Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada H3A 2B4
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ABSTRACT |
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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-
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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INTRODUCTION |
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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)-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 AND METHODS |
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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). [-32P]ATP (3,000 Ci/mmol), [
-32P]dCTP (3,000 Ci/mmol), and
[
-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-
B p65 subunit antibody were from
Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids.
Mouse COX-2 promoter constructs pGC815 and pGC815NF-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-
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
-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
-glycerophosphate, pH
7.6, 10 mM MgCl2, 1 mM Na3VO4, 20 µM ATP, and 5 µCi [
-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 [-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-B consensus binding site (5'-AGTTGAGGGGACTTTCCCAGG-3') was
radiolabeled by use of T4 polynucleotide kinase and
[
-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-
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-
B p65 subunit antibody was incubated with
the reaction mixture for 10 min before addition of radiolabeled NF-
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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RESULTS |
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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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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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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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Complement-induced COX-2 upregulation is not mediated by NF-B.
Another pathway that has been reported to regulate COX-2 is NF-
B. To
monitor activation of NF-
B, GEC were incubated with antibody and
complement, and translocation of NF-
B from the cytosol to the
nucleus was evaluated by EMSA, using 32P-labeled
NF-
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-
B p65
antibody, indicating that these bands are specific to NF-
B. The
bottom band was not supershifted by this antibody,
suggesting that it is nonspecific (Fig. 5B).
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DISCUSSION |
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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- isoform increased COX-2
expression in rat mesangial cells (29). In other systems,
the classic PKC-
isoform contributes to COX-2 regulation
(51). Rat GEC were shown to express PKC-
, -
, -
,
and -
isoforms (20). Because the atypical PKC-
isoform is not responsive to PMA, the results of the present study
suggest that PKC-
, -
, and/or -
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-B binding site(s) (9, 23, 46, 55).
Therefore, it was surprising that although NF-
B was activated by
complement in GEC (Fig. 5), NF-
B did not contribute to COX-2
upregulation. However, such a result is not without precedent. For
example, the NF-
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-
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/EBP
),
AP-2, SP1, and NF-
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-
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.
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ACKNOWLEDGEMENTS |
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We thank Drs. Kei Yamamoto, Shozo Yamamoto, Roger Davis, Albert Descoteaux, and JoAnne Richard for gifts of plasmids.
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FOOTNOTES |
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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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