INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (GR_r) and appears to bind irreversibly to the other (GR_irr). GR_irr sites are associated with inositol phospholipid (IP) turnover, increased intracellular calcium, and activation of protein kinase C (PKC), whereas GR_r sites are associated with platelet shape change and increased intracellular calcium levels, presumably from an IP₃-insensitive source (36). Platelet activating factor heterologously downregulates GR_irr but not GR_r 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

View larger version (41K): [in this window] [in a new window]   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.

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.

View larger version (17K): [in this window] [in a new window]   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.

View larger version (18K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

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).

View larger version (30K): [in this window] [in a new window]   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.

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).

View larger version (46K): [in this window] [in a new window]   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.

View larger version (46K): [in this window] [in a new window]   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.

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).

View larger version (38K): [in this window] [in a new window]   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.

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.

View larger version (19K): [in this window] [in a new window]   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.

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).

View larger version (38K): [in this window] [in a new window]   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.

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.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (26K): [in this window] [in a new window]   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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.