11-Deoxy,16,16-Dimethyl Prostaglandin E2 Induces Specific Proteins

Maria D. Person, Daniel E. Mason, Daniel C. Liebler, Terrence J. Monks, and ... D. Person, Herng-Hsiang Lo, Kelly M. Towndrow, Zhe Jia, Terrence J. Mo...
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Chem. Res. Toxicol. 2003, 16, 312-319

11-Deoxy,16,16-Dimethyl Prostaglandin E2 Induces Specific Proteins in Association with Its Ability to Protect Against Oxidative Stress Kelly M. Towndrow,†,‡ Zhe Jia,‡ Herng-Hsiang Lo, Maria D. Person, Terrence J. Monks, and Serrine S. Lau* Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712 Received June 21, 2002

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE2, and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE2 (DDM-PGE2), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE2-stimulated LLCPK1 cells to identify candidate cytoprotective proteins. DDM-PGE2 selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins: endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1R-1 (EF-1R), heat shock protein 90β (HSP90β), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1R, HSP90β, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE2-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

Introduction 1

PGs have diverse intercellular and intracellular effects and are involved in numerous physiological and pathophysiological processes. PGs offer cytoprotection against various cellular stresses, although most studies have focused on the protective effects of PGs in liver (1) and gastric mucosa (2). The mechanism(s) underlying PGmediated cytoprotection is unknown, but the ability of PGs to protect cells in culture against toxic insults implies that this mechanism(s) is cellular in nature (3). One potential mechanism of cytoprotection may involve PG-mediated regulation of cytoskeletal organization, * To whom correspondence should be addressed. Tel: 512-471-5190. Fax: 512-471-5002. E-mail: [email protected]. † Present address: Department of Investigative Toxicology, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, IN 46140. ‡ These authors contributed equally to this work. 1 Abbreviations: acetonitrile, ACN; 11-deoxy,16,16-dimethyl prostaglandin E2, DDM-PGE2; 16,16-dimethyl prostaglandin E2, dmPGE2; elongation factor, EF; HPLC-electrospray tandem mass spectrometry, ESI MS/MS; glucose-regulated protein, GRP; heat shock protein, HSP; membrane-organizing extension spike protein, moesin; endoplasmic reticulum, ER; [1S-[1R,2R(Z),3β(1E,3S),4R]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid, IBOP; [1S-[1R,2β(Z),3R,5R]]-7-[3-[[(4-iodophenyl)sulfonyl]amino]-6,6dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid, ISAP; matrix-assisted laser desorption/ionization, MALDI; protein kinase C, PKC; prostaglandin, PG; 2,3,5-tris-(glutathion-S-yl)hydroquinone, TGHQ; thromboxane, TX; time-of-flight, TOF; 12-O-tetradecanoyl-phorbol-13-acetate, TPA; unfolded protein response, UPR.

since the combination of PGE2 and a PGI2 analogue facilitates the repair of ischemic ileum epithelium (4). This cytoprotection is associated with elevations in cAMP and Ca2+ leading to cytoskeletal-mediated tight junction closure, likely via actin filament relaxation, which may confer cytoprotection by regulating membrane permeability (4). The effects of PGE2 on cell shape have been noted in many systems, including cultured osteoblasts (5). As an example, PGE1 and PGE2 induce morphological changes and the selective breakdown of actin microfilaments but not microtubules or vimentin filaments (5). The coupling of bisphosphonates to PGE2 to form synthetic PGE2-bisphosphonate conjugates may be potentially therapeutic in the treatment of osteoporosis (6). Such conjugates may be useful due to their ability to selectively target bone and to be slowly hydrolyzed into two therapeutically useful compounds, bisphosphonates and PGE2, which are potential bone resorption inhibitors and bone formation stimulators, respectively (6). Moreover, the protection of Caco-2 cells against ethanolinduced damage by 16,16-dimethyl PGE2 correlates with increased PKC activity and Ca2+ efflux and a subsequent stabilization of microtubules (7). Other possible PG cytoprotective mechanisms may include alterations in blood flow, enhanced regeneration, alterations in toxicant metabolism, and increased membrane stability (1).

10.1021/tx020048l CCC: $25.00 © 2003 American Chemical Society Published on Web 02/04/2003

DDM-PGE2-Induced Proteins in Renal Cells

PGE2 generally acts via G-protein-coupled cell surface receptors, designated EP1-EP4, each of which is coupled to different signal transduction systems. However, the complexity of PG signaling mechanisms is illustrated by the fact that (i) PGs can be transported into cells via a PG transporter (8-9), (ii) EP receptors can also be localized at the nuclear membrane (10), and (iii) some PGs (i.e., 11-deoxy-∆12, 14-PGJ2) are ligands for other receptors, such as the peroxisome proliferator-activated receptor family of nuclear receptors (11). Thus, PG signaling is multifaceted and complicated by autocrine and paracrine effects. DDM-PGE2 and PGE2 protect renal proximal tubular epithelial (LLC-PK1) cells against TGHQ-mediated cytotoxicity (12). This cytoprotective response is apparently not mediated via known EP receptor subtypes, since known EP agonists do not confer protection. The DDMPGE2 protective response is mediated through a PKCcoupled pathway since (i) TPA, an activator of PKC, also induces cytoprotection; (ii) DDM-PGE2 increases TRE binding activity; and (iii) cytoprotection is overcome by PKC-inhibiting concentrations of H-89 (12). The cytoprotective response to DDM-PGE2 is mediated by a TX A2 receptor (13), as both U46619 and IBOP, TXA2 receptor agonists, also protect against TGHQ-mediated cytotoxicity. Furthermore, DDM-PGE2-mediated cytoprotection and TRE and NF-κB binding activity are inhibited by the TX receptor antagonists, SQ29548 and ISAP (13). Sulfalazine, a TX A2 synthase inhibitor, also blocks cytoprotection and NF-κB binding activity induced by DDM-PGE2, indicating divergent downstream signaling pathways (13). Although it is clear that DDM-PGE2 is cytoprotective through a receptor-mediated pathway, the mechanism(s) underlying this response remains unknown. The studies described herein were conducted to identify proteins expressed in DDM-PGE2-stimulated LLC-PK1 cells that may participate in DDM-PGE2mediated cytoprotection.

Experimental Procedures Cell Culture. LLC-PK1 cells were purchased from the American Type Culture Collection (CL101) and cultured in Dulbecco’s modified Eagle medium with 4.5 g/L glucose (DMEM; GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) in a 37 °C/5% CO2-humidified incubator. 35S-Methionine Protein Labeling. Postconfluent LLC-PK1 cells were exposed to 1 or 2 µM DDM-PGE2 (Caymen Chemicals, Ann Arbor, MI), an ethanol vehicle control, 10 µM U46619 (Caymen Chemicals), 10 ng/mL TPA (Calbiochem, San Diego, CA), or 0.1% (v/v) dimethyl sulfoxide (DMSO) vehicle control in DMEM (methionine/cysteine free) with 25 mM HEPES and 10% FBS medium (pH 7.4) containing 35S-methionine (ICN; 0.1 mCi/ ml) for 24 h. At the end of the experiment, cells were washed three times with PBS, scraped, and collected in PBS. Cells were pelleted at 200 g (10 min) and lysed in buffer (80:10:10 stock buffer [10 mM Tris, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 0.1% NP-40, pH 7.4], 10 mg/mL PMSF, 100 mM sodium orthovanadate; 1 complete mini protease inhibitor cocktail tablet [containing antipain dihydrochloride (50 µg/mL), bestatin (40 µg/mL), chymostatin (60 µg/mL), E-64 (10 µg/mL), leupeptin (0.5 µg/mL), pepstatin (0.7 µg/mL), phosphoramidon (300 µg/mL), pefabloc SC (1 mg/mL), EDTA disodium salt (0.5 mg/mL), and aprotinin (2 µg/mL)] in 10 mL) for 15-30 min on ice. After lysis, the samples were subjected to one freeze/thaw cycle and centrifuged at 16 000g (10 min), and the supernatant was collected. The concentration of 35S-methionine-labeled proteins

Chem. Res. Toxicol., Vol. 16, No. 3, 2003 313 was determined by the Bradford method (14), and proteins were separated on an 8% denaturing polyacrylamide gel (16 cm × 16 cm SDS-PAGE), stained with Coomassie Blue R, dried, and analyzed by autoradiography. In-Gel Protein Tryptic Digestion. Differentially expressed protein bands were identified by autoradiography and then selected for in-gel digest after Coomassie Blue staining. 35SMethionine-labeled proteins were separated by SDS-PAGE and processed as described above, except without drying the gel. Typically, pooled individual protein bands originating from a combined total of 300-600 µg of cellular lysate were a sufficient amount for LC-MS/MS analysis. In-gel tryptic digestion was based on a modification of the method of Shevchenko et al. (15). Prior to in-gel digest, individual bands were cut into 1 mm pieces and destained in 5% acetic acid and 50% methanol to remove the Coomassie Blue. Gel pieces were dehydrated with ACN, and residual ACN was evaporated in a SpeedVac. Proteins were then reduced with 10 mM DTT in 100 mM NH4HCO3 at room temperature for 1 h. Residual DTT was removed, and cysteines were alkylated with 50 mM iodoacetamide (in 100 mM NH4HCO3) for 1 h. After the residual iodoacetamide was removed, gel pieces were subjected twice to washing (100 mM NH4HCO3 for 10 min) and dehydration (5 min in ACN). Gels were dried for 2-3 min in a SpeedVac and rehydrated on ice with 20 ng/µL sequencing grade modified trypsin (Promega, Madison, WI; in 50 mM NH4HCO3) for 10-15 min. Excess trypsin was removed, 20 µL of 50 mM NH4HCO3 was added, and gel pieces were digested overnight at 37 °C. After digestion, peptides were extracted twice in 75 µL of 5% formic acid/50% ACN. Peptide Sequencing by LC-ESI-MS/MS. Peptides were analyzed with an electrospray ion trap mass spectrometer (ThermoFinnigan LCQ, San Jose, CA) coupled to a microbore HPLC (Magic 2002, Michrom BioResources, Auburn, CA). Samples were injected into a MAGIC MS C18 (5 µm, 200 Å, 0.5 mm × 50 mm) column and eluted with a 5-95% mobile phase B (90:10:0.09:0.02 ACN:H2O:acetic acid:trifluoroacetic acid) over 45 min followed by 95% B for 10 min. Mobile phase A consisted of 2:98:0.1:0.02 ACN:H2O:acetic acid:trifluoroacetic acid. A flow rate of 20 µL/min was used, and a mass range of 400-2000 Da was recorded for the full scan, which was followed by a single Zoom scan and MS/MS of the most intense peak. Individual peptide sequences were identified with SEQUEST incorporated into ThermoFinnigan BIOWORKS software to match MS/MS spectra to amino acid sequences in the National Center for Biotechnology Information (NCBI) or OWL protein database. Samples from several experiments were analyzed, and only proteins seen in multiple runs are reported here. The two high molecular weight bands (bands 1 and 2 in Figure 1) were also analyzed by microspray LC-MS/MS. A resistive splitter (Magic Variable Splitter, Michrom BioResources) was used to split the precolumn flow from a 20 µL/min input to 0.35 µL/min through the column. A custom-built microspray interface, according to the design of Gatlin et al. (16), was mounted on the LCQ, using a PicoFrit (New Objective, Cambridge, MA) 75 µm i.d. x 5 cm column with a 15 µm tip filled with BioBasic C18 material (5 µm). A 30 min linear gradient from 5 to 65% B followed by a 15 min wash at 95% B was used to elute the digested peptides, with A (0.5% acetic acid, 0.005% trifluoroacetic acid in water) and B (0.5% acetic acid, 0.005% trifluoroacetic acid, 90% ACN, 10% water). The LCQ acquired a single MS over the m/z range 360-2000 followed by two datadependent MS/MS scans with dynamic exclusion. Peptide sequences were identified using the TurboSEQUEST software as described above. Peptide Mapping by MALDI-MS. Peptides were directly analyzed with a MALDI TOF mass spectrometer (PerSeptive Biosystems Voyager De-Str, Framingham, MA). Spectra were acquired in positive ion mode using the reflectron detector over a mass range of 700-3600 Da. The sample and R-cyano-4hydroxycinnamic acid (Agilent Technologies, Palo Alto, CA) matrix were mixed 1:1 and drop-dried on the target. A mixture of 1 ng/µL cze standards (Bio-Rad, Hercules, CA) and 2.5 ng/µL

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Towndrow et al. antibodies (1:3000 dilution for all blots except GRP78, which was 1:10 000) were purchased from the Santa Cruz company. Protein expression was visualized using enhanced chemiluminescence (Amersham, Arlington Heights, IL) according to the manufacturer’s specifications.

Results

Figure 1. Time-dependent induction of DDM-PGE2 responsive proteins. LLC-PK1 cells were exposed to vehicle (ethanol; E) or 1 µM DDM-PGE2 (D) for 4, 8, 12, and 24 h in the presence of 35S-methionine. Cell lysates (1 × 106 CPM/lane) were separated by SDS-PAGE and analyzed by autoradiography as described in the Experimental Procedures section. adrenocorticotropic hormone was used for external calibration of MALDI spectra. To minimize salt interference and enhance sensitivity, protein samples were desalted using a C18 ZipTip (Millipore, Bedford, MA) washed and eluted with 0.1% formic acid and 0.1% formic acid/50% ACN, respectively. The peptide mass list for each sample was entered in the protein identification program, MS-Fit, in the Protein Prospector Suite (www.prospector.ucsf.edu). The OWL database was searched using a 75 ppm peptide mass tolerance for tryptic digest and a maximum of one missed cleavage and carbamidomethylation of the cysteine residues. To enhance search sensitivity, a mass tolerance of 50 ppm coupled with an external calibration, at a position adjacent to the sample spot on the MALDI target, was utilized with mass searches for protein sample one. Western Blot Analysis. Cells were washed three times with ice cold PBS, scraped, and lysed in buffer (50 mM Tris-HCL; pH 7.5, 100 mM NaCL, 10 mM sodium fluoride, 5 mM EDTA, 1% Triton X-100, 40 mM β-glycerophosphate, 0.5 mM sodium orthovanadate, 0.25 mM PMSF, 1 complete mini protease inhibitor cocktail tablet in 10 mL) for 15-30 min on ice. The cell lysates were centrifuged at 14 000g for 20 min at 4 °C. The supernatant containing the total protein was collected and stored at -80 °C. Protein concentration was measured by the Bio-Rad DC protein assay kit (Bio-Rad Laboratories). Proteins were separated on a 10% denaturing polyacrylamide gel and transferred to a nitrocellulose membrane by electroblotting (wet transfer). Primary antibodies with different dilution factors and manufacturers are listed as follows: elongation factor 2 (EF2; 1:250, Santa Crutz Biotechnologies, Santa Cruz, CA); HSP90β (1:4000, Stressgene Biotechnologies, Victoria, BC, Canada); GRP78 (1:40 000, a generous gift from Dr. James Stevens, Eli Lilly & Co); elongation factor 1R-1 (EF-1R; 1:1000, Upstate Biotechnology, Lake Placid, NY); actin (1:10 000, Oncogene Research Products, Boston, MA). GAPDH (1:1000, a generous gift from Dr. Kline, University of Texas at Austin) was used as the housekeeping protein for loading normalization. Secondary

DDM-PGE2-Mediated Induction of Protein Synthesis. A 24 h pretreatment with DDM-PGE2 (1 or 2 µM) is required to protect LLC-PK1 cells from TGHQ (300 µM)-mediated cytotoxicity (12). However, the mechanism(s) underlying this cytoprotective response is unknown. 35S-Methionine labeling of newly synthesized proteins was utilized to identify candidate cytoprotective proteins induced during a 24 h exposure of LLC-PK1 cells to DDM-PGE2. DDM-PGE2 (1 or 2 µM, 24 h) increases (120 ( 6%; mean ( SE) overall protein synthesis in LLCPK1 cells, and at least eight proteins were selectively induced by DDM-PGE2 (Figure 1) as determined by increased levels of 35S-methionine incorporation. Induction of proteins 1, 5, and 8 was evident as early as 4 h following DDM-PGE2 treatment and persisted to 24 h (Figure 1). Proteins 3, 4, and 7 were only slightly induced at 4 h but were clearly elevated by 8, 12, and 24 h. A weaker induction of protein 6 was detected at 12 and 24 h after exposure of LLC-PK1 cells to DDM-PGE2. A less robust induction was seen with protein 2, which was induced by 8 h (Figure 1). Consistent with the hypothesis that DDM-PGE2 may be acting through a TX or TX-like receptor (13), U46619 (10 µM), a TX A2 receptor agonist, and the phorbol ester, TPA (10 ng/mL), were also found to increase total protein synthesis (119 ( 6 and 122 ( 1%; mean ( SE, respectively). Furthermore, induction of specific proteins, in particular, proteins 1, 4, 5, 7, and 8, were similar in LLCPK1 cells after treatment of DDM-PGE2, U46619, and TPA (Figure 2). Mass Spectrometric Analysis of DDM-PGE2Induced Proteins. To determine the identity of proteins elevated by DDM-PGE2, a mass spectrometric strategy involving in-gel tryptic digestion of SDS-PAGE separated 35S-methionine-labeled proteins was employed. The eight proteins induced during the 24 h pretreatment window (Figure 1) were targeted for tryptic in-gel digestion and LC-ESI-MS/MS analysis. Eight peptides were identified from an in-gel tryptic digest of protein 1 (Table 1). A computer-generated search of the human subset of the OWL database identified protein 1 as the 280.7 kDa endothelial actin-binding protein (ABP-280; also called nonmuscle filamin; Table 1). These eight peptides comprise 3.5% of the amino acid sequence of ABP-280 (i.e., 3.5% amino acid coverage). Six peptides from protein band 2 were identified to cover 5.4% of the amino acid sequence for myosin. Four peptides (comprising 5.7% of protein 3) were identified to be derived from EF-2, with a molecular mass of 95.3 kDa (Table 1). Digests of protein 4 produced eight peptides, which were attributed to HSP90β, a 83.2 kDa protein (with an amino acid coverage of 11.8%; Table 1). In one instance, peptides for both HSP90β and HSP90R were found (data not shown). Database searching of the complete OWL database revealed 13 peptides from protein 5 (27.8% amino acid coverage), which were identified as the 78 kDa GRP (GRP78) (Table 1). Searching the porcine subset of OWL determined that six peptides from protein 6 are derived

DDM-PGE2-Induced Proteins in Renal Cells

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Discussion

Figure 2. DDM-PGE2, U46619 (both TX receptor agonists), and TPA produce a similar induction of protein synthesis in LLC-PK1 cells. LLC-PK1 cells were exposed to vehicle (DMSO or ethanol (ETOH)), 1 µM DDM-PGE2, 10 µM U46619, or 10 ng/mL TPA for 24 h in the presence of 35S-methionine. Cell lysates (1 × 106 CPM/lane) were separated by SDS-PAGE and analyzed by autoradiography as described in the Experimental Procedures section.

from the 67.5 kDa moesin. Five peptides isolated from protein 7 were identified as EF-1R. Protein 8 was identified as cytoplasmic actin from 11 peptides comprising 27.7% amino acid coverage. One set of in-gel protein digests was confirmed by MALDI-TOF-MS peptide mapping. Both LC-ESI-MS/MS peptide sequencing and MALDI-MS peptide mass mapping techniques resulted in the same protein identification for all samples (data not shown). The MS/MS fragmentation pattern for the peptide identified as THINIVVIGHVDSGK from the EF-1R protein band is shown in Figure 3. Several other peptides isolated from the EF-1R band gave similar high quality MS/MS spectra, adding further confidence in the protein identification. Figure 4 is a representative MALDI-MS spectrum of the tryptic peptides identified from the EF-1R band. The tryptic fragment assignments for the EF-1R sequence, trypsin autolysis, and keratin contaminant peaks from sample handling are labeled. Both ESI-MS/MS and MALDI-TOF show complete agreement on the identification of the DDM-PGE2-induced proteins, although it remained possible that only the dominant protein(s) in each band had been identified. Therefore, to validate the mass spectral identification, expression of inducible proteins was subsequently confirmed by western blot analysis (Figure 5). Elevations in EF-1R and EF-2 expression were evident as early as 4 h and were sustained through 24 h of DDM-PGE2 exposure. HSP90β was maximally induced at 4 h and represented a plateau through 24 h of DDM-PGE2 exposure. In addition, a delayed temporal induction of GRP78 and actin, following 8-24 h of DDM-PGE2 treatment, was observed.

Although DDM-PGE2 induces overall protein synthesis, it also selectively stimulates the synthesis of specific proteins (Figure 1), including filamin, myosin, EF-2, HSP90β, moesin, GRP78, EF-1R, and actin (Table 1). The cytoprotective effects observed in LLC-PK1 cells following DDM-PGE2 treatment are coupled to a TX-like receptor (13) linked to the PKC pathway (12). Consistent with these findings, DDM-PGE2 and U46619, the TX receptor agonists, and TPA, the PKC stimulator, all induce a similar spectrum of proteins in LLC-PK1 cells (Figure 2). HPLC-ESI-MS/MS analyses provided the identity of eight proteins induced following treatment of LLC-PK1 cells with DDM-PGE2, with at least four peptides identified for each protein sample by peptide fragmentation and database searching, ensuring correct identification of the dominant protein in each gel slice (Table 1). MALDI-MS peptide mass mapping confirmed the identity of proteins identified by multiple HPLC-ESI-MS/MS experiments. With available antibodies, western blot analysis further confirmed the mass spectrometric identification of EF-1R, EF-2, HSP90β, GRP78, and actin (Figure 5). Unfortunately, currently available commercial antibodies against endothelial actin binding proteins and myosin were not specific, giving equivocal results. Moreover, although 35S-methionine labeling experiments showed weak induction of protein 6, identified as moesin by mass spectral analysis, western analysis failed to show induction (data not shown). It is possible that a low abundant, yet unknown cytoprotective protein(s) induced by DDMPGE2 comigrated with moesin on gel electrophoresis. On the other hand, the weak induction of protein 6 varied from experiment to experiment. Whether moesin plays an important role in DDM-PGE2-induced cytoprotection remains to be determined. The induction of endothelial actin binding protein ABP280 (filamin), myosin, EF-2, EF-1R, HSP90β, GRP78, moesin, and actin by DDM-PGE2 (Table 1) provides insight into the mechanism of DDM-PGE2-mediated cytoprotection. The known cellular functions and subcellular localization of these proteins are listed in Table 2. These proteins are interrelated in the following way: (i) ABP-280, EFs (17, 18), and HSP90β (19) are all actin binding proteins; (ii) ABP-280, myosin, moesin, and actin are associated with the cytoskeleton; (iii) EFs are critical components of the protein synthetic machinery, which resides in close association with the cytoskeleton (18, 20); (iv) EF-1R has other nonprotein synthetic functions, including involvement in cytoskeletal organization (21); and (v) GRP78 functions in nascent protein translocation and protein processing in the ER (22). Thus, DDM-PGE2mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an ER stress response as discussed below. Cyclooxygenase products have been implicated in mitogenic signaling pathways (23-26). Likewise, DDMPGE2 increases overall protein synthesis but also induces the synthesis of specific proteins (Figure 1). Furthermore, DDM-PGE2 induces DNA synthesis in LLC-PK1 cells (data not shown). EFs are critical components of the protein synthesis machinery (Table 2). EF-2, like EF-1R, is involved in peptide elongation, specifically by translocation of the nascent protein chain between the A and the P sites within the ribosome. EF-1R is an actin binding

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Table 1: Summary of Results from LC-ESI-MS/MS Analysis sample

a

mol mass (kDa)

1

280.7

2

226.5

3

95.3

4

83.2

5

72.3

6

67.5

7

50.1

8

41.7

peptide sequence LVSIDSK AIVDGNLK AGVAPLQVK GAGTGGLGLAVEGPSEAK VTVLFAGQHIAK LLGWIQNK DAGEGLLAVQITDPEGKPK LIALLEVLSQK HSQAVEELAEQLEQTKR VSHLLGINVTDFTR NLPIYSEEIVEMYK IRELESQISELQEDLESER SGFEPASLKEEVGEEAIVELVENGKK LQQELDDLLVDLDHQR NPADLPK VFSGLVSTGLK LDSEDKDKEGKPLLK AYLPVNESFGFTADLR LGIHEDSTNR YESLTDPSK SLVSVTK SIYYITGESK SLTNDWEDHLAVK GVVDSEDLPLNISR ALLFIPR HSQFIGYPITLYLEK IQQLVK VLEDSDLK TWNDPSVQQDIK TKPYIQVDIGGGQTK NQLTSNPENTVFDAK SQIFSTASDNQPTVTIK ITPSYVAFTPEGER KSDIDEIVLVGGSTR VTHAVVTVPAYFNDAQR IINEPTAAAIAYGLDKR IEWLESHQDADIEDFK AKFEELNMDLFR DNHLLGTFDLTGIPPAPR IQVWHEEHR FVIKPIDKK FVIKPIDK ESPLLFK EDAVLEYLK LFFLQVK EVSTYIK QLIVGVNK IGGIGTVPVGR LPIQDVYK THINIVVIGHVDSGK ILTER IIAPPERK AGFAGDDAPR LDLAGR IIAPPER QEYDESGPSIVHR GILTLK IWHHTFYNELR AVFPSIVGRPR VAPEEHPVLLTEAPLNPK SYELPDGQVITIGNER

A.Aa (%)

OWL ref

protein (H)b

3.5

ABP2_HUMAN

5.4

MYSN_HUMAN

myosin, heavy chain, nonmuscle type A

5.7

EF2_HUMAN (H)

EF-2

11.8

HS9B_HUMAN (H)

HSP90β (HSP84)

27.8

GR78_MESAU

78 KDa glucose-regulated protein (GRP78; BiP)

7.1

MOES_PIG (P)

membrane organizing extension spike protein (moesin)

10.6

EF11_HUMAN

EF-1R-1

27.7

ACTB_HUMAN (H) ACTG_HUMAN (H)

actin, cytoplasmic 1 (β-actin) actin, cytoplasmic 2 (γ-actin)

endothelial actin-binding protein (ABP-280; nonmuscle filamin)

Amino acid coverage (%). b Species OWL database subset; p, porcine; h, human.

protein that facilitates the GTP-dependent transfer of aminoacyl-tRNA to the ribosome (21). The upregulation of EF-2 and EF-1R (Figures 1 and 5, Table 1) is consistent with the increased protein synthesis observed following treatment of LLC-PK1 cells with DDM-PGE2. The increase in actin (whether F-actin or G-actin cannot be determined) by DDM-PGE2 is likely coupled to the induction of EF-1R, which has a role in cytoskeletal reorganization, including the regulation of microtubule stability and actin filament bundling (21). Thus, EF-1R

and actin are linked via their cytoskeletal and protein synthesis roles, respectively. A number of proteins identified from DDM-PGE2-stimulated LLC-PK1 cells exhibit cytoskeletal functions (Table 2). Actin is the main component of the cytoskeleton, which functions to maintain cell shape and integrity. Banan et al. (27) demonstrated that dmPGE2 increased the fraction of F-actin (polymerized) to G-actin (unpolymerized), prevented collapse of the actin cytoskeleton, and protected a rat intestinal cell line from ethanol-mediated toxicity. Similarly,

DDM-PGE2-Induced Proteins in Renal Cells

Figure 3. LC-ESI-MS/MS spectrum of EF-1R peptide THINILVVIGHVDSGK. The MS/MS spectrum of one peptide from the LC-ESI-MS/MS analysis of protein band 7 is shown. The peptide was identified as a tryptic peptide from EF-1R, and the b and y fragmentation ions are labeled next to their corresponding peaks. The data are collected in the centroid mode.

dmPGE2 reversed toxicity in Caco-2 cells by increasing PKC activity and microtubule stability (7). Moreover, carbacyclin (PGI2 analogue) and PGE2 together protected ileum intestinal mucosa from ischemic injury by initiating cytoskeletal-mediated closure of tight junctions, which was proposed to be protective by maintaining proper membrane permeability (4). Interestingly, disruption of the actin cytoskeleton of proximal tubules due to ischemic or other injurious agents is well-documented (28-31). Thus, maintenance of cytoskeletal integrity is likely an important component of the cytoprotective properties of DDM-PGE2. Proximal tubular cells express distinct apical and basolateral membrane domains, which can be disorganized during injury, resulting in a loss of cell polarity (32). Spectrin and ankrin link cortical actin to basolateral specific proteins, such as the Na, K-ATPase, thereby

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maintaining normal basolateral membrane composition (32). Filamin is a spectrinlike protein that functions to cross-link actin microfilaments to the plasma membrane (32) and is induced in DDM-PGE2-stimulated cells (Table 1). Moreover, the apical brush border is maintained by actin cross-linking via actin binding proteins such as ezrin (28), which can mediate survival of LLC-PK1 cells (33). In contrast, dissociation of ezrin has been observed following anoxia in rabbit proximal tubules (34). Taken together, the data suggest that DDM-PGE2 may be cytoprotective by upregulating moesin and filamin expression and maintaining proximal tubular cell polarity. HSP90 is an abundant (1-2% of total protein) cytosolic molecular chaperone that is involved in the correct folding of proteins (35-37). HSP90 production is induced following heat stress, but its specific role is not as wellcharacterized as that of HSP70 (35-37). The two isoforms of HSP90, R and β, share significant homology but appear to have different cellular functions. HSP90R is an actin and tubulin binding protein (38), and HSP90β is structurally related to the human microtubule interacting protein, MIP-90 (39) that may protect microtubules during stress. DDM-PGE2-mediated elevations in HSP90β may therefore prevent cytoskeletal damage induced by TGHQ through its association with cytoskeletal structures and/or by facilitating repair of damaged proteins. Toxicants induce ER stress proteins during proximal tubular epithelial cell death (40-42). Prior induction of ER stress (termed ER tolerance) and overexpression of ER chaperones protect proximal tubular epithelial cells from subsequent chemical insult (40-42). Induction of critical ER chaperones (termed unfolded protein response; UPR), such as GRP78 (BiP), GRP94, and calreticulin, is essential to this cytoprotective response. Presumably, overexpression of ER chaperones is cytoprotective via increasing the ER’s ability to deal with

Figure 4. MALDI-MS spectrum of EF-1R peptides identified from protein band 7. The EF-1R tryptic fragments, trypsin autolysis, and keratin contaminant peptides identified from the MALDI-MS spectrum of the gel digest of band 7 are labeled next to their corresponding peaks. The inset shows the sequence for EF-1R with the tryptic peptides detected in bold. The human sequence is used because the porcine sequence is not available.

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Towndrow et al.

Table 2: Function and Localization of Proteins Identified from DDM-PGE2-Stimulated LLC-PK1 Cellsa protein endothelial actin-binding protein (ABP-280; nonmuscle filamin) nonmuscle myosin, heavy chain type A EF-2 HSP90β 78 kDa glucose-regulated protein (GRP78) EF-1R actin a

function

subcellular localization

promotes actin filament branching and links actin filaments to membrane glycoproteins appears to play a role in cytokinesis, cell shape, and specialized functions such as secretion and capping causes GTP-dependent nascent protein chain translocation between the A and P sites of the ribosome molecular chaperone; refolding of denatured proteins ER stress protein

peripheral cytoplasm cytoplasmic cytoplasmic cytoplasmic ER lumen

mediates GTP-dependent aa-tRNA translocation to the A site of the ribosome cytoskeletal

cytoplasmic cytoplasmic

Adapted from http://prowl1.rockefeller.edu/prowl/proteininfo.html.

stimulated LLC-PK1 cells described herein is consistent with the ability of PGs to alter stress protein expression, the structure-activity relationship for PG-mediated effects on stress protein expression is unclear, since DDMPGE2 is a cyclopentanone PG. In summary, DDM-PGE2 stimulates the synthesis of specific proteins, each of which is likely involved in the overall cytoprotective response. Mass spectrometric analysis of proteins isolated from DDM-PGE2-stimulated LLCPK1 cells identified several potentially cytoprotective proteins, namely, filamin, myosin, moesin, EF-2, HSP90β, GRP78, EF-1R, and actin. Western analysis confirmed a temporal induction of the latter five proteins. Induction of these particular proteins suggests potential DDMPGE2-mediated cytoprotective mechanisms involve cytoskeletal changes, maintenance of proximal tubular epithelial cell polarity, activation of UPR, and possibly increased proximal tubular epithelial regenerative capacity.

Acknowledgment. This work was supported in part by awards from the National Institute of General Medical Sciences to S.S.L. (GM 56321), the National Institute of Environmental Health Sciences Center Grant (P30ES07784), and NIEHS Training Grant T32-ES07247 to K.M.T. MALDI spectra were acquired in the Mass Spectrometry Facility of the University of California at San Francisco under the direction of Dr. Al Burlingame and supported by NIH Grant NCRR RR01614. Figure 5. Time-dependent induction of EF-1R, EF-2, HSP90β, GRP78, and actin following exposure of LLC-PK1 cells to DDMPGE2. LLC-PK1 cells were exposed to vehicle (ethanol; EtOH) or 2 µM DDM-PGE2 for 4-24 h. (A) Protein lysates were separated by SDS-PAGE and blotted, and protein levels were determined as described in the Experimental Procedures section. GAPDH was used as a housekeeping protein for normalization of sample loading. (B) Quantification of protein expression by densitometry.

unfolded proteins and Ca2+ depletion following toxicant insult (43, 44). PGs induce GRP78 expression, and ∆12-PGJ2 induces GRP78 gene expression in HeLa cells via the UPR response element (45). Induction of GRP78 gene expression was observed following exposure of normal rat kidney cells to a synthetic PG with a cyclopentenone structure but not a synthetic cyclopentanone PG (46). While the identification of GRP78 from DDM-PGE2-

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