DDM-PGE2-mediated cytoprotection in renal
epithelial cells by a thromboxane A2 receptor coupled
to NF-
B
Thomas J.
Weber,
Terrence J.
Monks, and
Serrine S.
Lau
Division of Pharmacology and Toxicology, College of Pharmacy,
University of Texas at Austin, Austin, Texas 78712-1074
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ABSTRACT |
The present studies
were conducted to determine the pharmacological nature of a
cytoprotective 11-deoxy-16,16-dimethyl-PGE2 (DDM-PGE2) receptor in LLC-PK1 cells.
DDM-PGE2-mediated cytoprotection against
2,3,5-(trisglutathion-S-yl)hydroquinone (TGHQ)-mediated cytotoxicity can be reproduced using thromboxane A2
(TXA2) receptor (TP) agonists (U46619 and
IBOP), and the cytoprotective response to
DDM-PGE2 and TP agonists is inhibited by TP antagonists
(SQ-29,548 and ISAP). Western blot analysis using an
antipeptide antibody against the human platelet TP receptor (55 kDa)
identified a particulate associated 54-kDa protein.
DDM-PGE2-mediated 12-O-tetradecanoyl phorbol-13-acetate (TPA) responsive element (TRE) binding activity is
not inhibited by cyclooxygenase inhibitors (aspirin and
indomethacin) or a TXA2 synthase inhibitor
(sulfasalazine), suggesting that the biological response to
DDM-PGE2 is not dependent on de novo TXA2
biosynthesis. Peak DDM-PGE2- and U46619-mediated TRE
binding activity and nuclear factor-
B (NF-
B) binding activity are
inhibited by SQ-29,548. The full cytoprotective response to
DDM-PGE2 requires an 8-h pulse with agonist.
DDM-PGE2-mediated TRE and NF-
B binding activity remain
elevated in the presence of agonist and rapidly decay following agonist
washout, suggesting a direct correlation between
DDM-PGE2-mediated cytoprotection and persistent DNA binding activities. TPA, a protein kinase C activator, induces cytoprotection and a persistent increase of NF-
B binding activity.
DDM-PGE2-mediated cytoprotection and NF-
B binding
activity but not TRE binding activity are inhibited by sulfasalazine.
We conclude that the DDM-PGE2 receptor is a TP receptor and
that the cytoprotective response may be mediated in part by NF-
B.
quinone-thioether; TP receptor; protein kinase C; kidney
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INTRODUCTION |
A SINGLE GENE ENCODES the thromboxane A2
(TXA2) receptor (TP), and two alternative splice variants,
termed TP
and TP
, have been identified (23). Alternative splicing
occurs selectively in the carboxy terminus and confers association with
different G proteins, supporting the idea that these receptors couple
to different signal transduction pathways (13, 14). The tissue distribution of TP subtypes has been investigated using a variety of
techniques.
{1S-[1a,2b(5Z),3a(1E,3S)4a}-7- {3-[3-hydroxy-4(p-iodophenoxy)-1-butenyl]-7-oxabi-cyclo[2.2.1]hept-2-yl}-5-heptanoic acid ([125I]BOP; IBOP) is a widely used
TP agonist that detects a single high-affinity site on cultured
human vascular smooth muscle cells, a high-affinity and a low-affinity
site on human platelets, and a low-affinity site on K562 chronic
myelogenous leukemia cells (7). IBOP is also a
high-affinity agonist for a renal TP subtype (7). Consistent with
agonist binding studies, two platelet binding sites have been
identified using the TP antagonist GR-32191 (36). GR-32191 dissociates
rapidly from one site (GRr) and appears to bind
irreversibly to the other (GRirr). GRirr sites
are associated with inositol phospholipid (IP) turnover, increased
intracellular calcium, and activation of protein kinase C (PKC),
whereas GRr sites are associated with platelet shape change
and increased intracellular calcium levels, presumably from an
IP3-insensitive source (36). Platelet activating factor
heterologously downregulates GRirr but not GRr
sites on human platelets (24). TP subtypes are differentially
desensitized by phorbol ester, a potent activator of PKC, further
supporting the dissociation of these receptors (39).
In platelets and smooth muscle cells, TP-related signal transduction is
associated with increased intracellular calcium levels, IP turnover,
activation of PKC, and increased mitogen-activated protein kinase
(MAPK)-related activity (2, 15, 25, 30). In addition, TP-related signal
transduction is associated with activation of Ras in platelets (31) but
not in smooth muscle cells (17). PKC represents a family of at least 11 different isoforms that regulate diverse cellular functions from the
cell membrane to the nucleus. Of importance to the present work, PKC isoforms are known to regulate the activity of a number of
transcription factors, including activator protein-1 (AP-1) and nuclear
factor-
B (NF-
B; 16, 19, 21). AP-1 is a heterodimeric complex of
c-jun (c-Jun, JunB, JunD) and c-fos (c-Fos, Fos B,
Fra-1) protooncogene family members, as either a Jun:Jun
homodimer or Jun:Fos heterodimer (16, 26). NF-
B DNA binding activity
is associated with at least five different NF-
B family members:
NF-
B1 (p105/p50), NF-
B2 (p100/p52), RelA (p65), RelB, and c-Rel
(37). The most common NF-
B dimers consist of RelA (p65) and NF-
B1
(p50) or NF-
B2 (p52) subunits (32).
We have recently reported that PGE2 and
11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) induce
protection against
2,3,5-(trisglutathion-S-yl)hydroquinone (TGHQ)- mediated cytotoxicity in renal proximal tubule
epithelial cells (LLC-PK1; see Ref. 38). The
DDM-PGE2 receptor is coupled to PKC, as evidenced by the
induction of 12-O-tetradecanoyl phorbol-13-acetate (TPA)
responsive element (TRE) binding activity, inhibition of DDM-PGE2-mediated TRE binding activity by a PKC inhibitor,
and induction of cytoprotection by a PKC activator (TPA). Although DDM-PGE2 is a stable PGE2 analog, established
agonists for the known PGE2 receptor subtypes
(EP1, EP2, EP3, EP4)
failed to induce cytoprotection or TRE binding activity, suggesting
that the DDM-PGE2 receptor was unrelated to the presently
known EP subtypes. The present studies were conducted to determine the
pharmacological nature of the DDM-PGE2 receptor, and to
investigate a putative transcriptional requirement for the
cytoprotective response to DDM-PGE2. Our data suggest that
the cytoprotective response of renal epithelial cells to
DDM-PGE2 is mediated by a TP receptor coupled to NF-
B.
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MATERIALS AND METHODS |
Chemicals. TGHQ was synthesized as previously described (20)
and was greater than 98% pure as determined by HPLC.
DDM-PGE2, 17-phenyltrinor-PGE2, sulprostone,
PGE1, U46619, and SQ-29,548 were obtained from Cayman
Chemical (Ann Arbor, MI). Formaldehyde, glacial acetic acid, glycerol,
and ethanol were from Fisher Scientific (Houston, TX). TRE, NF-
B,
and AP-2 consensus sequences were purchased from Promega
(Madison, WI). [
-32P]ATP (3,000 Ci/mmol) was
obtained from New England Nuclear (Beverly, MA). Poly D(I-C) was from
Boehringer Mannheim (Indianapolis, IN). All other chemicals were from
Sigma Chemical (St. Louis, MO).
Cell culture. LLC-PK1 cells were obtained from the
American Type Culture Collection (CL101) at passage 181. Cells were
maintained in DMEM (JRH Biosciences, Lenexa, KS) supplemented with 4 g/l D-glucose (Sigma) and 10% FBS (Atlanta Biologicals,
Norcross, GA) in 5% CO2-95% air at 37°C. Cells were
subcultured by trypsinization, and all experiments were conducted with
5 day postconfluent cultures at passage levels 187-200.
Pretreatment of LLC-PK1 cells with
prostanoids. The protocol for PG-mediated cytoprotection has
previously been described (38). Briefly, LLC-PK1 cells are
seeded in 24-well plates and maintained in 10% FBS-DMEM until 5 days
postconfluent, with media replacement every 2 days. Cultures are then
rinsed three times with PBS and exposed to prostanoids in 10% FBS-DMEM
for 1-24 h. Prior to TGHQ challenge, media is aspirated, and cell
monolayers were rinsed three times with PBS to remove residual prostaglandin.
Cell viability. Measurements of cell viability were determined
by a neutral red assay as described (38). Briefly, vehicle or
prostaglandin-pretreated cells are rinsed three times with PBS and
exposed to 300 µM TGHQ in 0.1% FBS-DMEM and 25 mM HEPES (pH 7.4) for
2 h in a final volume of 0.5 ml. Following chemical challenge, cells
are washed three times with PBS and exposed to 50 µg/ml neutral red
in 0.1% FBS-DMEM and 25 mM HEPES (pH 7.4) for 1 h. Monolayers are
washed once with 1 ml of a 1% formaldehyde/1% calcium chloride
solution, and neutral red was extracted from the cells
with 1 ml of a 1% glacial acetic acid/50% ethanol solution for 15 min
at room temperature while protected from light. The extracted dye is
quantified spectrophotometrically at 540 nm, and results were expressed
as percent of control.
Aspirin, indomethacin, and sulfasalazine treatment. Aspirin (1 mM), indomethacin (10 µM), or sulfasalazine (2 mM) was solubilized in
10% FBS-DMEM with gentle sonication, and the media were then sterile
filtered (0.2 µm). LLC-PK1 cells were pretreated with aspirin, indomethacin, or sulfasalazine for 30 min prior to addition of
DDM-PGE2 (1 µM, 24 h). Following DDM-PGE2
treatment, cells were exposed to TGHQ (300 µM, 2 h), and cell
viability was determined as described above.
Electrophoretic mobility shift assays. Electrophoretic mobility
shift assays (EMSAs) were carried out as described previously (38).
LLC-PK1 cells are collected and lysed in a HEGD buffer [25 mM HEPES, pH 7.6, 1.5 mM EDTA, 10% glycerol, 1 mM DTT, and 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF)] using 20 strokes with a Dounce homogenizer. Homogenates are centrifuged at 12,000 g in an Eppendorf microcentrifuge at 4°C for 5 min, and the
supernatant was discarded. The remaining pellet is centrifuged for 10 s, and the residual supernatant was aspirated. The pellet is extracted with 40 µl HEGDK buffer (25 mM HEPES, pH 7.6, 1.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.1 mg/ml PMSF, and 0.5 M KCl) for 1 h on ice.
Extracted pellets are centrifuged at 16,000 g for 20 min at
4°C, and the supernatant was designated as the nuclear
extract. Protein concentrations are determined by the
method of Bradford (5) with BSA as standard. For mobility shift assays,
10 µg of nuclear extract were incubated in a reaction mixture
consisting of 18.8 mM HEPES, 40 mM KCl, 1.1 mM EDTA, 7.5% glycerol,
0.75 mM DTT, and 62.5 ng/µl poly D(I-C) for 15 min at 25°C to
reduce interference by nonspecific DNA binding proteins.
[
-32P]ATP-labeled TRE or NF-
B (3.5 nM)
probe is added for 15 min to determine DNA binding activity. Bound DNA
is separated on a 5% polyacrylamide nondenaturing gel for 2 h at 120 V. Specificity for the binding reaction is confirmed by addition of
excess target or nontarget DNA, which competitively eliminates the
inducible band or is without effect, respectively. Gels are dried and
exposed to Hyperfilm-MP (Amersham) for autoradiography or quantified by electronic autoradiography using a Packard Instant Imager.
Statistics. Individual comparisons were made using the
Student's t-test or ANOVA with a post hoc Student-Newman-Keuls
test, as appropriate. P
0.05 was accepted as significant.
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RESULTS |
The DDM-PGE2 receptor is pharmacologically distinct from
the currently known EP subtypes (38). Studies were conducted to investigate a role for the TP receptor in the cytoprotective response to DDM-PGE2. LLC-PK1 cells were cotreated with
1 µM DDM-PGE2 and 0.01-1.0 µM SQ-29,548 (TP
antagonist) for 24 h, then subsequently treated with a moderately toxic
concentration of TGHQ (300 µM) for 2 h, and cell viability was
determined. Pretreatment of cells with DDM-PGE2 protected
against TGHQ-mediated cytotoxicity, and SQ-29,548 fully inhibited
DDM-PGE2-mediated cytoprotection in a
concentration-dependent manner (Fig. 1).

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Fig. 1.
Inhibition of 11-deoxy-16,16-dimethyl-PGE2
(DDM-PGE2)-mediated cytoprotection by SQ-29,548.
LLC-PK1 cells were cotreated with 1 µM
DDM-PGE2 and 0.01-1.0 µM SQ-29,548 for 24 h,
subsequently challenged with 300 µM
2,3,5-(trisglutathion-S-yl)hydroquinone (TGHQ) for 2 h, and
cell viability was determined as described in MATERIALS AND
METHODS. Values are means ± SE (n = 3). *Significantly
different from control, P 0.05. Significantly different from TGHQ-treated group,
P 0.05. ¶Significantly different from cells pretreated with
DDM-PGE2 and subsequently treated with TGHQ,
P 0.05. Similar results were observed in two
separate experiments.
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To further investigate the specificity of this response, the
cytoprotective property of structurally distinct TP
pharmacons were evaluated. Treatment of
LLC-PK1 cells with 0.05-10 µM U46619 (TP agonist)
for 24 h induced cytoprotection against TGHQ-mediated (300 µM, 2 h)
cytotoxicity in a concentration-dependent fashion (Fig.
2). The TP antagonist
[1S-[1a,2b(Z),3a,5a]]-7-[3-[[(4-iodophenyl)sulfonyl]amino]-6,6-dimethylbi-cyclo[3.1.1]hept-2-yl]-5-heptenoic acid (ISAP; 1 µM) inhibited the cytoprotective response
of LLC-PK1 cells to DDM-PGE2 and U46619 (Fig.
3). Pretreatment of LLC-PK1 cells with the TP agonist IBOP for 24 h afforded protection against TGHQ-mediated (300 µM, 2 h) cytotoxicity in a concentration-dependent fashion, and this response was inhibited by cotreatment with ISAP and
SQ-29,548 (1 µM, Fig. 4). Collectively,
these data indicated a role for thromboxane pharmacology in the
cytoprotective response to DDM-PGE2. However, it is
possible that DDM-PGE2 induces the cyclooxygenase-dependent
biosynthesis of TXA2 and is not a direct ligand for the
putative TP receptor. To test this hypothesis, we examined the
induction of DDM-PGE2-mediated TRE binding activity, a
marker of receptor activation (38), in the presence and absence of
cyclooxygenase inhibitors. LLC-PK1 cells were pretreated
for 30 min with 10 µM indomethacin and 1 mM aspirin, then
subsequently treated with DDM-PGE2 for 2 h, and nuclear
extracts were prepared as described in MATERIALS AND
METHODS. Inhibition of cyclooxygenase activity by aspirin and
indomethacin was verified using a PGE2 radioimmunoassay
(RIA) (NEN DuPont, Boston, MA; data not shown). Pretreatment with
aspirin and indomethacin did not modulate peak DDM-PGE2-mediated TRE binding activity (Fig.
5), suggesting this response is not
dependent on cyclooxygenase activity. Of interest, our results contrast
historical reports indicating that LLC-PK1 cells have low
cyclooxygenase activity. Although the specific reason for this
discrepancy cannot be determined, there are several plausible
explanations. The PGE2 RIAs used over 20 years ago
demonstrate PGE2 detection in the nanogram-per-milliliter
range, whereas the PGE2 RIAs used in the present studies
can detect PGE2 in the picogram-per-milliliter range. Thus
the detection of PGE2 synthesis in our studies may be
related to state-of-the-art for prostaglandin
measurements. Alternatively, the LLC-PK1 cell line used in
earlier studies was at low passage (passage 5-30), whereas the
LLC-PK1 cells used in this present study are high passage
(passage 187-200). Therefore, it is also feasible that in vitro
selection has resulted in a LLC-PK1 phenotype with enhanced
prostanoid biosynthetic capabilities.

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Fig. 2.
U46619-mediated cytoprotection against TGHQ-mediated cytotoxicity.
LLC-PK1 cells were treated with 0.05-10 µM U46619
for 24 h, subsequently challenged with 300 µM TGHQ for 2 h, and cell
viability was determined as described in MATERIALS AND
METHODS. Values are means ± SE (n = 3). *Significantly
different from control, P 0.05. Significantly different from TGHQ-treated group,
P 0.05. Similar results were observed in four separate
experiments.
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Fig. 3.
Inhibition of DDM-PGE2- and U46619-mediated cytoprotection
by ISAP. LLC-PK1 cells were cotreated with 1 µM
DDM-PGE2 or U46619 and 1.0 µM ISAP for 24 h, subsequently
challenged with 300 µM TGHQ for 2 h, and cell viability was
determined as described in MATERIALS AND METHODS. Values
are means ± SE (n = 3). *Significantly different from
control, P 0.05. Significantly different
from TGHQ-treated group, P 0.05. ¶Significantly different
from cells pretreated with DDM-PGE2 or U46619 and
subsequently treated with TGHQ, P 0.05. Similar results were
observed in two separate experiments.
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Fig. 4.
Inhibition of IBOP-mediated cytoprotection by ISAP and SQ-29,548.
LLC-PK1 cells were cotreated with 0.01-1 µM IBOP and
1.0 µM ISAP or SQ-29,548 for 24 h, subsequently exposed to 300 µM
TGHQ for 2 h, and cell viability was determined as described in
MATERIALS AND METHODS. Values are means ± SE (n = 3). *Significantly different from control, P 0.05. Significantly different from TGHQ-treated group,
P 0.05. ¶Significantly different from cells pretreated with
IBOP and subsequently treated with TGHQ, P 0.05. Similar
results were observed in two separate experiments.
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Fig. 5.
DDM-PGE2-mediated
12-O-tetradecanoylphorbol-13-acetate (TPA) responsive
element (TRE) binding activity in aspirin- and indomethacin-pretreated
LLC-PK1 cells. LLC-PK1 cells were pretreated
for 30 min with 1 mM aspirin and 10 µM indomethacin, subsequently
treated with 1 µM DDM-PGE2 for 2 h, and nuclear extracts
were prepared. Nuclear extracts were incubated with a
32P-labeled TRE in a standard electrophoretic mobility
shift assay (EMSA) as described in MATERIALS AND METHODS.
Protein-DNA complexes were separated on a 5% native polyacrylamide gel
and visualized by autoradiography. Specificity for the binding reaction
was confirmed by addition of excess unlabeled target DNA, which
competitively eliminated the inducible band, and addition of excess
unlabeled nontarget DNA, which was without effect (data not shown).
Similar results were observed in two separate experiments.
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Studies were conducted to determine the presence of a
particulate-associated TP receptor by Western blot using
an antipeptide antibody (P2) against the human platelet TP
receptor (kind gift of Dr. Guy Le Breton, University of Illinois at
Chicago; Ref. 3). Western blot analysis demonstrated the presence of a
particulate-associated 54-kDa protein that immunoreacts with the TP
antibody (Fig. 6A). Control
reactions with secondary antibody alone were included to determine
nonspecific binding and clearly demonstrated that detection of the
54-kDa protein was dependent on the anti-TP antibody (Fig. 6B).

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Fig. 6.
Western blot analysis of particulate LLC-PK1 proteins using
an antipeptide antibody against the human platelet TP receptor.
Proteins were separated on a 10% SDS-Page gel, transferred to
nitrocellulose, and Western blot analysis was conducted as described in
MATERIALS AND METHODS. A: immunoblot representing
primary and secondary antibody. B: immunoblot representing
secondary antibody alone to detect nonspecific binding.
Immunoreactivity was detected by enhanced chemiluminescence. Similar
results were observed in three separate experiments.
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We have previously demonstrated that the cytoprotective properties of
DDM-PGE2 and PGE2, but not
17-phenyltrinor-PGE2, sulprostone, or
PGE1, correlate with increased TRE binding activity in
LLC-PK1 cells (38). A standard EMSA was conducted to
determine whether these agonists also modulate NF-
B binding
activity. NF-
B binding activity was increased in LLC-PK1
cells treated for 2 h with 20 µM DDM-PGE2 and
PGE2, but not 17-phenyltrinor-PGE2,
sulprostone, PGE1, or vehicle (Fig.
7). The high dose (20 µM) used was to
ensure agonist concentration was not limiting. The NF-
B binding
response consists of two inducible complexes, the major binding
activity is termed "complex 1" and a minor binding activity
termed "complex 2." It is not known whether these complexes
represent the DNA binding activities associated with different NF-
B
subunits or with degradation products. Consistent with a
role for thromboxane pharmacology in the cytoprotective response, peak
DDM-PGE2- and U46619-mediated (1 µM) TRE binding and
NF-
B binding activity was inhibited by cotreatment of cells with 1 µM SQ-29,548 (Fig. 8).

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Fig. 7.
Nuclear factor- B (NF- B) binding activity in nuclear extracts from
LLC-PK1 cells treated with PT-PGE2,
DDM-PGE2, sulprostone, PGE1, or
PGE2. Nuclear extracts from prostaglandin-treated (20 µM,
2 h) LLC-PK1 cells were incubated with a
32P-labeled NF- B in an EMSA as described in
MATERIALS AND METHODS. Protein-DNA complexes were separated
on a 5% continuous polyacrylamide gel and visualized by
autoradiography.
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Fig. 8.
Inhibition of DDM-PGE2 and U46619-mediated TRE and NF- B
binding activity by SQ-29,548. LLC-PK1 cells were treated
with 1 µM DDM-PGE2 and U46619 in the presence or absence
of 1 µM SQ-29,548 for 2 h, and nuclear extracts were prepared.
Nuclear extracts were incubated with a 32P-labeled TRE or
NF- B consensus sequence in an EMSA as described in MATERIALS
AND METHODS. Protein-DNA complexes were separated on a 5% native
polyacrylamide gel and visualized by autoradiography. Specificity for
the binding reaction was confirmed by addition of excess unlabeled
target DNA, which competitively eliminated the inducible band, and
addition of excess unlabeled nontarget DNA, which was without effect.
Similar results were observed in two separate experiments.
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Inducible TRE binding activity was examined in LLC-PK1
cells treated with DDM-PGE2 and U46619 as either a
continuous exposure for up to 6 h, or as a 1-h pulse, followed by
washing to remove the prostanoid analog and incubation in
prostanoid-free medium for the remainder of the experiment. Treatment
of cells with 1 µM DDM-PGE2 and U46619 as a continuous
exposure resulted in the induction of peak TRE binding activity at ~2
h (Fig. 9: DDM-PGE2, solid circles; U46619, solid
squares), and TRE binding activity remained elevated in
the presence of these agonists for the time points examined (Fig.
9A). In contrast,
DDM-PGE2- and U46619-mediated TRE binding activities
rapidly decayed following agonist washout (Fig. 9:
DDM-PGE2, open circles; U46619, open
squares). An identical response is observed for
DDM-PGE2-mediated NF-
B binding activity (Fig.
9B). The NF-
B binding response was maintained in the
presence of DDM-PGE2 (1 µM) and rapidly decayed following
agonist washout (1-h pulse).

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Fig. 9.
Inducible DNA binding activity rapidly decays following removal of
agonist. LLC-PK1 cells were treated with 1 µM
DDM-PGE2 (squares) and U46619 (circles) for up to 6 h as a
continuous exposure (solid symbols) or as a 1-h pulse followed by a
wash to remove prostanoid and addition of prostanoid-free media for the
remainder of the experiment (open symbols). Nuclear extracts from
treated cells were incubated with a 32P-labeled TRE
(A) or NF- B (B) consensus sequence in an EMSA as
described in MATERIALS AND METHODS. Protein-DNA complexes
were separated on a 5% native polyacrylamide gel and visualized by
autoradiography. Similar results were observed in two separate
experiments.
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There was a clear association between the presence of agonist and
persistent DNA binding activities (Fig. 9). To determine the
significance of this association, we examined the relationship between
DDM-PGE2 exposure time and cytoprotection.
LLC-PK1 cells were exposed to 1 µM DDM-PGE2
at time 0. The media containing DDM-PGE2 was
removed at various times (0.5-8 h) thereafter, the cells were
washed with PBS, and control media (10% FBS-DMEM) was added for the
remainder of the 24-h pretreatment period. Following this treatment
regimen, cells were exposed to 300 µM TGHQ for 2 h, and cell
viability was determined. A 0.5-h pulse with DDM-PGE2 induced a marginal, but significant cytoprotection against
TGHQ-mediated cytotoxicity, whereas an 8-h pulse was required for
induction of maximal cytoprotective activity (Fig.
10). Collectively, these observations
demonstrated a direct correlation between the presence of agonist,
persistent DNA binding activities, and cytoprotection.

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Fig. 10.
Induction of cytoprotection correlates with DDM-PGE2
treatment time. LLC-PK1 cells were exposed to 1 µM
DDM-PGE2 at time 0, the media containing
DDM-PGE2 was removed at various times (0.5-8 h)
thereafter, the cells were washed with PBS, and control media (10%
FBS-DMEM) was added for the remainder of the 24-h pretreatment period.
Following this dosing regimen, cells were treated with 300 µM TGHQ
for 2 h, and neutral red uptake was determined as described in
MATERIALS AND METHODS. Values are means ± SE (n = 3). *Significantly different from control, P 0.05. Significantly different from TGHQ, P 0.05. Similar results were observed in two separate experiments.
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We have provided evidence that the cytoprotective response is
associated with PKC-related signal transduction, and can be induced by
a PKC activator (TPA; 38). TPA-mediated cytoprotection is associated
with a persistent induction of TRE binding activity, but we have not
examined whether TPA modulates NF-
B. LLC-PK1 cells were
treated with 10 ng/ml TPA or DMSO for up to 5 h, and NF-
B binding
activity was determined by EMSA. TPA increased NF-
B binding
activity, and this response was sustained at all times examined (Fig.
11).

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Fig. 11.
TPA-mediated NF- B binding activity in LLC-PK1 cells.
Nuclear extracts from TPA-treated (10 ng/ml, 1-6 h) or
DMSO-treated LLC-PK1 cells were incubated with a
32P-labeled NF- B in an EMSA as described in
MATERIALS AND METHODS. Protein-DNA complexes were separated
on a 5% continuous polyacrylamide gel and visualized by
autoradiography. Specificity for the binding reaction was confirmed by
addition of excess unlabeled NF- B, which competitively eliminated
the inducible band, and addition of excess unlabeled nontarget DNA,
which was without effect (data not shown). Similar results were
observed in two separate experiments.
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Sulfasalazine inhibits NF-
B binding activity, but not TRE binding
activity, and the dose-response relationship for this effect is steep
(0.2-2 mM; 37). In addition, sulfasalazine is also a TXA2 synthase inhibitor (IC50 of 0.9 mM; 34).
Therefore, sulfasalazine was used to investigate a differential
requirement for TRE or NF-
B binding activity, as well as
TXA2 synthase activity in the cytoprotective response to
DDM-PGE2. To verify the differential inhibitory effect of
sulfasalazine on TRE and NF-
B binding activity, LLC-PK1
cells were pretreated for 30 min with 2 mM sulfasalazine, then
subsequently treated with 1 µM DDM-PGE2 for 2 h, and
nuclear extracts were prepared as described in MATERIALS AND
METHODS. Sulfasalazine pretreatment inhibited
DDM-PGE2-mediated NF-
B but not TRE binding activity
(Fig. 12). The effect of sulfasalazine on
DDM-PGE2-mediated cytoprotection was then examined.
LLC-PK1 cells were pretreated with 2 mM sulfasalazine for
30 min followed by 1 µM DDM-PGE2 for 24 h. Cells were
then exposed to 300 µM TGHQ for 2 h and cell viability determined as
described in MATERIALS AND METHODS. Sulfasalazine
pretreatment fully inhibited the cytoprotective response to
DDM-PGE2 (Fig. 13).
Sulfasalazine alone did not modulate cell viability relative to
control, suggesting this response is not secondary to a cytotoxic
response to sulfasalazine. Collectively, these observations
specifically implicate NF-
B binding activity in the cytoprotective
response.

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Fig. 12.
Inhibition of DDM-PGE2-mediated NF- B but not TRE binding
activity by sulfasalazine. LLC-PK1 cells were pretreated
with 2 mM sulfasalazine for 30 min, subsequently treated with 1 µM
DDM-PGE2 for 2 h, and nuclear extracts were prepared.
Nuclear extracts were incubated with a 32P-labeled NF- B
(open bars) or TRE (solid bars) consensus sequence in an EMSA as
described in MATERIALS AND METHODS. Protein-DNA complexes
were separated on a 5% continuous polyacrylamide gel and quantified by
densitometry using NIH Image.
|
|

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Fig. 13.
Inhibition of DDM-PGE2-mediated cytoprotection by
sulfasalazine. LLC-PK1 cells were pretreated with 2 mM
sulfasalazine for 30 min, followed by 1 µM DDM-PGE2 for
24 h. Following DDM-PGE2 treatment, cells were exposed to
300 µM TGHQ for 2 h, and cell viability was determined as described
in MATERIALS AND METHODS. Groups shown are, from
left to right, control (open bar), sulfasalazine, TGHQ,
DDM-PGE2 + TGHQ, and sulfasalazine + DDM-PGE2 + TGHQ. Values are means ± SE (n = 3). *Significantly different
from control, P 0.05. Significantly
different from TGHQ-treated group, P 0.05. ¶Significantly
different from cells pretreated with DDM-PGE2 and
subsequently treated with TGHQ, P 0.05. Similar results were
observed in two separate experiments.
|
|
Recently, evidence has been presented for the existence of a novel
isoprostane receptor and a number of laboratories are currently attempting to dissociate the isoprostane response from the TP receptor
(8, 9, 22). Two ligands have generally been used to investigate
isoprostane function, namely 8-iso-PGF2
and 8-iso-PGE2, and the biological response to these agents is
cell-type specific. Treatment of LLC-PK1 cells with
8-iso-PGF2
and 8-iso-PGE2 (1 µM, 2 h) did
not modulate TRE- or NF-
B binding activity or induce cytoprotection
in LLC-PK1 cells (data not shown), suggesting that the
DDM-PGE2 receptor is unrelated to the putative isoprostane receptor.
 |
DISCUSSION |
The mechanism of prostaglandin-mediated cytoprotection is not known,
but cellular and systemic components have been reported (10, 11, 28,
29). We have previously demonstrated that DDM-PGE2 induces
cytoprotection against quinone-thioether-mediated cytotoxicity in
LLC-PK1 cells via a receptor that is pharmacologically distinct from the known EP subtypes (38). The present work extends our
original observations and suggests that the DDM-PGE2
receptor is a TP receptor coupled to TRE and NF-
B binding activity.
A number of observations suggest that DDM-PGE2 elicits its
cytoprotective effect through a TP receptor, including 1)
DDM-PGE2-mediated cytoprotection is inhibited by the TP
antagonists SQ-29,548 and ISAP (Figs. 1 and 3), 2) TP agonists
(U46619 and IBOP) induce cytoprotection (Figs. 2 and 4),
and 3) TP agonist-mediated cytoprotection is inhibited by TP
antagonists (Fig. 3 and 4). SQ-29,548 inhibits DDM-PGE2-
and U46619-mediated TRE binding activity (Fig. 8), suggesting these
pharmacons interact with a common receptor.
DDM-PGE2-mediated TRE binding activity is not sensitive to
cyclooxygenase or TXA2 synthase inhibitors, indicating this
response is not dependent on de novo TXA2 synthesis.
Collectively, these observations suggest that DDM-PGE2 is a
ligand for the LLC-PK1 TP receptor. Western blot analysis
indicates the presence of a particulate-associated 54-kDa protein in
LLC-PK1 cells that immunoreacts with an antipeptide antibody against the human platelet TP receptor (55 kDa; Fig. 6), and
the TP receptor has been detected in this cell type in vivo (35).
Although DDM-PGE2 is a stable PGE2 analog, we
have previously shown that EP receptor agonists do not induce TRE
binding activity, suggesting the DDM-PGE2 receptor is
unrelated to the presently known EP subtypes (38). Consistent with this
observation, EP agonists (17-phenyltrinor-PGE2,
sulprostone, and PGE1) do not modulate NF-
B binding
activity (Fig. 6). In addition, a unique class of arachidonic acid
metabolites termed the isoprostanes have been associated with
thromboxane pharmacology, although there is evidence suggesting the
existence of a unique isoprostane receptor (8, 9, 22). However,
8-iso-PGE2 and 8-iso-PGF2
did not modulate
TRE- or NF-
B binding activity in LLC-PK1 cells (data not
shown), suggesting that the DDM-PGE2 receptor is unrelated to the putative isoprostane receptor. We have evidence that the isoprostanes modulate target biological responses in other cell types,
suggesting the lack of response to isoprostanes is not related to
chemical stability or handling issues (unpublished observations).
The TP receptor is known to couple to the PKC and/or MAPK pathways (2,
15, 25, 30). Inducible TRE binding activity is a marker of AP-1
activation, which is considered a nuclear third messenger in the PKC
cascade. We have previously demonstrated that
DDM-PGE2-mediated TRE binding activity and cytoprotection are inhibited by a PKC inhibitor (38). In the present study, we extend
these observations and show that DDM-PGE2 and TP agonists increase TRE- and NF-
B binding activity in LLC-PK1
cells, and this response is inhibited by TP antagonists (Fig. 8). Thus
the molecular response of LLC-PK1 cells to
DDM-PGE2 is consistent with TP-related signal transduction.
Interestingly, DDM-PGE2-mediated DNA binding activities
remain elevated in the presence of agonist but rapidly decay following
agonist washout (Fig. 9). This observation suggests that
LLC-PK1 cells lack a negative feedback leading to receptor
desensitization, and differential desensitization of TP subtypes has
been reported (39). Additional studies are warranted to define the
regulation of DNA binding activities by the DDM-PGE2 receptor.
Western blot analysis using a TP antibody (kind gift of Dr. Guy Le
Breton) detected a discrete 54-kDa band in the particulate fraction of
LLC-PK1 cells. A number of plausible interpretations for
the lower mobility and discrete nature of this band have been identified. Differences in the molecular mass of the TP receptor from
different species and tissues (ranging from 52-58 kDa) have been
reported (4, 14) and may account for the slight difference in molecular
mass for the human platelet and porcine renal epithelial TP receptor.
Alternative splicing also produces TP receptors with different apparent
molecular masses (14). The platelet preparation is used as a positive
control, and meaningful comparisons cannot be made for relative signal
intensities. Alternatively, we have provided evidence that the
LLC-PK1 TP receptor is not desensitized following agonist
challenge, an event associated with posttranslational modification of
the target TP receptor subtype (12, 33). Lack of posttranslational
modification could account for the discrete nature of the band observed
by Western blot, however, additional studies are required to validate
this hypothesis. Importantly, the presence of a particulate-associated
protein that immunoreacts with an anti-TP antibody in the predicted
molecular mass range supports the existence of a TP receptor in
LLC-PK1 cells and is consistent with the observed pharmacology.
At the molecular level, DDM-PGE2- and U46619-mediated TRE
and NF-
B binding activities are inhibited by a TP antagonist (Fig. 8), consistent with the suggestion that these agonists interact with a
common receptor and implicating transcriptional activities in the
cytoprotective response. DDM-PGE2 is a stable prostanoid analog, and exposure of LLC-PK1 cells to this agonist
results in a persistent increase of DNA binding activities (Fig. 9). In fact, a continuous exposure (8-h pulse) to DDM-PGE2 is
required for the induction of maximal cytoprotection (Fig. 10). In
experiments where agonist is washed out after a short-term pulse (1 h),
the DNA binding response rapidly decays to control values (Fig. 9) and
the cytoprotective response is dramatically reduced (Fig. 10).
Consistent with a requirement for persistent DNA binding activity in
the cytoprotective response, TPA-mediated cytoprotection is associated
with a persistent increase of TRE binding (38) and NF-
B binding
(Fig. 11) activities. To directly test a requirement for NF-
B
binding activity in the cytoprotective response, we pretreated
LLC-PK1 cells with sulfasalazine, an NF-
B but not TRE
binding activity inhibitor (37). Sulfasalazine inhibits NF-
B but not
TRE binding activity in LLC-PK1 cells (Fig. 12) and fully
inhibits DDM-PGE2-mediated cytoprotection (Fig. 13). These data suggest an important role for NF-
B binding activity in the cytoprotective response to DDM-PGE2. Although sulfasalazine
did not inhibit TRE binding activity, the role of TRE binding activity cannot be determined from these studies. For example, if the
cytoprotective response is dependent on a gene regulated by both
NF-
B and TRE binding activity, then loss of either would result in
loss of target gene expression and cytoprotection. Thus additional
studies are required to determine the role of TRE binding activity in the cytoprotective response.
It is important to recognize that TXA2 biosynthesis is
largely implicated in renal pathophysiology (1). Increases in
TXA2 biosynthesis contribute to renal pathologies
characterized clinically by progression to end-stage failure, including
diabetic nephropathy or loss of renal parenchymal mass (6).
TXA2 is also implicated in the pathophysiology of
nephritis, allograft transplantation rejection, and urinary tract
obstruction. The adverse effects of TXA2 in the kidney are
largely attributed to dietary constituents and/or hemodynamic
alterations within the glomerulus (6). Inhibition of thromboxane
synthase activity or antagonism of TP receptors prevents the
exacerbation of renal injury caused by these diseases (for a review see
Ref. 27). These observations have provided a rationale for pursuing
TXA2-blocking strategies through drug development or
dietary intervention.
Few investigators have considered a beneficial role for TP agonists
against chemical-induced injury. With the emergence of TP subtypes and
putative novel TXA2 binding receptors such as the
isoprostane receptor, it remains to be determined whether the adverse
effects of TXA2 will be associated with a specific receptor
subtype or localized to a target cell type. Although preventing
TXA2 function may be beneficial to pathologies involving the deregulation of renal hemodynamics or in the progression of glomerular nephropathies, there may be adverse side effects to these
strategies. Our data raise the possibility that renal proximal tubule
epithelial cells express a TP receptor that induces protection against
chemical-induced injury, an observation that is inconsistent with the
association of TXA2-related signaling with renal pathology. Although additional studies are required to determine the regulation of
this pathway in vivo, inhibition of TXA2-related
pharmacology could interfere with the cellular defense/repair
mechanisms of renal proximal tubular epithelial cells acting through
the DDM-PGE2 receptor. Alternatively, the
DDM-PGE2 receptor could be exploited for its protective
properties through the development of a selective agonist. This
proposal is consistent with a recent report describing TP subtype
selective agonists (AGN-191976, AGN-192093) with differential activity
on TP receptor-mediated events in platelets and smooth muscle
preparations (18).
In summary, studies were conducted to determine the pharmacological
nature of the DDM-PGE2 receptor in LLC-PK1
cells. Our data suggest that the cytoprotective response to
DDM-PGE2 is mediated by a TP receptor. Inhibition of
DDM-PGE2-mediated TRE- and NF-
B binding activity by TP
antagonists, coupled with the insensitivity of
DDM-PGE2-mediated TRE binding activity to cyclooxygenase
and thromboxane synthase inhibitors, suggests DDM-PGE2 is a
direct ligand for the putative TP receptor. In addition,
DDM-PGE2- and U46619-mediated cytoprotection
is associated with the induction of multiple DNA binding activities,
raising the possibility that transcriptional activity is required for
the cytoprotective response. Sulfasalazine fully inhibits
DDM-PGE2-mediated cytoprotection and NF-
B binding
activity supporting a transcriptional component in
DDM-PGE2-mediated cytoprotection.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institute of General
Medical Sciences Award GM-56321 (to S. S. Lau) and by National Institute of Environmental Health Sciences Awards P30-ES07784 (Center
Grant) and T32-ES-07247 (to T. J. Weber).
 |
FOOTNOTES |
Present address of T. J. Weber: Molecular Biosciences Department, 902 Battelle Blvd, P7-56, Richland, WA 99352.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. S. Lau,
Division of Pharmacology and Toxicology, College of Pharmacy,
Univ. of Texas at Austin, Austin, Texas 78712-1074 (E-mail:
slau{at}mail.utexas.edu).
Received 20 May 1999; accepted in final form 19 August 1999.
 |
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