulation.

c-Jun NH2-terminal kinase; eicosanoid; lipid mediators; membranous nephropathy; signal transduction


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CYCLOOXYGENASE (COX) plays a key role in the metabolism of arachidonic acid to the important inflammatory mediators, prostaglandins (PGs) and thromboxane A2 (47). Two isoforms of COX, namely COX-1 and COX-2, have been characterized so far. Although both isoforms have similar structures, enzymatic properties, and intracellular distribution, their modes of regulation are distinct. COX-1 is expressed constitutively in most mammalian cells, whereas COX-2 is not expressed in most tissues under normal physiological conditions but is induced in certain cell types in response to various stimuli (47). The regulation of COX-2 gene expression and the role of COX-2 in pathophysiology have received considerable attention in recent years. COX-2 is usually induced as an immediate early gene by mitogenic or inflammatory stimuli, as well as by stimuli that act via G protein- and protein kinase C (PKC)-mediated pathways (18). More recently, mitogen-activated protein (MAP) kinase (MAPK) signaling cascades have also been implicated in the regulation of COX-2 (15, 53). There are at least three MAPK pathways: 1) the extracellular signal-regulated kinase (ERK)-1/2 (p44/p42) pathway, typically activated by growth factors; 2) the c-Jun NH2-terminal kinase (JNK), or stress-activated protein kinase pathway; and 3) the p38 pathway, activated by diverse stimuli, including stress (45). All three MAPK pathways may contribute to COX-2 gene regulation (13, 53, 54); however, involvement of the pathways appears to be cell or stimulus specific, and there appears to be cross talk among the MAPK pathways, as well as with other signaling pathways, including PKC (12, 14, 28, 54).

The complement system plays an important role in mediating inflammation, cytolysis, and phagocytosis. Activation of the complement cascade near a cell surface leads to assembly of terminal components, exposure of hydrophobic domains, and insertion of the C5b-9 membrane attack complex into the lipid bilayer of the plasma membrane (21, 30, 34). Nucleated cells require multiple C5b-9 lesions for lysis, but, at lower doses, C5b-9 induces sublytic injury and various metabolic effects. These may include activation of phospholipases and protein kinases and induction of certain genes, e.g., growth factors, nuclear factor (NF)-kappa B, and c-Jun (5, 6, 16, 22, 30, 34-36, 38, 41). An example of C5b-9-mediated injury in vivo is passive Heymann nephritis (PHN) in the rat, a well-established experimental model of human membranous nephropathy. In PHN, C5b-9 induces nonlytic injury of glomerular visceral epithelial cells (GEC) in association with altered GEC morphology and proteinuria (3, 43). A number of studies have demonstrated that COX-derived metabolites of arachidonic acid (eicosanoids) play an important role in the pathogenesis of proteinuria in membranous nephropathy. Specifically, PG and thromboxane A2 production is enhanced in glomeruli isolated from rats with PHN, and inhibition of enzymes involved in prostanoid synthesis may lead to amelioration of proteinuria (4, 33, 59). Fish oil diet also decreased proteinuria in PHN by shifting production of certain endogenous glomerular eicosanoids away from dienoic prostanoids to inactive metabolites (52). We have recently reported that the activity of cytosolic phospholipase A2 (cPLA2), the key enzyme that provides substrate for eicosanoid synthesis, is increased in glomeruli of rats with PHN (7).

Previously, we have employed well-differentiated rat GEC in culture to characterize biochemical changes induced by sublytic C5b-9, which include arachidonic acid release and metabolism. We have shown that sublytic C5b-9 activates cPLA2 (5, 6, 38). This activation is dependent on a rise in intracellular Ca2+ concentration and occurs secondarily to the activation of PKC. Although C5b-9 also activated ERK2, cPLA2 activation occurred independently of ERK2. Free arachidonic acid released by cPLA2 was further converted to bioactive eicosanoids, including PGs and thromboxane A2. Cultured rat GEC express COX-1 constitutively but not COX-2 (49). Stimulation of GEC with sublytic C5b-9 upregulates COX-2 but does not affect COX-1 expression. Both COX isoforms contribute to complement-mediated eicosanoid generation in GEC. We have also reported that COX-1 and -2 are upregulated in glomeruli of rats with PHN, compared with control rats, and that both isoforms contribute to eicosanoid generation in PHN (49). The recent development of COX-2-selective inhibitors provides an opportunity for blockade of COX-2 activity without significant alteration of the effects of COX-1. However, this approach requires a better understanding of the roles of the COX isoforms in kidney physiology as well as in pathological conditions, such as glomerulonephritis. Because several studies support a role for COX isoforms in the pathogenesis of complement-mediated glomerular injury in vivo, it is important to understand the mechanisms of C5b-9-induced expression of COX-2. The present study addresses complement-mediated COX-2 regulation in cultured rat GEC. We demonstrate that COX-2 expression is regulated at the transcriptional level and is mediated via PKC and JNK pathways.


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Materials. Tissue culture media and T4 nucleotide kinase were purchased from GIBCO BRL (Burlington, ON). NuSerum was from Collaborative Research (Bedford, MA). C8-deficient human serum (C8D), purified human C8, PGE2, phorbol 12-myristate 13-acetate (PMA), and pyrrolidinedithiocarbamate (PDTC) were from Sigma (St. Louis, MO). Protease inhibitor cocktail and FuGENE 6 were from Roche Diagnostics (Laval, QC). The gel shift assay system, luciferase assay system, and chloramphenicol acetyltransferase (CAT) enzyme assay system were from Promega (Madison, WI). [gamma -32P]ATP (3,000 Ci/mmol), [alpha -32P]dCTP (3,000 Ci/mmol), and [alpha -32P]CTP (3,000 Ci/mmol) were from New England Nuclear (Boston, MA). PD-98059 and JNK assay kit were from New England BioLabs (Mississauga, ON). SC-68376 and MG-132 were from Calbiochem (La Jolla, CA). Rabbit anti-COX-2 antiserum was from Cayman Chemical (Ann Arbor, MI). This antiserum was raised against a synthetic peptide corresponding to amino acids 584-598 of murine COX-2. It recognizes COX-2 as a 72- to 74-kDa protein by immunoblotting and does not cross-react with COX-1. Rabbit anti-MAP/ERK kinase kinase (MEKK) antiserum and monoclonal anti-NF-kappa B p65 subunit antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmids. Mouse COX-2 promoter constructs pGC815 and pGC815NF-kappa BM, which contain the -815 to +123 sequence of the COX-2 gene, were gifts from Drs. Shozo Yamamoto and Kei Yamamoto (Tokushima Univ., Tokushima, Japan) (58). pcDNA3-Flag-JNK1(apf) (kinase-inactive form of JNK1) was from Dr. Roger Davis (Univ. of Massachusetts Medical School, Worcester, MA) (8). pFC-MEKK, the constitutively active form of MEKK-1, was purchased from Stratagene (La Jolla, CA). The rat COX-2 promoter construct rCOX2-2.7-CAT, which contains the -2698 to +32 sequence of the COX-2 gene, was from Dr. JoAnne Richard (Baylor College of Medicine, Houston, TX) (46). rCOX2-1.9-luc, which contains the -1878 to +32 sequence of the rat COX-2 gene, was generated by subcloning a XbaI fragment of the rat COX-2 gene into the NheI site of the pGL3-basic vector (Promega). NF-kappa B-luciferase was a gift from Dr. Albert Descoteaux (Institut Armand-Frappier, Montreal, QC).

GEC culture, activation of complement, and induction of PHN. Culture and characterization of rat GEC were described previously (5, 49). A subclone of GEC, which grows on plastic substratum in serum-replete K1 medium, was used in this study. Rabbit antiserum to GEC (5) was used to activate complement on GEC membranes. Briefly, GEC were incubated with antiserum (5% vol/vol) for 40 min at 22°C. GEC were then incubated with normal human serum (NS; 2.5-5.0% vol/vol) or heat-inactivated (decomplemented) human serum (HIS; 56°C, 30 min, 2.5-5.0% vol/vol) in controls for the indicated times at 37°C. In some experiments, antibody-sensitized GEC were incubated with C8D (5.0% vol/vol) 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 (5). Sublytic concentrations of complement (<= 5% NS) were established previously (5). Previous studies have shown that in GEC, complement is not activated in the absence of antibody (5). PHN was induced in male Sprague-Dawley rats (150-175 g body wt; Charles River, St. Constant, QC) by intravenous injection (400 µl/rat) of sheep anti-Fx1A antiserum as described previously (49). Preparation of anti-Fx1A antiserum was described previously (42). Rats developed significant proteinuria 14 days after injection (~250 mg/day; normal rats excrete <10 mg protein/day). At the 14-day time point, rats were killed, and glomeruli were isolated by differential sieving as described previously (49).

Assay of JNK activity. Confluent GEC in 6-cm culture plates were incubated with complement as described above for 20-40 min. Cells were washed with ice-cold homogenization buffer [50 mM HEPES, 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 20 µM leupeptin, 20 µM pepstatin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.40] and lysed with 100 µl of cold lysis buffer [0.1% Triton X-100, 25 mM HEPES, 20 mM beta -glycerophosphate, pH 7.7, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM 1,4-dithiothreitol (DTT), 20 µM leupeptin, 20 µM pepstatin, 0.2 mM PMSF, and 1 mM Na3VO4]. After centrifugation (10,000 g, 10 min), protein concentration was quantified and adjusted to 4 mg/ml. Fifty microliters of cell lysate (200 µg) were incubated with glutathione S-transferase (GST)-c-Jun-agarose (100 µl, 10% suspension) for 2 h at 4°C. After four washes, pellets were resuspended in 40 µl of kinase buffer (50 mM beta -glycerophosphate, pH 7.6, 10 mM MgCl2, 1 mM Na3VO4, 20 µM ATP, and 5 µCi [gamma -32P]ATP) and incubated for 20 min at 30°C. The kinase reaction was terminated with 20 µl of 3× Laemmli reducing buffer. Samples were boiled and subjected to SDS-PAGE and autoradiography. In some experiments, JNK activity was measured by use of the JNK assay kit. Samples were processed in an analogous manner, and GST-c-Jun phosphorylated at Ser63 was detected by immunoblotting with anti-phospho-c-Jun antibody. Equal loading was verified by immunoblotting with anti-c-Jun antibody. Signals were quantified by use of scanning densitometry (NIH Image software).

Transfection of GEC and reporter assays. Different transfection methods and reporter constructs were used to achieve optimal transfection efficiency and protein expression of specific plasmids. Confluent GEC were harvested, washed with ice-cold serum-free culture medium, and resuspended in ice-cold serum-free medium (2.4 × 107 cells/ml). Cell suspensions (0.5 ml) were placed in electroporation cuvettes (0.4 cm) and mixed with indicated amounts of DNA by pipetting. Cuvettes were placed on ice for 10 min, and electroporation was performed with the use of Gene-Pulser II (Bio-Rad) at a voltage of 200 V and a capacitance of 975 µF. The time constant was typically 32-35 ms. Cuvettes were placed on ice for 10 min before cells were transferred to 35-mm plates (cells from 1 cuvette were divided into 8 plates). Cells were incubated with antibody and complement 2-3 days after electroporation. At the end of 6 h, cells were washed with PBS and harvested in 0.4 ml of reporter lysis buffer (Promega). After the freezing and thawing, cell lysates were centrifuged at 14,000 g for 5 min, and 25 µl of supernatant were mixed with 100 µl of luciferase reagent (Promega). Luciferase activity was measured in a luminometer.

In some experiments, GEC were transfected with plasmid DNA using FuGENE 6 transfection reagent according to the manufacturer's instructions. For gene reporter assays, GEC were passed into 35-mm plates at 3 × 105 cells/plate. On the following day, plasmids were mixed with FuGENE 6 at a ratio of 1:6 (micrograms of plasmid to microliters of FuGENE 6) in 100 µl of serum-free medium and were then added to each plate containing 2 ml of K1 medium. Cells were harvested after 24 h, and luciferase was assayed as described above. For immunoblotting, GEC were passaged into 6-cm plates at 6 × 105 cells/plate. On the following day, plasmids were mixed with FuGENE 6 at a ratio of 1:9 (micrograms plasmid to microliters FuGENE 6) in 200 µl of serum-free medium and added to each plate containing 4 ml of K1 medium for 24 h.

The CAT assay was performed using the CAT enzyme assay system (Promega) according to the manufacturer's instructions.

RNase protection assay, Northern blotting, and immunoblotting. RNase protection assay and Northern blotting were performed as described previously (49). In brief, for Northern blotting, RNA (15 µg) was separated by gel electrophoresis on 1% agarose gels containing 1.9% formaldehyde and was transferred to a nylon membrane. The coding region of rat COX-2 cDNA (1.8 kb) was radiolabeled with [alpha -32P]dCTP and hybridized with membranes for 16 h. Membranes were washed and exposed to X-ray film with an intensifying screen at -85°C for 48-72 h. An RNase protection assay was performed using a probe corresponding to a 241-bp fragment of rat COX-2 cDNA (bp 291-531 in the coding region). Total RNA (5-8 µg) was hybridized with 32P-labeled antisense cRNA probes for 16 h at 55°C. Unhybridized probes were digested with RNaseA and RNase T1, and then the RNases were digested with proteinase K. After phenol/chloroform extraction and ethanol precipitation, the hybrids were denatured at 85°C for 3 min and electrophoresed on 6% polyacrylamide gels. After being dried, gels were exposed to X-ray film at -85°C for 24-48 h. The amount of mRNA was quantitated by use of scanning densitometry (NIH Image software). Immunoblotting of COX-2 was performed with the use of nuclear fractions, which are enriched in COX-2 (48). Cell fractions were prepared as described previously (38). Equal amounts of protein (30 µg of nuclear fraction for COX-2 and 50 µg of postnuclear supernatant for MEKK immunoblots) were separated by 10% SDS-PAGE under reducing conditions. Proteins were electrophoretically transferred to nitrocellulose membrane, blocked with 5% dry milk, and incubated with antisera to COX-2 or MEKK for 16 h at 4°C. After three washes, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase, and horseradish peroxidase activity was detected by enhanced chemiluminescence.

Preparation of nuclear extracts. Nuclear extracts of GEC were prepared as described by Richardson et al. (40) with minor modifications. Briefly, GEC cultured in 6-cm plates were collected in ice-cold PBS and centrifuged. Pellets were resuspended in 0.4 ml of hypotonic buffer containing 10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT. Nonidet P-40 was added to a final concentration of 0.5%, and cell suspensions were incubated for 10 min on ice. After being vortexed for 10 s, nuclei were sedimented by centrifugation for 10 min at 1,500 g. The nuclear pellets were extracted with 100 µl of high-salt extraction buffer (20 mM HEPES, pH 7.4, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 20% glycerol, and 0.5 mM DTT) for 10 min on ice with frequent agitation. The nuclear debris was discarded by centrifugation at 12,000 g for 10 min, and the nuclear extracts were collected and stored at -85°C.

Electrophoretic gel mobility shift assay. An electrophoretic gel mobility shift assay (EMSA) was performed with the use of the gel shift assay system (Promega) according to the manufacturer's instructions. Double-stranded oligonucleotide for the human NF-kappa B consensus binding site (5'-AGTTGAGGGGACTTTCCCAGG-3') was radiolabeled by use of T4 polynucleotide kinase and [gamma -32P]ATP and was purified by centrifugation through G-25 Sephadex spin columns. Ten micrograms of nuclear protein were incubated with 50,000-200,000 counts/min (cpm) of 32P-labeled NF-kappa B probe for 20 min at room temperature in binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris · HCl, pH 7.5, and 0.25 mg/ml poly(dI-dC)]. Samples were loaded onto 7% polyacrylamide gels with Tris-borate buffer (45 mM Tris-borate and 1 mM EDTA), and electrophoresis was performed at 200 V for 3 h. The gel was dried under vacuum and exposed to X-ray film for 16 h. For supershift assays, anti-NF-kappa B p65 subunit antibody was incubated with the reaction mixture for 10 min before addition of radiolabeled NF-kappa B probe.

Statistics. Data are presented as means ± SE. The t-statistic was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t-statistic and by adjusting the critical value according to the Bonferroni method.


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Complement-induced COX-2 upregulation is partially dependent on PKC. Previously, we demonstrated that sublytic C5b-9 activates PKC and ERK and upregulates COX-2, but not COX-1, expression in cultured rat GEC (49). In keeping with these results, when GEC were stimulated with antibody and complement (NS), COX-2 mRNA and protein were upregulated compared with control (HIS; Fig. 1A). C8D, which forms C5b-7 and is biologically inactive in GEC (49), did not cause significant COX-2 mRNA upregulation, whereas C8D reconstituted with purified C8 clearly upregulated COX-2 mRNA, indicating that formation of C5b-9 is required for COX-2 upregulation (Fig. 1A). Next, we addressed the role of PKC and ERK in complement-induced COX-2 expression. Prolonged incubation of GEC with PMA (2 µg/ml for 18 h) downregulates PKC activity by 100% (6). In PKC-depleted cells, complement-induced COX-2 mRNA expression was inhibited by 45%, indicating that mRNA upregulation is, at least in part, mediated by PKC (Fig. 1, B and C). In contrast, PD-98059, a specific inhibitor of MAP/ERK kinase (MEK)-1 and -2 and thus of the ERK pathway, did not affect COX-2 upregulation at a concentration that inhibited complement-stimulated ERK2 activity by >90% (6). Thus the ERK pathway, although activated by complement, does not appear to have a major role in complement-induced COX-2 upregulation.


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Fig. 1.   Complement induces cyclooxygenase (COX)-2 mRNA, which is partially mediated via protein kinase C (PKC). A: glomerular epithelial cells (GEC) were incubated with anti-GEC antiserum and normal human serum (NS) to form C5b-9 for 100 min (mRNA) or 3 h (protein). In control incubations, heat-inactivated human serum (HIS) was used instead of NS. In other experiments, antibody-sensitized GEC were incubated with C8-deficient serum (C8D) with or without reconstitution with purified C8. COX-2 mRNA and protein were detected by RNase protection assay and immunoblotting, respectively. B: to deplete PKC (PKC dep), GEC were incubated with 2 µg/ml of phorbol 12-myristate 13-acetate (PMA) for ~18 h before complement incubation. The mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) inhibitor, PD-98059 (50 µM), was added together with anti-GEC antiserum and NS. RNA was analyzed by RNase protection assay. C: expression of COX-2 mRNA was quantified by densitometry and was normalized to beta -actin signal. Values are expressed as percentage of NS after subtraction of HIS. * P < 0.02 vs. NS; each bar represents 5-8 values.

Complement activates JNK in GEC. PKC appeared to mediate complement-induced COX-2 expression in GEC, but downregulation of PKC did not completely inhibit COX-2 upregulation. This result suggested involvement of additional mediators, which may include JNK. We therefore examined whether complement stimulates JNK activity in GEC. Incubation of GEC with antibody and complement (20-40 min) increased JNK activity 2.3- to 3.3-fold compared with control (HIS; Fig. 2A). To verify that C5b-9 assembly was actually required for JNK activation, antibody-treated GEC were incubated with C8D, with or without reconstitution with purified C8. C8D alone did not activate JNK compared with buffer. However, when C8D was reconstituted with purified C8, JNK activation was evident, indicating that formation of C5b-9 was required. It should also be noted that complement-induced JNK activation is unaffected by PKC depletion (data not shown).


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Fig. 2.   Complement activates the c-Jun NH2-terminal kinase (JNK) pathway in GEC. A: antibody-sensitized GEC were incubated with NS (HIS in controls) and C8D+C8 (C8D in controls), as in Fig. 1, or were exposed to ultraviolet (UV) light for 2 min (positive control). Cell lysates were incubated with glutathione S-transferase (GST)-c-Jun-agarose to absorb activated JNK. Complexes were incubated with [gamma -32P]ATP and were subjected to SDS-PAGE and autoradiography (32P). In some experiments, phospho-c-Jun and c-Jun were detected by immunoblotting (IB). Bottom panel shows the result of densitometric analysis (* P < 0.05 and ** P < 0.01 vs. control; N = 3-5 experiments). B: glomeruli were isolated from rats with passive Heymann nephritis (PHN; day 14) and controls. JNK activity was measured in glomerular lysates (as in A). Top 2 panels: immunoblot of 2 rats with PHN and 2 controls. Bottom panel: densitometry. * P < 0.05 vs. control; N = 5 rats/group.

It is important to verify that complement-induced JNK activation in cultured GEC is relevant to GEC injury in vivo, i.e., in the PHN model, where C5b-9 induces visceral GEC injury and proteinuria (49). JNK activity was studied in glomeruli isolated from rats with PHN on day 14, when the rats showed significant proteinuria. JNK activity in glomerular lysates was approximately four times higher in rats with PHN compared with control rats (Fig. 2B). Thus the JNK pathway is activated by C5b-9 both in cultured GEC and in vivo.

JNK mediates complement-induced COX-2 expression in GEC. Chemical inhibitors of the JNK pathway are currently not available. Thus we utilized a kinase-inactive form of JNK1, JNK1(apf) (8), and a COX-2 promoter-reporter construct to evaluate the contribution of the JNK pathway to complement-induced COX-2 upregulation. After transient transfection of GEC with the rat COX-2 promoter-luciferase construct, rCOX2-1.9-luc, a high basal luciferase activity was noted compared with cells transfected with vector (20-200 times baseline). This high basal luciferase activity does not reflect basal COX-2 mRNA expression in unstimulated GEC, which is trivial (49), and most likely is due to pronounced stability of the luciferase mRNA compared with the relatively unstable COX-2 mRNA. After GEC transiently transfected with rCOX2-1.9-luc were stimulated with complement, promoter activity (as reflected by luciferase activity) increased by ~82% (Fig. 3). In consideration of the high basal activity of this promoter, the complement-induced increase is actually substantial. The COX-2 promoter was also activated significantly by 6 h of stimulation with PMA (~42%) (Fig. 3A). Although promoter activation by PMA was relatively small in magnitude, incubation of GEC with PMA also induced expression of COX-2 mRNA and protein (Fig. 3B), implying that the effect of PMA on COX-2 induction is biologically significant. When rCOX2-1.9-luc was transiently cotransfected with JNK1(apf), the complement-induced promoter activation was markedly attenuated, indicating that JNK(apf) most likely acted as a dominant inhibitor of endogenous JNK and prevented activation of the COX-2 promoter (Fig. 3A). JNK(apf) did not affect the COX-2 promoter activation by PMA. These results strongly suggest that the JNK pathway is an important mediator of complement-induced COX-2 upregulation. We also attempted to establish a subclone of GEC that stably expresses JNK1(apf) to demonstrate directly a blunted COX-2 response of such clone to complement. However, we were not able to obtain a clone that had a high expression level of JNK1(apf), because dominant-negative JNK may have impaired GEC proliferation.


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Fig. 3.   Kinase-inactive mutant of JNK, JNK(apf), attenuates complement-mediated COX-2 gene transcription. A: GEC (1.2 × 107 cells) were transiently transfected with COX-2 promoter coupled to luciferase, rCOX2-1.9-luc (15 µg), plus JNK(apf) or vector (10 µg) by use of electroporation. Cells were plated into 35-mm plates and serum starved for 24 h before incubation with antibody+NS for 6 h (HIS in controls) or PMA (250 ng/ml) for 6 h (0.1% DMSO in controls; Ctrl). At the end of incubations, luciferase activity was measured in cell lysates. * P < 0.02 vs. control; N = 3-6 experiments conducted in duplicate. B: GEC were stimulated with PMA (250 ng/ml) for 100 min (for mRNA) or 5 h (for protein). COX-2 mRNA and protein expression were detected by Northern hybridization and immunoblotting, respectively.

To further confirm the role of the JNK pathway in COX-2 upregulation, we transfected GEC with a constitutively active MEKK1 (pFC-MEKK), a kinase upstream of JNK, and evaluated COX-2 promoter activity. Cotransfection of pFC-MEKK with rCOX2-2.7-CAT increased COX-2 promoter activity 2.2- to 2.4-fold compared with control (vector; Fig. 4, A and B). In keeping with the results in Fig. 3, the COX-2 promoter in vector-transfected cells was activated by 6 h of treatment with PMA (~40%; Fig. 4A). The effects of MEKK1 and PMA were additive, because stimulation of pFC-MEKK-transfected cells with PMA further activated the COX-2 promoter (Fig. 4A). PKC depletion did not inhibit MEKK1-induced COX-2 promoter activity, indicating that the effect of MEKK1 on the COX-2 promoter is not dependent on PKC (Fig. 4A). These results indicate that MEKK most likely induces COX-2 expression via JNK activation and that PKC acts in concert with the JNK pathway. PKC- and JNK-mediated COX-2 upregulation are independent of each other, and these two pathways likely act in an additive manner.


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Fig. 4.   MEK kinase (MEKK)-1 induces COX-2 expression via the JNK pathway. A: GEC in 35-mm plates were transiently transfected with COX-2 promoter coupled to chloramphenicol acetyltransferase (CAT), rCOX2-2.6-CAT (0.5 µg), plus constitutively active MEKK (pFC-MEKK) or vector (50 ng), using FuGENE 6. Left: after 24 h, cells were incubated with PMA (250 ng/ml) or vehicle (0.1% DMSO) for 6 h, and CAT activity in cell lysates was quantified. Right: to deplete PKC, cells were incubated with 2 µg/ml of PMA for ~18 h before transfection. Cells were transfected as above, and CAT activity in cell lysates was quantified after 24 h. * P < 0.002 vs. vector+DMSO; N = 3-6 experiments conducted in duplicate. B: vehicle (DMSO), PD-98059 (50 µM), or SC-68373 (10 µM) was added to the medium immediately after transfection. CAT activity in cell lysates was quantified after 24 h. * P < 0.05 and ** P < 0.01 vs. vector; each bar represents 4 values. C: GEC were stimulated with complement (NS) for 100 min in the presence of vehicle (DMSO), PD-98059 (50 µM), or SC-68373 (10 µM). Total RNA was analyzed for COX-2 mRNA expression by Northern hybridization. Equal loading was confirmed by ethidium bromide staining of the gel (not shown). D: GEC plated in 6-cm plates were transfected with the indicated amounts of pFC-MEKK, using FuGENE 6. After 24 h, nuclear fractions and postnuclear supernatants were analyzed for expression of COX-2 and MEKK1 protein, respectively, by immunoblotting.

Although MEKK primarily activates the JNK pathway, it has been reported that MEKK, when overexpressed, may also activate the ERK or p38 MAPK pathways (25). To verify the potential contribution of the ERK and p38 pathways to MEKK-induced COX-2 promoter activation, we utilized specific inhibitors of these two pathways. Neither PD-98059 (an inhibitor of the ERK pathway) nor SC-68376 (an inhibitor of the p38 pathway) inhibited MEKK-induced COX-2 promoter activity, indicating that these two pathways were unlikely to contribute to MEKK-induced COX-2 promoter activity (Fig. 4B). Furthermore, PD-98059 and SC-68376 did not inhibit complement-mediated COX-2 mRNA upregulation in GEC (Figs. 1 and 4C). It has also been reported that MEKK1 can activate the NF-kappa B pathway via activation of the Ikappa Balpha kinase complex (27). To rule out the potential contribution of the NF-kappa B pathway in MEKK-induced COX-2 promoter activity, we utilized a mouse COX-2 promoter in which the NF-kappa B binding site was mutated (pGC815NF-kappa BM). When the wild-type promoter pGC815 was cotransfected with pFC-MEKK, COX-2 promoter activity was increased 1.6 ± 0.2-fold (N = 5 experiments conducted in duplicate). Promoter activity of pGC815NF-kappa BM was increased to a similar extent by pFC-MEKK (1.8 ± 0.4-fold, N = 5 experiments conducted in duplicate), indicating that NF-kappa B is not involved in MEKK-induced COX-2 promoter activity. Taken together, these results indicate that activation of the JNK pathway by MEKK leads to promoter activation of the COX-2 gene in GEC.

To verify that MEKK-induced COX-2 promoter activity is biologically significant, i.e., associated with COX-2 protein expression, we transiently transfected GEC with pFC-MEKK and evaluated the expression of COX-2 protein by immunoblotting. COX-2 protein was clearly induced when the constitutively active MEKK was overexpressed (Fig. 4D).

Complement-induced COX-2 upregulation is not mediated by NF-kappa B. Another pathway that has been reported to regulate COX-2 is NF-kappa B. To monitor activation of NF-kappa B, GEC were incubated with antibody and complement, and translocation of NF-kappa B from the cytosol to the nucleus was evaluated by EMSA, using 32P-labeled NF-kappa B-binding oligonucleotide as probe. After stimulation with complement, at least three distinct bands were observed by EMSA (Fig. 5A). The two top bands were supershifted by preincubation with the anti-NF-kappa B p65 antibody, indicating that these bands are specific to NF-kappa B. The bottom band was not supershifted by this antibody, suggesting that it is nonspecific (Fig. 5B).


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Fig. 5.   Complement activates nuclear factor (NF)-kappa B. A: antibody-sensitized GEC were incubated with complement (NS) or HIS for 1 h. Nuclear extracts were analyzed by electrophoretic gel mobility shift assay (EMSA) to evaluate nuclear translocation of NF-kappa B. B: nuclear extracts from complement-stimulated GEC were subjected to EMSA with or without preincubation with rabbit anti-p65 antibody or nonimmune rabbit serum (NRS). The top 2 bands (labeled NF-kappa B) were supershifted by the antibody. The bottom band was not supershifted by the antibody, indicating that this band is nonspecific. (The spot in the lane farthest right is nonspecific.) C: GEC (1.2 × 107 cells) were transfected with NF-kappa B binding sequence coupled to luciferase (NF-kappa B-luc; 15 µg) by electroporation and were plated into 35-mm wells. After 48 h, cells were incubated with antibody and complement (NS) or PMA (250 ng/ml) for 6 h. Luciferase activity was measured in cell lysates. * P < 0.05, ** P < 0.005, and *** P < 0.002 vs. HIS; N = 6 experiments conducted in duplicate.

To determine whether complement can activate gene transcription via activation of NF-kappa B, GEC were transfected with the luciferase gene driven by the NF-kappa B binding sequence (NF-kappa B-luc), and the cells were incubated with antibody and complement for 6 h. GEC stimulated with 2.5% NS showed an ~2.2-fold increase in luciferase activity compared with control (HIS). NS (5% vol/vol) caused an ~3.3-fold increase, whereas PMA, a known stimulator of NF-kappa B, caused an ~8.4-fold increase compared with control (Fig. 5C). Thus complement activates gene transcription via NF-kappa B, although to a lesser extent than PMA.

To address the role of NF-kappa B in complement-mediated COX-2 expression, we utilized two inhibitors of the NF-kappa B pathway that are known to act via different mechanisms. PDTC is an antioxidant known to inhibit the NF-kappa B pathway in various systems (1). MG-132 is a proteosome inhibitor that inhibits the NF-kappa B pathway by inhibiting degradation of the inhibitory protein Ikappa B (37). Using EMSA, we confirmed that these two compounds inhibited complement-mediated nuclear translocation of NF-kappa B (Fig. 6A). However, when GEC were incubated with antibody and complement (100 min), neither PDTC (100 µM) nor MG-132 (20 µM) inhibited complement-induced COX-2 mRNA expression (NS+PDTC: 140 ± 16% of NS alone, 3 experiments; NS+MG-132: 150 ± 15% of NS alone, 3 experiments; mRNA levels were quantified by densitometry).


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Fig. 6.   Complement-induced COX-2 upregulation is independent of the NF-kappa B pathway. A: GEC were incubated with complement (NS) in the presence of pyrrolidinedithiocarbamate (PDTC; 100 µM) or MG-132 (20 µM) for 1 h. Nuclear extracts were prepared and analyzed for NF-kappa B by EMSA. Arrows point to specific NF-kappa B bands. B: GEC (1.2 × 107 cells) were transfected with the wild-type mouse COX-2 promoter-luciferase construct (pGC815; 15 µg) or a COX-2 promoter, where the NF-kappa B site is mutated (kappa BM; 15 µg), and were plated into 35-mm wells. After 48 h, cells were incubated with antibody and complement for 6 h, and luciferase activity was quantified in cell lysates. * P < 0.05 vs. HIS; N = 3 experiments conducted in duplicate.

To further assess the role of NF-kappa B in complement-mediated COX-2 upregulation, we utilized mouse COX-2 promoter-reporter constructs with or without a mutation at the NF-kappa B binding site. GEC were transfected with the mouse COX-2 promoter-luciferase construct (pGC815) or with a promoter in which the NF-kappa B binding site was mutated (pGC815NF-kappa BM). After incubation with antibody and complement, luciferase activity increased ~1.6-fold compared with control in GEC expressing the wild-type promoter and ~1.7-fold in GEC expressing pGC815NF-kappa BM (Fig. 6B). Similar results were obtained with the use of rat COX-2 promoters with or without a mutation at the NF-kappa B binding site (data not shown).

It has been reported recently that NF-kappa B is activated in GEC of rats with PHN and that PDTC (200 mg · kg-1 · day-1) effectively inhibits NF-kappa B activation (32). To determine whether NF-kappa B activation contributes to complement-mediated COX-2 upregulation in vivo, rats with PHN were treated with PDTC according to a similar protocol (200 mg · kg-1 · day-1 from day 7 to 14). Glomerular COX-2 mRNA expression was evaluated on day 14 with the use of RNase protection assay. Densitometric analysis showed that COX-2 mRNA expression in glomeruli from rats with PHN treated with vehicle (water) was 1.5 ± 0.1-fold greater compared with normal rats (N = 3), consistent with our previous study (49). PDTC treatment of PHN rats did not affect the increase in COX-2 mRNA, i.e., COX-2 mRNA expression was 2.0 ± 0.2-fold greater compared with normal rats (N = 3). Taken together, the results show that NF-kappa B does not contribute to complement-induced COX-2 upregulation in GEC both in vitro and in vivo.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study characterizes the regulation of the COX-2 gene by C5b-9. We have previously shown that in GEC, complement activates protein kinases, including PKC and ERK. Our present study demonstrates that complement activates JNK (Fig. 2A). Moreover, JNK activity was stimulated by C5b-9 in GEC in vivo, i.e., in PHN (Fig. 2B). To our knowledge, the present study is the first to demonstrate C5b-9-mediated JNK activation in vivo. Complement-induced COX-2 mRNA was upregulated via PKC, whereas the ERK pathway was not involved (Fig. 1). The rat COX-2 promoter was activated by complement, and this activation was inhibited significantly by blocking of the JNK pathway by overexpression of a kinase-inactive mutant of JNK1 (Fig. 3A). Inhibition of the JNK pathway did not, however, affect PKC-mediated COX-2 promoter activation (Fig. 3A). Stimulation of the JNK pathway using a constitutively active mutant of MEKK1 resulted in COX-2 promoter activation and protein expression, which was independent of PKC (Fig. 4, A, B, and D). Thus the JNK and PKC pathways contribute to COX-2 upregulation in GEC independently and additively. It is interesting to note that in GEC, PKC is involved in the regulation of eicosanoid production at two levels, including stimulation of cPLA2, thereby facilitating arachidonic acid release and induction of COX-2, which enhances arachidonic acid metabolism, whereas JNK, which does not activate cPLA2 (unpublished observations), regulates only COX-2.

Our results are in keeping with studies of Xie and Herschman (53, 54), who showed that the Ras-MEKK1-JNK signal transduction pathway contributed to v-src-, platelet-derived growth factor-, or serum-induced COX-2 promoter activation in mouse fibroblasts. The same investigators also reported a role for the JNK pathway in transcriptional regulation of the COX-2 gene in activated mast cells and the endotoxin-treated mouse macrophage cell line RAW 264.7 (39, 50). In these cells, the cAMP-response element (CRE) appears to be critical in COX-2 gene activation. Curiously, the rat COX-2 promoter does not contain the CRE, although the E-box element, which overlaps the CRE in mouse and human genes, is conserved in the rat and is critical in hormone-induced COX-2 expression in rat ovarian granulosa cells (31). The cis-acting elements required for the JNK pathway-mediated COX-2 regulation in rat GEC remain to be elucidated. Guan et al. (15) demonstrated that a constitutively active truncation mutant of MEKK1 increased COX-2 expression in mouse fibroblast cells (15). Although these results appear to be similar to our study, in that system, inhibition of p38 by SC-68376 completely abolished MEKK1-induced COX-2 induction, whereas in our study, SC-68376 inhibited neither MEKK1-induced COX-2 promoter activity nor complement-mediated COX-2 upregulation (Fig. 4, B and C). The reason for this discrepancy is not clear, but the effects of MEKK1 might be influenced by the cell type.

The mouse COX-2 gene was originally identified as a phorbol ester-inducible gene (24), and, since then, other studies have demonstrated that PKC is a potent inducer of COX-2 (17). However, surprisingly little is known about the mechanisms of PKC-induced COX-2 gene activation. In some reports, it was shown that PKC-dependent COX-2 upregulation may be mediated by activation of ERK (12, 19), although this is not the case in the present study (Fig. 1). Miller et al. (29) reported that overexpression of the atypical PKC-zeta isoform increased COX-2 expression in rat mesangial cells (29). In other systems, the classic PKC-alpha isoform contributes to COX-2 regulation (51). Rat GEC were shown to express PKC-alpha , -delta , -epsilon , and -zeta isoforms (20). Because the atypical PKC-zeta isoform is not responsive to PMA, the results of the present study suggest that PKC-alpha , -delta , and/or -epsilon mediates complement-induced COX-2 upregulation in GEC.

The promoter region of the COX-2 gene in all species examined so far is known to contain NF-kappa B binding site(s) (9, 23, 46, 55). Therefore, it was surprising that although NF-kappa B was activated by complement in GEC (Fig. 5), NF-kappa B did not contribute to COX-2 upregulation. However, such a result is not without precedent. For example, the NF-kappa B binding site was not relevant to COX-2 regulation in endotoxin-treated mouse macrophages and activated mouse mast cells (39, 50). On the other hand, a number of studies demonstrate a critical role for NF-kappa B in COX-2 regulation (57). One possible explanation for these conflicting results is that an extracellular stimulus activates a panel of transcription factors rather than a single transcription factor, and the stimulus-dependent combination of transcription factors would lead to a certain pattern of gene regulation. Transcription cofactors, such as p300/CBP, may orient certain transcription factors to stimulate gene transcription (10). Promoter regions of the human (23), mouse (9), chicken (55), and rat (46) COX-2 gene have been shown to contain multiple regions of homology, including putative binding sites for transcription factors such as CRE, E-box, NF-interleukin-6(C/EBPbeta ), AP-2, SP1, and NF-kappa B. C5b-9 has been reported to induce expression of several growth factors, cytokines, and extracellular matrix proteins, suggesting that C5b-9 can potentially activate a variety of distinct transcription factors. A specific pattern of transcription factor induction could upregulate COX-2. There might also be a redundancy of certain induced transcription factors, such as NF-kappa B in the case of COX-2. Recent studies have reported that COX-2 expression may also be regulated by stabilization of mRNA (26, 56). Assembly of C5b-9 could thus potentially enhance COX-2 mRNA stability in GEC. This might account for the discrepancy between relatively modest COX-2 promoter activation (~1.8-fold) and more pronounced mRNA upregulation (~4-fold) (49). The potential effect of complement on RNA stability should be addressed in future studies.

A number of studies support an important role for prostanoids in the mediation of proteinuria in experimental (49) and human membranous nephropathy (11). The potential role for COX-2 in the pathogenesis of proteinuria was highlighted by our earlier study (49), and it was recently reported that a COX-2-selective inhibitor, flosulide, reduced proteinuria in PHN, although the inhibitory action of flosulide may not have occurred solely via COX-2 inhibition (2). Elucidation of the pathways of C5b-9-induced regulation of COX-2 gene expression will provide further insights into the pathogenesis of membranous nephropathy and other forms of glomerulonephritis, as well as different types of C5b-9-mediated tissue injury involving prostanoids (30, 34, 44), and will eventually allow for development of novel therapeutic approaches.


    ACKNOWLEDGEMENTS

We thank Drs. Kei Yamamoto, Shozo Yamamoto, Roger Davis, Albert Descoteaux, and JoAnne Richard for gifts of plasmids.


    FOOTNOTES

First published July 12, 2001; 10.1152/ajprenal.0048.2001

This study was supported by grants from the Medical Research Council of Canada (to T. Takano and A. V. Cybulsky) and the Kidney Foundation of Canada (to T. Takano and A. V. Cybulsky). T. Takano is a recipient of a Medical Research Council of Canada Scholarship. A. V. Cybulsky is a recipient of a Senior Scholarship from the Fonds de la recherche en santé du Québec.

Address for correspondence: T. Takano, Nephrology Research, McGill Univ., 3775 Univ. St., Rm. 236, Montreal, Quebec, Canada H3A 2B4 (E-mail: ttomok{at}po-box.mcgill.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 February 2001; accepted in final form 25 June 2001.


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Am J Physiol Renal Fluid Electrolyte Physiol 281(5):F841-F850
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