0022-3565/00/2921-0271$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 292:271–279, 2000
Vol. 292, No. 1
Printed in U.S.A.
Diphenyleneiodium Chloride Blocks Inflammatory CytokineInduced Up-Regulation of Group IIA Phospholipase A2 in Rat
Mesangial Cells1
GLENN DORSAM, MOHINDDIN M. TAHER, KRISTOFFER C. VALERIE, NANCY B. KUEMMERLE, JAMES C. CHAN, and
RICHARD C. FRANSON
Departments of Biochemistry and Molecular Biophysics (G.D., R.C.F.), Radiation Oncology (M.M.T., K.C.V.), and Pediatric Nephrology (N.B.K.,
J.C.C.), School of Medicine, Virginia Commonwealth University, Richmond, Virginia
Accepted for publication August 27, 1999
This paper is available online at http://www.jpet.org
Phospholipases A2 (E.C. 3.1.1.4) are lipolytic enzymes that
catalyze bond cleavage at the sn-2 position of glycerolphospholipids that produce cis-unsaturated fatty-acids and the
corresponding lysoderivative (Dennis, 1997). Both enzymatic
products are membrane perturbing due to their amphipathic
characteristics and can be precursors for potent inflammatory mediators such as eicosanoids and platelet-activating
factor (Dennis, 1997). Phospholipase A2 (PLA2) enzymes are
either calcium dependent or independent with respect to
enzymatic activity and are expressed both as cell-associated
and secretory forms (Kramer, 1993; Dennis, 1994; Tischfield,
1997). In mammals, five calcium-dependent PLA2 isozymes
Received for publication June 10, 1999.
1
Funded in part by Azimuth Pharmaceuticals and the Department of
Biochemistry and Molecular Biophysics, Virginia Commonwealth University,
Richmond, VA 23298. The laboratory of Richard Franson was the principle
location were this work was performed.
test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state
levels of group IIA PLA2 mRNA and extracellular enzymatic
activity over time. DPI (0.02–20 mM), added 90 min before
cytokine activation, inhibited both group IIA PLA2 mRNA and
enzymatic activity in a concentration-dependent manner. By
electrophoretic mobility shift analysis, cytokine activation also
increased specific NF-kB binding to one of two NF-kB consensus elements in the rat group IIA PLA2 promoter and also was
suppressed by DPI pretreatment. Antibodies to NF-kB p65 (Rel
A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-kB species as the
classical p50/p65 heterodimer.
have been identified: the cytosolic group IV PLA2 (Kramer
and Sharp, 1997) and four closely related secretory forms
group I (Seilhamer et al., 1986), group IIA (Seilhamer et al.,
1989), group IIC (Tischfield, 1997), and group V PLA2
(Tischfield, 1997). Group III is found in bee venom, whereas
group IIB is found only in snake venom (Tischfield, 1997).
The 14-kDa group IIA PLA2 is up-regulated by inflammatory
cytokines such as interleukin 1b (IL-1b) and tumor necrosis
factor-a (TNF-a) (cytokine) in numerous cells, including astrocytes (Oka and Arita, 1991), vascular smooth muscle cells
(Nakama et al., 1990), chondrocytes (Kerr et al., 1989), hepatoma cells (Crowl et al., 1991), and mesangial cells (Nakazato et al., 1991). Only enzymatically active group IIA PLA2
is proinflammatory and elicits edema when injected into
animal paws (Vadas et al., 1989). In addition, group IIA PLA2
is mitogenic as demonstrated by the proliferation of synovial
cells when injected into joints of experimental animals (Va-
ABBREVIATIONS: PLA2, phospholipase A2; IL-1b, interleukin 1b; TNF-a; tumor necrosis factor-a; NF-kB, nuclear factor- kB; ROS, reactive
oxygen species; AA, arachidonic acid; DPI, diphenyleneiodium chloride; FBS, fetal bovine serum; GM, growth media; DMSO, dimethyl sulfoxide;
RT, room temperature; LSM, low-serum media; ACM, activation media; LDH, lactate dehydrogenase.
271
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ABSTRACT
Inflammatory cytokines, interleukin 1b and tumor necrosis factor-a, potently stimulate rat mesangial cells to express and
secrete group IIA phospholipase A2 (PLA2). Cytokine-induced
up-regulation of PLA2 has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kB (NF-kB),
suggesting a role for NF-kB in the regulation of group IIA PLA2
expression. Reactive oxygen species such as H2O2, which are
elevated in mesangial cells after cytokine activation, can mimic
cytokine-induced NF-kB activation. However, the source of
reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently,
tumor necrosis factor-a has been demonstrated to increase
superoxide radical generation in mesangial cells. Therefore, we
hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced
group IIA PLA2 up-regulation by attenuating NF-kB binding. To
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Dorsam et al.
p50/p65 NF-kB binding to one of two NF-kB elements in the
rat PLA2 promoter.
Experimental Procedures
Materials
Human recombinant IL-1b and TNF-a were purchased from R&D
Systems (Minniapolis, MN). Fetal bovine serum (FBS), glutamine,
penicillin, streptomycin, Trizol, and random priming kit were purchased from Life Technologies (Grand Island, NY). Human mesangial cells and media for their growth were purchased by Clonetics
(Walkersville, MD). All tissue culture flasks, dishes, and insulin/
transferin/selenium were purchased from Becton Dickinson (Franklin Lakes, NJ). Antibodies for cytokeritin, von Willebrand factor,
smooth muscle actin, and Thy 1.1 were purchased from Accurate
Scientific (Westbury, NY). DPI was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Standard saline citrate buffer (103) for
Northern analysis was purchased from Promega (Madison, WI). Nitrocellulose was purchased from Bio-Rad (Hercules, CA). Radionucleotides were purchased from Amersham Corp. (Piscataway, NJ).
Oligomers were synthesized by Genosys (The Woodlands, TX). Stainless steel meshes and all other chemicals were purchased from
Sigma Chemical Co. (St. Louis, MO).
Mesangial Cell Isolation
Kidneys from sacrificed male Lewis Sprague-Dawley rats (150 g)
were removed, placed in Hanks’ solution, and the capsule was removed. All steps were performed aseptically at 4°C unless otherwise
stated. The kidney was bivalved and the cortex was separated from
the medula. Also, corticol tissue was minced with a glass pestle in
Hanks’ solution and passed through a 280-mm stainless steel mesh.
The suspension of cortex tissue was passed 15 times through a
21-gauge needle and sequentially passed through staineless steel
meshes of 280, 190, 140, 90, and 73 mm, respectively. Isolated glomeruli that did not pass through the 73-mm mesh were washed three
times with Hank’s solution and collected by inverting the mesh and
washing with Hanks’ solution. By light microscopy, isolated glomeruli appeared spherical, and little-to-no tubular or arteriole tissue
was present. Glomeruli were centrifuged for 5 min at 750g and the
pelleted glomeruli saved. Two thousand glomeruli per milliliter were
digested with 0.1% collagenase for 30 min at 37°C to remove epithelial cells and centrifuged at 50g for 5 min. The pellet, containing
intact glomeruli void of most epithelial cells, was resuspended in
growth media (GM) consisting of 82% RPMI 1640, 17% fetal calf
serum, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenium, 2 mM
glutamine, 60 mg/ml penicillin, and 60 mg/ml streptomycin. Cells
were plated in a volume of media that covered only one-third the area
of a 100-mm tissue culture dish and incubated at 37°C with 5% CO2.
After 5 h, attached glomeruli (10 –20%) were washed with PBS, and
6 ml of fresh GM was added.
Mesangial Cell Selection and Culture
Isolated rat mesangial cells were grown in GM that was changed
every 48 h for the first three passages and every 72 h for passages 4
to 15. To pass cells, 0.025 to 0.5% trypsin in Hanks’ solution or PBS
was added to detach cells, centrifuged at 300g, and split 1:4. A
passage refers to approximately four to five doublings. To select for
mesangial cells, isolated cells were 1) grown in D-valine, which inhibits fibroblast proliferation until passage 6; 2) treated for 24 h with
10 mg/ml puromycin, which is toxic to epithelial and endothelial cells;
and 3) incubated twice with 0.5% FBS in RPMI 1640 media for 48 h
(twice), which detaches epithelial and endothelial cells. Surviving
cells in passages 9 to 15 were used for these experiments. Cells
(3–5 3 106) were frozen in 50% GM, 40% FBS, and 10% dimethyl
sulfoxide, and stored in liquid nitrogen. Cells used in these experiments were frozen up to three times with no noticeable change in
their ability to respond to cytokine.
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das et al., 1989), and the activation of p42/p44 MAP kinase in
mesangial cells (Sugiura, 1995).
Group IIA PLA2 (subsequently referred to as PLA2) is
thought to play a pivotal role in the pathogenesis of inflammatory kidney diseases such as glomerulonephritis (Wada et
al., 1997) due to the enzyme’s proinflammatory (Vadas et al.,
1989) and mitogenic properties (Vadas et al., 1989; Arita et
al., 1991). Indeed, a hallmark of glomerulonephritis is the
proliferation of mesangial cells, a resident kidney cell, within
the inflammed glomerulus (Sedor, 1992). Cytokines, which
may trigger the onset of glomerulonephritis (Lan et al.,
1995), up-regulate the synthesis and secretion of PLA2 by
cultured mesangial cells (Nakazato et al., 1991). Therefore,
delineating the intracellular signaling mechanism(s) for cytokine regulation of PLA2 may lead to future therapeutic
drugs aimed at ameliorating the toxic consequences of glomerulonephritis.
Nuclear factor-kB (NF-kB), a cytoplasmically sequestered
transcription factor, is activated by cytokines that up-regulate the expression of many inflammatory genes such as IL-b,
TNF-a, and IL-6 (Baeuerle and Baltimore, 1996). Cytokine
activation of cells, including mesangial cells, increases intracellular reactive oxygen species (ROS) such as O 2–. , H2O2, and
OH z (Radeke et al., 1990; Feng et al., 1995). Exogenously
added H2O2 activates NF-kB in cultured cells, indicating
ROS as second messengers for inflammatory cytokine signal
transduction (Radeke et al., 1990; Feng et al., 1995). Antioxidants and/or heavy metal chelators have successfully
blocked inflammatory cytokine-induced NF-kB activation
and PLA2 up-regulation (Walker et al., 1995). However, a
direct link demonstrating NF-kB as a necessary trans-acting
factor regulating transcriptional control for PLA2 has yet to
be demonstrated. Therefore, we investigated whether NF-kB
binds to the rat PLA2 promoter.
Cytokines have been demonstrated to increase ROS from
mitochondria by an acidic-sphingomylinase-dependent pathway (Schutze et al., 1992). However, embryonic fibroblast
cells lacking acidic sphingomylinase (asmase 2/2) nevertheless demonstrated activated NF-kB upon cytokine treatment
(Zumbansen and Stoffel, 1997). This finding suggests that
cellular sources other than mitochondrial enzymes may be
responsible for cytokine-induced ROS generation and subsequent NF-kB activation. Numerous other cellular sources
could potentially contribute to cytokine-induced ROS generation instead of mitochondrial enzymes, such as P-450 complex, nitric oxide synthetase, xanthine oxidase, arachidonic
acid (AA) metabolism, and NADPH oxidase (Johnson and
Nashr-Esfahani, 1993). Indomethacin (prostaglandin inhibitor) did not block cytokine-induced activation of NF-kB and
PLA2 expression in mesangial cells, implying that prostaglandin metabolism is not a major contributer to ROS generation (Vervoordeldonk et al., 1996). Conversely, TNF-a
treatment of mesangial cells has been demonstrated to generate superoxide radical (O 2–. ,) production (Radeke et al.,
1990). Therefore, we investigated whether a selective
NADPH oxidase inhibitor (O’Donnell et al., 1993), dipheneyleneiodium chloride (DPI), could block cytokine up-regulation
of PLA2 by inhibiting NF-kB activation.
In this study, a primary mesangial cell line provided evidence that DPI may block cytokine-induced up-regulation of
PLA2 in a concentration-dependent manner by suppressing
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2000
DPI Blocks Cytokine-Induced Up-Regulation of Group IIA PLA2
273
Measurement of PLA2 Activity
Confluent rat mesangial cells grown in 35-mm dishes were fixed
with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and
stored at 4°C until processed. The fixed cells were washed twice for
5 min at 4°C with 0.1 M sodium cacodylate buffer, pH 7.4. Cells were
postfixed for 1 h at 4°C with 2% osmium tetroxide in 0.1 M cacodylate
buffer, pH 7, followed by one wash (as above). Cells were then
dehydrated with incubations of 50, 70, 80, and 95% ethanol for 10
min at 4°C and then with 100% ethanol for 20 min at room temperature (RT), repeating twice.
Cells were immersed three times with hexamethyldisilizane for 20
min at RT, and air dried. The cells were mounted on scanning
electron microscope stubs with silver paint, coated with gold-palladium, and stored in a desiccator.
PLA2 activity was measured by an established method that used
autoclaved 1-[14C] oleate-labeled Escherichia coli (E. coli; Dorsam et
al., 1995). The reaction mixtures contained: 10 nmol of labeled E. coli
phospholipid (4000 – 6000 cpm), 1 mM CaCl2, 100 mM Tris, pH 7.4,
and 100 ml of conditioned mesangial cell media. Media was diluted
appropriately with ACM to ensure linear kinetics. Samples were
extracted by the following procedure. Reactions were stopped with 3
ml of chloroform/methanol (1:2 v/v) and vortexed briefly. One milliliter of chloroform and 4 ml of H2O were added and vortexed for 15 s.
The samples were centrifuged at 500g for 5 min at RT. The organic
layer was collected and dried under nitrogen gas at 37°C. The remaining lipid residue was dissolved in 65 ml of chloroform/methanol
(1:9 v/v) and separated on silica plates with a running buffer of 78.5%
petroleum ether, 19% ethyl ether, and 2.5% glacial acetic acid.
The regions corresponding to fatty acid and phospholipid were
scraped into scintilation vials containing nonaqueous scintillation
cocktail, and the counts per minute of 14C was measured. Hydrolysis
was calculated as the counts per minute of oleate divided by the sum
of counts per minute of oleate and E. coli phospholipid. This ratio
was multiplied by the dilution factor, 10 nmol of phospholipid, time
(minutes), and volume assayed (milliliters) to obtain an enzymatic
rate with units of nanomoles of phospholipid hydrolyzed per minute
per milliliter.
Immunohistochemistry
Northern Analysis
Para-formaldehyde-perfused rat kidney cortex cross sections were
used as positive staining controls for cytokeratin, von Willebrand
factor, smooth muscle actin, and Thy 1.1, whereas fixed human
mesangial cells were used for positive vimentin staining (data not
shown). Isolated rat mesangial cells grown on glass plates were fixed
with 4% para-formaldehyde in 0.1 M phosphate buffer, pH 7.2, and
stained by the following procedure. Fixed cells were washed three
times with PBS for 5 min, blocked with 1.5% serum (horse for mouse
monoclonal antibodies, goat for rabbit polyclonal antibodies, 5% BSA
for von Willebrand factor), 0.3% Triton X-100 for 1 h, and washed as
described above. Primary monoclonal and polyclonal antibodies were
used at a concentration between 1 and 5 mg/ml in PBS, 0.5% serum
(1.7% BSA for von Willebrand factor), and incubated for 1 h at RT.
Cells were washed (as described above) and the appropriate biotinylated secondary antibody applied for 1 h. Hydrogen peroxide
quenching was performed with 0.3% H2O2 for 30 min. A biotinylated
avidin complex was incubated next for 1 h, washed as described
above, and washed again with 0.1 M Tris, pH 7.6, three times.
One-half milligram per milliliter 3,3-diaminobenzidine in 0.05%
H2O2, Tris, pH 7.6, was incubated with cells for 2 to 10 min and
immediately dehydrated, rehydrated, counterstained with hematoxylin, dehydrated, and mounted. Peroxidase staining was visualized
by a reversed-phase Nikon microscope.
Confluent mesangial cells in T-75 flasks were treated with cytokine for the indicated time interval, which were lysed by TRIsol, and
total RNA isolated per manufacturer’s instructions. RNA concentration was measured by spectrophotometry (260 nm) and 10 to 25 mg of
total RNA was loaded per lane on a 1.25% agarose, 2.2 M formaldehyde gel, and electrophoresed overnight with 15 V (constant voltage)
for 16 h at RT. The RNA gel was removed, blotted (16 h) by capillary
action with 103 standard saline citrate buffer onto nitrocellulose
and baked for 2 h at 80°C. The distance of ribosomal RNA migration
was measured. Group IIA PLA2 transcript is ;800 base pairs.
Twenty nanograms of group IIA PLA2 cDNA and b-actin (control
gene) were labeled with alpha [32P]dCTP by the random priming
procedure. Percentage of incorporation of radiolabeled nucleotide
was between 15 and 50%, and the entire synthesized probe was used
for hybridization studies.
The nitrocellulose membrane was blocked with salmon sperm
DNA in 40 mM phosphate buffer, 1% SDS, and 5 mM EDTA for 1 h
at 65°C in a hybridization oven (25a). The buffer was decanted and
fresh buffer with alpha [32P]dCTP-labeled probe was added to the
membrane and incubated for 16 h. Membranes were exposed to
X-ray film with an intensifying screen at 280°C until developed (24
h) or by phosphoimager.
Transmission Electron Microscopy
Cells were immersed three times in 100% propylene oxide for 20
min at RT, 50% propylene oxide in 50% poly/bed 812 embedding
media (v/v) overnight with constant mixing, followed by pure poly/
bed 812 embedding media (v/v) overnight with constant mixing.
These prepared cells were embedded into capsules and heated at
60°C for 3 days.
Scanning Electron Microscopy
Mesangial Cell treatment with IL-1b and TNF-a
Confluent mesangial cells between passages 9 and 15 were grown
to confluency with GM in 24-well plates, T-25 or T-75 flasks. GM was
aspirated and cells washed twice with PBS, pH 7.4. Low-serum
media (LSM), consisting of MsBM media, 0.5% FBS, and 12.5 mM
HEPES, was added to cells for 48 h. LSM was aspirated and cells
were washed twice with PBS. Activation media (ACM), consisting of
LSM but with 0.1% BSA (fraction V) substituted for FBS, was added
with 100 pM IL-1b and TNF-a or PBS and 0.1% BSA, for the
indicated time intervals. For experiments with DPI, cells were incubated before the addition of cytokine in ACM with freshly prepared
DPI (0.3% DMSO) or 0.3% DMSO for 90 min, aspirated, washed
twice with PBS, and then fresh ACM was added. After cytokine
incubation, media was collected, centrifuged at 2000g for 5 min at
4°C, and stored at 220°C until assayed. For total RNA isolation, cells
were washed twice with PBS, released with 0.5% trypsin and lysed
with TRIsol per manufacturer’s instructions.
Electrophoretic Mobility Shift Assay
Annealing of Oligomers. Synthesized sense and antisense DNA
oligomers were annealed by placing equal molar amounts in a 1.5-ml
microfuge tube, heated to 90°C, and allowed to cool slowly. The
annealed oligo was electrophoresed on a 20% acrylamide gel with 150
V. Probe was visualized by UV-shadowing and eluted in 10 mM Tris
and 1 mM EDTA (TE buffer) overnight at 4°C, and absorbance
values (260 nm) were measured to determine oligonucleotide concentration. Probe was stored at 220°C until needed.
Isolation of Nuclear Extracts. Confluent rat mesangial cells in
T-75 flasks were treated with cytokine for the indicated time intervals. Cells were washed, scraped with ice-cold PBS, and collected by
centrifugation (300g). All subsequent steps were conducted at 4°C
unless otherwise noted. Cells were lysed with 100 ml of lysis buffer
containing 20 mM Tris, pH 7.4, 140 mM NaCl, 1.5 mM MgCl2, 1 mM
EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.5% NP-40, 0.5 mM
sodium orthovandadate, 1 mM aprotinin, 1 mM leupeptin, and 1 mM
phenylmethylsulfonyl fluoride, and centrifuged at 12,000g for 5 min.
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Electron Microscopy
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Dorsam et al.
Viability Analysis
Lactic Dehydrogenase Activity Assay. Lactate dehydrogenase
(LDH) activity in rat mesangial cellular extracts and mesangial cells
conditioned media was measured. Briefly, 0.1 M NADH, 0.1 mg/ml
sodium pyruvate, PBS, pH 7.4, was incubated with mesangial cellular extracts or mesangial cells conditioned media (5–10% total volume) for 20 min at RT, and the decrease in absorbance at 340 nM
was monitored with a spectrophotometer. All measurements ensured
linear kinetics because two enzyme concentrations were analyzed
per sample. Twenty micromolar DPI pretreatment (90 min) did not
significantly modulate LDH activity levels compared with control
and were always ,10% total LDH activity (standardized for cell
number).
Trypan Blue Exclusion. On ice, cells were resuspended in
RPMI, diluted (1:2) with 0.2% trypan blue, and counted on a hemocytometer. Up to a 20 mM DPI pretreatment (90 min) did not significantly modulate the extent of trypan blue exclusion compared with
control after 24 h, and all experiments consisted of $85% viable cells.
Statistical Analysis
SAS institute (version 6) statistical procedures were preformed by
the Department of Biostatistics at Virginia Commonwealth University. Percentage of inhibition by DPI verses control was analyzed for
statistical significance by one-way parametric analysis. A P value of
,.05 was considered statistically significant.
Results
Mesangial Cell Characterization. Isolated glomerular
cells used for these experiments required extensive characterization to properly identify a mesangial cell phenotype
and to determine the homogeneity of the cell culture. Surviving cells in passages 9 to 15 showing an appropriate stellate
mesangial cell morphology (Radeke et al., 1990) were characterized ultrastructurally and immunochemically. Consistent with a mesangial phenotype, scanning and transmission
electron micrographs in Fig. 1 confirmed a large nucleus to
cytoplasm ratio, numerous nucleoli, a distinct rough endoplasmic reticulum, and Golgi apparatus (Lan et al., 1995).
Mesangial cells are mesanchymal in origin and have smooth
muscle characteristics and therefore express vimentin and
smooth muscle actin (Radeke et al., 1990). In addition, it has
recently been reported that rat mesangial cells are positive
for the plasma membrane protein Thy 1.1 (Radeke et al.,
1990). Table 1 shows the high percentage of cells stained
positive for vimentin, smooth muscle actin, and Thy 1.1.
Endothelial and epithelial cell markers, von Willebrand factor, and cytokeratin did not stain these cells compared with
control. All aforementioned antibodies positively stained per-
Fig. 1. Electron microscopy of isolated
mesangial cells. A, scanning electron micrograph of a confluent mesangial cell
monolayer. Arrows indicate large nucleus
(circle) with numerous nucleoli (smaller
circles). B, transmission electron micrograph of two mesangial cells. Arrows indicate the presence of a distinct Golgi aparatus and endoplasmic reticulum. All of
these characteristics are representative of
a mesangial cell phenotype.
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The pelleted nuclei were washed twice with 1 ml of lysis buffer
lacking NP-40 and resuspended in 50 ml of nuclear extraction buffer
containing 250 mM Tris, pH 7.8, 60 mM KCl, 1 mM EDTA, 1 mM
EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride,
and 0.5 mM sodium orthovanadate. After freezing and thawing three
times, the nuclei were centrifuged at 12,000g for 15 min and the
supernatants (nuclear extracts) were frozen until assayed. Protein
concentrations were determined by the Bradford method.
DNA Binding Assay. NF-kB consensus sequences (90% homologous NF-kB sequence, 59-GGAAAGGGGAAATTACCAGG-39; 80%
homologous NF-kB sequence, 59-GCGTTGGGTATGCCCGTCTG-39;
Promega NF-kB consensus sequence, 59-GGAAAGGGACTTTCCGTCTG-39) were labeled with T4 polynucleotide kinase with
[g-32P]ATP and incubated for 30 min at RT. Unincorporated nucleotide was removed by phenol/chloroform extraction and ethanol precipitation. The binding reaction consisted of 5 mg of protein, 5%
glycerol, 1 mg of poly(dI-dC), and 0.1 ng 32P-labeled NF-kB oligonucleotide (binding reaction), which was incubated for 30 min at RT.
The protein-DNA complexes were then resolved on a 5% polyacrylamide gel electrophoresis gel, 0.5 3 Tris-borate-EDTA buffer with 40
V. Gels were dried in a glycerol/ethanol mixture and exposed to X-ray
film or a phosphoimager screen.
Competitive studies were conducted by adding increasing concentrations of unlabeled probe to the binding reaction. The effect of DPI
on NF-kB binding was carried out by exogenously adding DPI to the
binding reaction (minus labeled probe) for 10 min at RT before
adding the labeled probe. Supershift analysis used 1 mg of anti-p65,
-p50, or normal rabbit IgG added to the binding reaction for 30 min
at RT.
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DPI Blocks Cytokine-Induced Up-Regulation of Group IIA PLA2
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TABLE 1
Immunochemical characterization of isolated primary rat mesangial
cells
Three cellular proteins were used as markers to establish a mesangial cell phenotype
(Thy 1.1) with a mesanchymal (vimentin) and myogenic (smooth muscle actin) origin.
von Willebrand factor and cytokeratin, endothelial, and epithelial cell markers
respectively, were used as negative controls. Percentage of cells is the ratio of
positively stained cells per 100 nuclei (hematoxylin-stained) multiplied by 100.
%
Vimentin
Smooth muscle actin
Thy 1.1
Von Willebrand factor
Cytokeratin
96
98
97
#3
#3
Fig. 2. Biochemical characterization of mesangial cells. Confluent mesangial cell monolayers cultured in 24-well plates or T-75 flasks were
incubated with 0.5% serum for 48 h. Mesangial cells were treated with
100 pM IL-1b and TNF-a for various time intervals. A, accumulation of
group IIA PLA2 enzymatic activity in conditioned media over time (50 h).
Results are shown as means 6 S.E. from three independent experiments.
B, accumulation of group IIA PLA2 steady-state mRNA levels over time
(50 h). Group IIA PLA2 message is ;800 base pairs. This blot was
subsequently probed for b-actin and is a representative of four similar
experiments. C, concentration curve for IL-1b, TNF-a, and both IL-1band TNF-a -induced up-regulation of group IIA PLA2 enzymatic activity.
Cytokine concentrations between 1 and 1000 pM were incubated with
mesangial cells for 24 h and group IIA PLA2 enzymatic content measured.
Results are shown as means 6 S.E. from four independent experiments.
for directly enhancing the transcriptional rate of PLA2, we
analyzed 500 base pairs of the rat PLA2 promoter region for
known transcription factor-binding motifs (Seilhamer et al.,
1989). This analysis revealed two potential NF-kB-binding
sites located at 112 and 131 base pairs upstream from the
transcriptional start site (Fig. 4A). The 2112 motif is 80%
homologous, and the 2131 motif is 90% homologous to the
known NF-kB (p50/p65) consensus sequence. Interestingly,
as seen in Fig. 4C, electrophoretic mobility shift analysis
demonstrated no detectable retardation signal with the 2112
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fused rat kidney sections or human mesangial cells with an
appropriate staining pattern (data not shown).
Biochemical Characterization of Mesangial Cells.
Biochemical characterization of cultured mesangial cells was
performed by treatment with 100 pM IL-1b and TNF-a for
50 h. Figure 2A illustrates the enzymatic increase in PLA2
activity from conditioned medium as measured by 1-[14C]
oleate-labeled E. coli. Secreted PLA2 activity was not detected from cells treated with vehicle alone. PLA2 mRNA
levels showed a time-dependent increase similar to PLA2
enzymatic activity as measured by Northern analysis (Fig.
2B), indicating that the increase in secreted enzymatic activity is associated with an elevation in steady-state levels of
PLA2 mRNA. Figure 2C illustrates the concentration dependence of cytokine on secreted PLA2 enzymatic activity. IL-1b
or TNF-a alone increased the expression of PLA2, whereas
both IL-1b and TNF-a up-regulated enzyme expression and
subsequent secretion to a greater extent than either one
alone. Synergistic up-regulation by both cytokines for PLA2
(mRNA, protein mass and enzymatic activity) has been previously demonstrated (Konieczkowski and Sedor, 1993). IL-2
and IL-4 did not activate rat mesangial cells based on secreted PLA2 enzymatic levels in conditioned media (data not
shown).
Diphenyleneiodium Chloride Inhibits Cytokine-Induced PLA2 Expression. Vervoordeldonk et al. (1997) have
reported that cytokine-induced up-regulation of PLA2 in rat
mesangial cells is blocked by the antioxidant pyrrolidine
dithiocarbamate, a known NF-kB inhibitor. Pyrrolidine dithiocarbamate lowered nuclear NF-kB binding to its consensus sequence by presumably neutralizing the elevation of
ROS generation in response to cytokine and inhibiting
NF-kB activation (Baeuerle and Baltimore, 1996). However,
the cellular source for cytokine-induced ROS generation in
mesangial cells is not clearly defined. Recently, NADPH oxidase has been implicated as a potential source for cytokineinduced generation of ROS (Radeke et al., 1990). Therefore,
DPI, a selective inhibitor of NADPH oxidase (O’Donnell et
al., 1993), was used and its effect on cytokine-induced upregulation of PLA2 was investigated. Figure 3A illustrates
the effects of a 90-min exposure to DPI on the up-regulation
of PLA2 in a range of 0.02 to 20 mM. The decrease in PLA2
enzymatic activity agreed well with a concomitant reduction
in mRNA transcript as seen in lanes 3 and 4 of Fig. 3B. These
data demonstrate that DPI pretreatment suppress steadystate levels of PLA2 mRNA, resulting in a decrease in subsequent enzyme synthesis and secretion.
Mechanism of Inhibition for DPI on PLA2 Up-Regulation. To determine whether NF-kB was a viable candidate
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Dorsam et al.
sequence from cells activated with cytokine. However, the
2131 sequence as well as a commercially available NF-kB
consensus probe (Fig. 4, B and D; Promega) resulted in the
detection of a retardation signal that peaked at ;40 min and
persisted for 24 h. Cells pretreated with DPI (0.2 and 20 mM)
and then activated with cytokine for 1 and 24 h showed a
decrease in this retardation signal when the 2131 probe and
the NF-kB probe were used (Fig. 4, B and D; lanes 5 and 6
compared with 4, and lanes 8 and 9 compared with 7). In
addition, the decrease in NF-kB binding observed at 60 min,
due to DPI pretreatment, may account for the decrease in
steady-state levels of PLA2 mRNA and enzymatic activity as
seen in Fig. 3, A and B. These data suggest that DPI blocks
protein binding to an NF-kB element found in the rat PLA2
promoter and may explain the observed inhibition by DPI on
cytokine-induced up-regulation of this mitogenic and proinflammatory enzyme.
Fig. 4. Inhibitory effect of DPI on NF-kB binding. A, schematic diagram
of the rat group IIA PLA2 promoter region indicating the location and
sequence of two potential NF-kB-binding sites sharing 90 (2131) and 80%
(2112) homology to the classical p50/p65 NF-kB consensus sequence.
B–D, mesangial cells were pretreated with 0.3% DMSO or DPI (0.3%
DMSO) for 90 min, washed thoroughly with PBS (twice), and incubated
with 100 pM IL-1b and TNF-a for the indicated time intervals in minutes
(lanes 1– 6) and hours (lanes 7–9). Lanes 5 and 6 and 8 and 9 are nuclear
extracts from cells pretreated, before cytokine addition, with 0.2 and 20
mM DPI, respectively. Nuclear extracts were isolated as described in
Experimental Procedures and incubated with g[32P]ATP-labeled 2131
sequence (B), 2112 sequence (C), and NF-kB consensus sequence (D).
Identification of Protein Species Binding to 2131
NF-kB Sequence. Competition and supershift studies were
conducted to identify the protein(s) responsible for the observed retardation signal. Nuclear extracts were isolated
from cells pretreated with DPI or vehicle for 90 min, washed
to remove media, and treated with 100 pM IL-1b and TNF-a
for 40 min. Protein binding to the 2131 sequence was competed with unlabeled NF-kB consensus sequence and 2131
sequence (Fig. 4, B–D) but not with unlabeled 2112 sequence
(data not shown), indicating the specificity of this protein/
DNA interaction. Polyclonal antibodies for p50 and p65
NF-kB subunits supershifted this retardation signal,
whereas normal rabbit IgG did not (Fig. 5B). These data
demonstrate that the protein species binding to the 2131
sequence in the rat PLA2 promoter is the classical NF-kB
heterodimer p50/p65, and suggest that DPI suppresses
NF-kB binding at this site, thereby decreasing transcriptional activation.
Discussion
The response of mesangial cells to individual cytokines is
well documented. Typically, these studies use nanomolar
concentrations. In contrast, both IL-1b and TNF-a were used
in these experiments instead of a single cytokine treatment
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 3. Inhibitory effect of DPI on cytokine-induced up-regulation of
group IIA PLA2. Confluent mesangial cell monolayers grown in T-75
flasks were incubated with 0.5% serum for 48 h for this and all subsequent experiments. Mesangial cells were washed and pretreated with
0.3% DMSO or varying concentrations of DPI (0.3% DMSO; 0 –20 mM) for
90 min, washed thoroughly with PBS (twice), and treated with 100 pM
IL-1b and TNF-a for 24 h. A, effect of DPI on group IIA PLA2 enzymatic
activity. Conditioned media were collected and assayed for group IIA
PLA2 enzymatic activity as discussed in Experimental Procedures. Shown
are the means 6 S.E. from eight independent experiments (*P , .05
versus cytokine alone). B, effect of DPI on group IIA PLA2 steady-state
mRNA levels. Total RNA was isolated with Trizol, separated by size on a
1.25% agarose gel electrophoresis, and hybridization to group IIA PLA2
message was analyzed. Lanes 3 and 4 represent cells pretreated for 90
min before cytokine activation with 0.2 and 20 mM DPI, respectively. The
blot was subsequently probed for b-actin and is a representative of three
similar results.
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2000
DPI Blocks Cytokine-Induced Up-Regulation of Group IIA PLA2
for two reasons. First, both cytokines are direct mediators of
glomerulonephritis and are present simultaneously during
the progression of this disorder (Sedor, 1992; Lan et al.,
1995). Therefore, the addition of IL-1b and TNF-a at subnanomolar concentrations is not only physiologically relevant
but also mimics more closely the environment of the inflammed glomerulus (Sedor, 1992). Second, although there
are many similarities between IL-1b and TNF-a-induced signal transduction, there are definite differences pertaining to
elicited cellular responses (Feng et al., 1995). Thus, inhibiting a cellular response induced by IL-1b may not inhibit
TNF-a responses, and conversely. However, inhibiting the
synergistic effects induced by both cytokines may be more
meaningful in regard to glomerulonephritis.
The classical p50/p65 NF-kB binds to a 90% homologous
NF-kB element located at position 2131 in the rat PLA2
promoter but not to an 80% homologous NF-kB element
located at position 2112. To our knowledge, this is the first
demonstration of specific NF-kB binding to identical DNA
sequences found in the promoter of rat PLA2. The affinity of
p50/p65 for the 2131 sequence appears less than that for the
NF-kB consensus sequence as demonstrated in the competition study because less unlabeled NF-kB consensus sequence
was needed to completely ablate the retardation signal compared with unlabeled 2131 sequence. This is an expected
consequence due to the 2131 sequence sharing only 90%
homology with the NF-kB consensus sequence. Furthermore,
Konieczkowski and Sedor (1993) demonstrated that IL-1b
increased the transcriptional rate for PLA2 only modestly
(2-fold) but was amplified by a prolonged half-life for its
mRNA transcript. An extended half-life of Cox II mRNA
message, also up-regulated by IL-1b, also has been observed
(Srivastava et al., 1994). Therefore, a single low-affinity NFkB-binding site directing PLA2 transcription may account for
the moderate transcriptional increase observed for PLA2
when activated by cytokine. However, because only 500 nucleotides of the rat PLA2 promoter sequence were analyzed, it
is possible that additional NF-kB-binding elements exit further upstream that may contribute to NF-kB-directed transcription.
The biphasic inhibition by increasing concentrations of
DPI on p50/p65 binding may indicate that NF-kB is necessary but not sufficient for cytokine-induced up-regulation of
PLA2, and thus other mechanisms besides NF-kB may contribute to cytokine-induced PLA2 expression. Because the
PLA2 mRNA message is completely ablated at 20 mM, perhaps different transcription factors such as activator protein-1 (AP-1) need to form a suitable transcription complex
for RNA polymerase II-initated transcription. Scheinman et
al. (1995) have demonstrated that NF-kB associates with
other transcription factors that are necessary for transcriptional activation. A second explanation may be that multiple
signaling pathways, simultaneously activated by cytokine,
need to converge for NF-kB-directed transcriptional activation. This idea is supported by the observation made by
Bergmann et al. (1998) that TNF-a-induced NF-kB translocation is not sufficient for NF-kB-directed transcriptional
activity. A third explanation could be that the stability of
PLA2 mRNA (Sedor et al., 1993) may be significantly attenuated by higher concentrations of DPI, thus not allowing for
a modest increase in transcriptional activity (2-fold) to be
measured by Northern analysis. There is no evidence, however, to suggest that DPI can alter mRNA stability.
NF-kB binding was induced by IL-1b and TNF-a within 20
min and persisted through 24 h. Moreover, NF-kB binding
peaked at 40 to 60 min, declined between 2 and 6 h, and
subsequently peaked for a second time at 18 and 24 h (data
not shown) However, another group also has demonstrated
NF-kB binding 24-h postcytokine activation of rat mesangial
cells (Vervoordeldonk et al., 1997). Consequently, these data
may suggest an autocrine activation mechanism initiated by
inflammatory cytokines and perpetuated by the up-regulation of IL-1b and TNF-a. A perpetual activation of NF-kB
may explain, at the molecular level, the inflammatory loop
taking place within mesangial cells during glomerulonephritis. Uncoupling this overzealous inflammatory reaction may
unlock the glomerulus from this autocrine loop and allow the
healing process to begin.
However, targeting NF-kB in an attempt to regulate
chronic inflammation may cause inadvertant complications.
Targeting instead, mechanisms other than NF-kB activation,
such as mRNA stability of target transcripts, for example,
although maintaining an intact NF-kB pathway and high
expression of necessary antiapoptotic proteins [(TRAF 1,
TRAF 2, C-IAP1, C-IAP2, and IEX-1L (Irani et al., 1997)]
also may regulate chronic inflammation with fewer deleterious side effects. Recently, it has been demonstrated that
TNF-a-induced activation of NF-kB up-regulates the expression of antiapoptotic proteins capable of rescuing cells from
programmed cell death (Irani et al., 1997) Hence, the blocking of NF-kB translocation to the nucleus may induce an
undesired apoptotic event in cells proximal to an inflammatory foci.
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Fig. 5. Competition and supershift analyses. Mesangial cells were
treated for 40 min with PBS/0.1% BSA or 100 pM IL-1b and TNF-a.
Nuclear extracts were isolated as described in Experimental Procedures.
The 2131 sequence from the group IIA PLA2 promoter was labeled with
g[32P]ATP and used as probe for all samples. A, decreasing concentrations of unlabeled 2131 sequence (lanes 3–5) and NF-kB sequence (lanes
8 –10) were coincubated with the binding reaction as indicated. B, cells
were incubated with 100 pM IL-1b and TNF-a for 1 min (lanes 1, 3, 5, and
7) and 40 min (lanes 2, 4, 6, and 8). Nuclear extracts were isolated as
described in Experimental Procedures and paired into four groups. These
groups were incubated with labeled 2131 sequence and PBS, anti-p65,
anti-p50, and normal rabbit IgG, respectively.
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Dorsam et al.
NF-kB p50/p65 to the 2131 sequence located in the rat PLA2
promoter. Furthermore, our study supports that ROS generation induced by cytokine-receptor interaction may be generated by several cellular enzymes such as NADPH oxidase
in addition to respiratory mitochondrion enzymes. Therefore,
drugs directed at inhibiting NADPH oxidase activity may
lead to novel therapeutic strategies for the treatment of inflammatory kidney diseases.
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2000
DPI Blocks Cytokine-Induced Up-Regulation of Group IIA PLA2
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Send reprint requests to: Glenn Dorsam, Ph.D., Division of Allergy and
Immunology, University of California Medical School, 533 Parnassus Ave.,
Room UB8B, Box 0711, San Francisco, CA 94143-0711. E-mail:
[email protected]
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