Identification of Novel Protein Targets for Modification by 15-Deoxy-12,14-Prostaglandin J2 in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton
Konstantinos Stamatakis,
Francisco J. Sánchez-Gómez and
Dolores Pérez-Sala
Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Madrid, Spain
Address correspondence to: Dr. Dolores Pérez-Sala, Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, C.S.I.C., Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: +34-91-5346623; Fax: +34-91-5360432; E-mail: dperezsala{at}cib.csic.es
Received for publication March 30, 2005.
Accepted for publication October 1, 2005.
The cyclopentenone prostaglandin 15-deoxy-12,14-PGJ2 (15d-PGJ2)has been shown to display protective effects against renal injuryor inflammation. In cultured mesangial cells (MC), 15d-PGJ2inhibits the expression of proinflammatory genes and modulatescell proliferation. Therefore, cyclopentenone prostaglandins(cyPG) have been envisaged as a promise in the treatment ofrenal disease. The effects of 15d-PGJ2 may be dependent on orindependent from its role as a peroxisome proliferator–activatedreceptor agonist. It was shown recently that an important determinantfor the peroxisome proliferator–activated receptor–independenteffects of 15d-PGJ2 is the capacity to modify proteins covalentlyand alter their function. However, a limited number of proteintargets have been identified to date. Herein is shown that abiotinylated derivative of 15d-PGJ2 recapitulates the effectsof 15d-PGJ2 on the stress response and inhibition of induciblenitric oxide synthase levels and forms stable adducts with proteinsin intact MC. Biotinylated 15d-PGJ2 was then used to identifyproteins that potentially are involved in cyPG biologic effects.Extracts from biotinylated 15d-PGJ2–treated MC were separatedby two-dimensional electrophoresis, and the spots of interestwere analyzed by mass spectrometry. Identified targets includeproteins that are regulated by oxidative stress, such as heat-shockprotein 90 and nucleoside diphosphate kinase, as well as proteinsthat are involved in cytoskeletal organization, such as actin,tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 bindingto several targets was confirmed by avidin pull-down. Consistentwith these findings, 15d-PGJ2 induced early reorganization ofvimentin and tubulin in MC. The cyclopentenone moiety and thepresence of cysteine were important for vimentin rearrangement.These studies may contribute to the understanding of the mechanismof action and therapeutic potential of cyPG.
Prostaglandins with cyclopentenone structure are endogenouseicosanoids that are generated by nonenzymatic dehydration ofarachidonic acid metabolites. Cyclopentenone prostaglandins(cyPG) exert varied biologic actions, including inhibition ofcell proliferation in several cancer cell lines and anti-inflammatoryand antiviral activities. The molecular basis for these variedeffects is multiple. The common feature of these prostaglandinsis the presence of an unsaturated carbonyl group in the cyclopentanering (cyclopentenone; see Figure 1). This structure was foundearly to be an important requirement for the antitumoral andantiviral effects of cyPG (1). This moiety confers cyPG thecapacity to form covalent adducts with thiol groups in glutathioneor in proteins by Michael addition (Figure 1). Modificationof critical cysteine residues in signaling proteins can modulatecell function. Moreover, some cyPG, such as 15-deoxy-12,14-PGJ2(15d-PGJ2), may act as ligands for the transcription factorsof the nuclear receptor superfamily known as peroxisome proliferator–activatedreceptors (PPAR), which have been reported to play importantroles in the regulation of lipid metabolism (2) and in cardiovascularand renal pathophysiology (3,4). For these reasons, the potentialuse of cyPG as therapeutic agents has been explored in numerousstudies using cellular and animal models of inflammation orinjury. Protective effects of micromolar concentrations of 15d-PGJ2against ischemia-reperfusion injury and multiple-organ failurecaused by endotoxic shock have been reported (5–7). Thesebeneficial effects were associated with an inhibition of theinflammatory response and of the activation of transcriptionfactors NF-B and activator protein 1 (AP-1). Anti-inflammatoryeffects of 15d-PGJ2 have also been evidenced in cultured cells.In mesangial cells (MC), 15d-PGJ2 inhibited cytokine-elicitedinduction of cyclo-oxygenase-2 (8) and monocyte chemoattractantprotein-1 (9). By using an analog of 15d-PGJ2 that retains fullPPAR agonist activity but lacks the cyclopentenone structure,we showed recently that the capacity of 15d-PGJ2 to modify covalentlycellular thiols plays a key role in the inhibition of proinflammatorygenes such as inducible nitric oxide (iNOS), cyclo-oxygenase-2,and ICAM-1 in MC (10). Several proteins that are involved inthe activation of NF-B and AP-1, as well as components of thetranscription factors themselves, have been identified as targetsfor modification by 15d-PGJ2. Modification of IKK reduces NF-Bactivation (11,12), whereas 15d-PGJ2 addition to critical cysteinesin the DNA binding domains of NF-B and AP-1 proteins resultsin inhibition of DNA binding (13,14). CyPG also modulate cellproliferation. In MC, a biphasic effect of 15d-PGJ2 has beenreported, with low concentrations promoting cell proliferationand higher concentrations inducing cell death (15). On thisbasis, a potential for the use of cyPG in the restoration ofglomerular architecture in progressive glomerular disease hasbeen postulated (15). Another important feature of the effectof 15d-PGJ2 is the induction of a heat-shock response in manycell types, including MC (16,17). This stress response, whichrequires the cyclopentenone moiety, could contribute to thebeneficial effects of 15d-PGJ2 in renal cell injury during inflammationor ischemia (17). Therefore, the modification of protein thiolsby cyPG, which could be referred to as protein prostanylation,seems to play an important role in 15d-PGJ2 protective effects.Nevertheless, the potential for cytotoxic effects of 15d-PGJ2should also be considered. The identification of proteins thatare susceptible to be modified by cyPG addition could help definenovel targets for therapeutic intervention and identify potentialadverse effects. In a previous study, we observed the presenceof multiple targets for modification by biotinylated 15d-PGJ2in MC (10). Here we address the identification of the modifiedproteins by proteomic approaches (Figure 1) and report the bindingof biotinylated 15d-PGJ2 to several previously unknown targets.In addition, we provide evidence for the potential involvementof protein prostanylation in the regulation of MC cytoskeletalorganization.
Figure 1. Strategy for the detection and identification of proteins that were modified by biotinylated 15-deoxy-12,14-PGJ2 (15d-PGJ2). Intact mesangial cells (MC) are incubated with the biotinylated analog of 15d-PGJ2. This analog binds to cysteine residues in certain proteins by Michael addition. Duplicate samples from cell lysates that contained modified proteins are analyzed in parallel by two-dimensional (2D) electrophoresis followed by Western blot or total protein staining. Biotin-positive spots are analyzed by mass spectrometry techniques.
Materials
15d-PGJ2 was from Calbiochem-Novabiochem (San Diego, CA) orfrom Cayman Chemical (Ann Arbor, MI). 15d-PGJ2 biotinylatedat the carboxyl group was provided by Dr. F.J. Cañada(Centro de Investigaciones Bilógicas, Madrid, Spain).Recombinant human IL-1 (5 x 107 U/mg) was from Roche DiagnosticsS.L. (Barcelona, Spain). TNF- was from Serotec (Oxford, UK).Polyclonal anti-iNOS (sc-651) and anti-RhoGDI (sc-360) werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Horseradishperoxidase (HRP)-conjugated anti-rabbit Ig were from Dako (Glostrup,Denmark). Anti-vimentin, HRP-conjugated streptavidin, and enhancedchemiluminescence reagents were from Amersham Biosciences (Barcelona,Spain). Anti–heat-shock protein 90 (anti-Hsp90) was fromStressgen (Victoria, BC, Canada). The monoclonal anti-tubulinantibody was the gift of Dr. I. Barasoaín (Centro deInvestigaciones Biológicas, Madrid, Spain). Secondaryantibodies for immunofluorescence anti-rabbit–Texas Red,anti-mouse–Alexa488, and Phalloidin-Alexa546 were fromMolecular Probes (Invitrogen Life Technologies S.A., Barcelona,Spain). All other reagents were of the highest purity availablefrom Sigma Chemical Co. (St. Louis, MO).
Cell Culture
Cell culture media and supplements were from Invitrogen. RatMC were obtained as reported earlier (18). Cells were grownin RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100U/ml penicillin, and 100 µg/ml streptomycin. Throughoutthis study, passages 7 to 18 were used. Cell treatments wereperformed in serum-free medium. CyPG were added in DMSO. FinalDMSO concentration was 0.1% (vol/vol). Control cells receivedan equivalent amount of DMSO. For cytokine stimulation, confluentMC were incubated in serum-free medium for 24 h before the additionof a combination of 3 ng/ml IL-1 plus 37 ng/ml TNF-.
Incorporation of Biotinylated 15d-PGJ2 into MC Proteins
MC were incubated with biotinylated 15d-PGJ2 for 2 h in serum-freemedium. These conditions were found to yield maximal proteinlabeling. Lysates were obtained by disrupting cells in 50 mMTris (pH 7.5); 0.1 mM EDTA; 0.1 mM EGTA; 0.1 mM -mercaptoethanol;0.5% SDS that contained 2 µg/ml of each of the proteaseinhibitors leupeptin, pepstatin A, and aprotinin; and 1.3 mMPefablock (Roche). Biotin incorporation was assessed by Westernblot.
Protein Electrophoresis and Identification
For two-dimensional electrophoresis, cells were lysed in 20mM Hepes (pH 7.2), 50 mM NaCl, 1% NP-40, 0.3% sodium deoxycholate,and 0.1% SDS plus protease inhibitors. Aliquots of cell lysatesthat contained 600 µg of protein were precipitated with10% TCA, resuspended in 260 µl of IEF sample buffer (4%CHAPS, 2 M thiourea, 7 M urea, 100 mM dithiothreitol, and 0.5%Bio-lyte ampholytes), split in two aliquots and loaded on ReadyStripIPG Strips (pH 3 to 10; Bio-Rad, Hercules, CA) for isoelectricfocusing on a Protean IEF cell (Bio-Rad), following the instructionsof the manufacturer. For the second dimension, strips were loadedon duplicate 15% polyacrylamide SDS gels. Gels were stainedwith GelCode Blue (Pierce, Rockford, IL). One of the gels wassubsequently transferred to Immobilon P membrane (Millipore,Bedford, MA) and used for localization of biotinylated 15d-PGJ2–labeledspots by Western blot (10). The Coomassie-stained spots thatco-migrated with the biotin-positive proteins were excised fromthe duplicate gel. The accuracy of the procedure was confirmedby the disappearance of the biotin signal in the Western blotof the gel used for picking. The confirmed spots were subjectedto in-gel digestion with trypsin (19) and analysis by matrix-assistedlaser desorption ionization–time of flight mass spectrometry(MALDI-TOF MS) using -cyano-4-hydroxy-cinnamic acid as the matrix.Mass spectra were calibrated internally using the peptide mixresulting from trypsin autolysis. Proteins were identified withthe MASCOT (Matrix Science, London, UK) searching algorithmsusing the monoisotopic peptide masses and a peptide mass toleranceof ±50 ppm. When indicated, protein identity was confirmedby MALDI-TOF MS-MS analysis of selected peptides using the MALDI-tandemTOF mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems,Framingham, MA).
Avidin Pull-Down Assays
MC were incubated in the presence of 5 µM 15d-PGJ2 orbiotinylated 15d-PGJ2. Cells were lysed, and biotinylated proteinswere purified by adsorption onto Neutravidin beads (Pierce)following the manufacturer’s instructions. Proteins ofinterest were detected in the eluate by Western blot.
Fluorescence Microscopy
Cells that were grown on glass coverslips were treated withvarious agents for 2 h. For immunofluorescence, cells were fixedwith 3.5% formaldehyde and permeabilized with 0.1% Triton X-100.After blocking with 1% BSA, they were incubated with primaryantibodies at 1:200 dilution. Subsequently, coverslips werewashed with PBS and incubated with secondary antibodies at 1:200dilution and/or with DAPI for 1 h. Coverslips were mounted withFluorsafe (Calbiochem) and images were obtained with a LeicaTCS-SP2-AOBS-UV confocal inverted microscope, using a x63/1.4objective.
Plasmids and Transfections
Full-length human cDNA vimentin (Origene, Rockville, MD) wascloned into the EcoRI, SmaI sites of the pEGFP-C1 vector (Clontech,Palo Alto, CA) to obtain GFP-vimentin-wt. Cysteine 328 was mutatedto serine using the Quickchange XL site-directed mutagenesiskit from Stratagene (La Jolla, CA) and primers forward 5'-GGTGCAGTCCCTCACCTCTGAAGTGGATGCCC-3'and reverse 5'-GGGCATCCACTTCAGAGGTGAGGGACTGCACC-3' to obtainGFP-vimentin-C328S. Cells that were grown on glass coverslipswere transfected with constructs that were purified with Endofreeplasmid kit (Qiagen, Valencia, CA) using Lipofectamine 2000(Invitrogen). After 24 h, cells were treated as described above.
Effects of Biotinylated 15d-PGJ2 on the Stress and Inflammatory Responses of MC
The protective effects of cyPG have been attributed to theirability to induce a cell stress response and attenuate the inflammatoryresponse. Covalent protein modification is important for theseeffects (10,20). To substantiate the use of biotinylated 15d-PGJ2as a tool to identify potential targets for cyPG action, weassessed its ability to mimic the effects of 15d-PGJ2. The inductionof Hsp70 is a hallmark of the heat-shock response. Therefore,we assessed Hsp70 protein levels in cyPG-treated MC. As previouslyreported (17), micromolar concentrations of 15d-PGJ2 potentlyelicited Hsp70 expression. This effect was mimicked by the biotinylatedanalog (Figure 2A). We have previously shown that micromolarconcentrations of 15d-PGJ2 abolish cytokine-elicited iNOS inductionin MC (10). Treatment of MC with biotinylated 15d-PGJ2 alsoprovoked a marked inhibition of iNOS levels (95% reduction with5 µM and undetectable levels with 10 µM biotinylated15d-PGJ2, respectively; Figure 2B). These observations showthat the biotinylated analog recapitulates the anti-inflammatoryand stress-inducing effects of 15d-PGJ2 in MC.
Figure 2. Effects of biotinylated 15d-PGJ2 in MC. (A) MC were incubated in the presence of the indicated concentrations of cyclopentenone prostaglandins (cyPG) or vehicle for 16 h and the levels of constitutive and inducible heat-shock protein 70 (HSC70/Hsp70) were assessed by Western blot. (B) The induction of inducible nitric oxide synthase (iNOS) was elicited by treatment of MC with a cytokine mixture (Ck) for 16 h. CyPG were added to the medium 2 h before the addition of cytokines. Levels of iNOS were detected by Western blot. The levels of actin were used as a control for intersample variability. Results shown are representative of three experiments with similar results.
Binding of Biotinylated 15d-PGJ2 to Cellular Proteins in Intact MC
The effects of biotinylated 15d-PGJ2 on MC responses were associatedwith the modification of cellular proteins (Figure 3A). Bindingof cyPG to cellular proteins occurs through the formation ofadducts with free cysteine residues by Michael addition (Figure 1).Using radioactively labeled cyPG, this binding has beenshown to be stable under reducing conditions; however, it canbe hydrolyzed by alkali (21). As shown in Figure 3B, bindingof biotinylated 15d-PGJ2 to proteins in MC lysates was resistantto treatment with 10 mM dithiothreitol, but it was clearly reducedafter treatment with 0.1 N NaOH, thus showing the same susceptibilityas the binding of nonmodified cyPG. Taken together, these resultssupport the use of biotinylated 15d-PGJ2 as a tool to identifyprotein targets for covalent modification potentially involvedin the biologic effects of cyPG.
Figure 3. Labeling of MC proteins with biotinylated 15d-PGJ2. (A) MC were incubated with biotinylated 15d-PGJ2 as above, and the incorporation of the biotin label into MC proteins was assessed by Western blot and detection with horseradish peroxidase (HRP)-conjugated streptavidin using enhanced chemiluminescence. (B) Lysates from control or biotinylated 15d-PGJ2–treated cells were incubated in the presence of 10 mM dithiothreitol or 0.1 N NaOH for 30 min at room temperature. After desalting by gel filtration, lysates were analyzed by SDS-PAGE and Western blot, as above. To ensure even protein loading, membranes were stripped and rehybridized with anti-actin antibody. Results shown are representative of three assays.
Identification of Biotinylated 15d-PGJ2–Modified MC Proteins
To identify the proteins that are modified by 15d-PGJ2 in MC,we analyzed lysates from biotinylated 15d-PGJ2–treatedcells by two-dimensional electrophoresis. The patterns thatwere given by Coomassie staining and detection of biotin withHRP-streptavidin are shown in Figure 4. After superimpositionof both patterns, the Coomassie-stained proteins that coincidedwith the biotin-positive spots were excised and analyzed bytryptic digestion and MALDI-TOF MS. Figure 5 shows a representativeMALDI-TOF mass spectrum and peptide mass fingerprinting analysisthat corresponds to spot 3 from Figure 4, identified as vimentin.Table 1 displays a list of the proteins identified along witha summary of the identification data. As control for the selectivityof cyPG addition, we analyzed several spots that were clearlydetected with Coomassie but gave no signal with HRP-streptavidin.However, several biotin-positive spots were detected by Westernblot and could not be matched to any of the Coomassie-stainedspots and may represent less abundant proteins that get modifiedin a high proportion.
Figure 4. Analysis of biotinylated 15d-PGJ2–modified proteins by 2D electrophoresis. Cell lysates from MC incubated with 5 µM biotinylated 15d-PGJ2 for 2 h were analyzed by 2D electrophoresis as described in Materials and Methods. (Upper panel) Total protein staining with colloidal Coomassie blue. (Lower panel) Western blot and detection of modified proteins by incubation with HRP-streptavidin. Spots that were excised are indicated by numbers in the upper panel and the position of the co-migrating spots in the lower panel is indicated by arrowheads. A similar pattern of biotin staining was obtained in five independent experiments.
Figure 5. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis of spot 3. Spot 3 from Figure 4 was digested in gel with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS as detailed in the experimental section. (A) Typical mass spectrum from a representative experiment. (B) List of the monoisotopic masses of some of the peptides identified showing their position in the vimentin sequence (MSO, compatible with oxidation of methionine residues).
Table 1. Proteins identified by trypsin digestion and mass spectrometrya
The binding of biotinylated 15d-PGJ2 to some of the proteinsidentified was confirmed by pull-down assays using Neutravidin-agarose.Figure 6 shows that Hsp90, vimentin, tubulin, and actin, presentin lysates from MC that were treated with biotinylated 15d-PGJ2,were selectively retained on avidin beads. In contrast, theabundant cytosolic protein RhoGDI was not detected in the avidin-bindingfraction, thus suggesting that this protein is not a targetfor modification by cyPG. The various proteins that were foundin the avidin-binding fraction were present in different proportionswith respect to their levels in total lysates. Vimentin andtubulin were the proteins retained in a higher proportion, whereasonly a minor fraction of total Hsp90 was retained on avidinbeads. This may reflect the different degree of modificationof these targets by biotinylated 15d-PGJ2.
Figure 6. Retention of biotinylated 15d-PGJ2–modified proteins on avidin beads. Cell lysates from MC incubated with 5 µM 15d-PGJ2 or biotinylated 15d-PGJ2 for 2 h were subjected to pull-down assays with neutravidin agarose beads. The levels of the proteins of interest in total cell lysates and in the avidin-binding fractions were assessed by Western blot. Results shown are representative of at least three assays for every protein.
Interactions of 15d-PGJ2 with Cytoskeletal Proteins in MC
Several of the biotinylated 15d-PGJ2–modified proteinsidentified are constituents of microfilaments, microtubules,or intermediate filaments and are involved in cell architectureand dynamics. Therefore, we explored the effect of 15d-PGJ2on MC cytoskeletal organization by immunofluorescence (Figure 7).Incubation of MC with 15d-PGJ2 induced marked morphologicchanges in the vimentin filament network. Control MC showeda typical vimentin pattern characterized by abundant fine filamentsextending from the nuclear periphery toward the plasma membrane(Figure 7A). The position and the integrity of cell nuclei wereshown by DAPI staining. Treatment with 15d-PGJ2 resulted inthe disappearance of vimentin filaments from the cell peripheryand accumulation in the perinuclear region. For better evidencingthis effect, cells were stained with an antibody against thecytosolic protein RhoGDI. This clearly showed the loss of vimentinstaining from extensive areas of the cytoplasm. The organizationof tubulin also showed early changes after treatment with 15d-PGJ2,consisting of a reduction in the density and the length of microtubules,compatible with a disruption of the tubulin network. In contrast,actin fibers were not appreciably affected by 15d-PGJ2 treatmentunder these conditions. The reorganization of vimentin and tubulinnetworks was not accompanied by changes in the levels of theseproteins as assessed by Western blot (Figure 7B). It is interestingthat 9,10-dihydro-15d-PGJ2, an analog of 15d-PGJ2 that lacksthe cyclopentenone moiety and displays reduced ability to bindto proteins (10), did not induce vimentin or tubulin redistribution,thus suggesting that thiol modification is important for theeffects of 15d-PGJ2 on MC cytoskeletal organization. Consistentwith this, biotinylated 15d-PGJ2 induced a marked redistributionof intermediate vimentin filaments and microtubules that resembledthe effects of nonmodified 15d-PGJ2 (Figure 7C).
Figure 7. Effects of 15d-PGJ2 on cytoskeletal organization in MC. (A) MC were treated for 2 h in the absence or presence of 5 µM 15d-PGJ2, as indicated, and stained with antibodies against vimentin (green), tubulin (green), or RhoGDI (red) or with phalloidin to visualize filamentous actin (orange). Nuclei were stained with DAPI (blue). (B) Levels of total vimentin and tubulin were assessed by Western blot. (C) MC were treated for 2 h with 5 µM 9,10-dihydro-15d-PGJ2 or biotinylated 15d-PGJ2. After fixation, cells were stained with antibodies against vimentin or tubulin, as indicated, and DAPI. Bar = 47.62 µm. Confocal fluorescence microscopy images shown are maximum projections of series acquired at 0.5-µm intervals and are representative of three experiments with similar results.
To confirm the importance of cysteine modification in the effectsof cyPG, GFP-vimentin-wt and C328S mutant constructs were transfectedin MC. GFP-vimentin was incorporated into the endogenous intermediatefilament network, as it has been characterized previously (22).Cells that were transfected with GFP-vimentin-wt showed markedvimentin collapse in response to 15d-PGJ2 treatment (Figure 8).It is interesting that the proportion of cells that underwentvimentin reorganization was significantly lower in cells thatwere transfected with the GFP-vimentin-C328S mutant. These resultsindicate that mutation of this cysteine residue partially protectsthe vimentin network against 15d-PGJ2–elicited disruption.
Figure 8. Effect of 15d-PGJ2 on the reorganization of wild-type and mutant GFP-vimentin. MC transfected with plasmids GFP-vimentin-wt or GFP-vimentin-C328S were treated in the absence or presence of 5 µM 15d-PGJ2 and observed by confocal fluorescence microscopy. (A) Images were acquired as in Figure 7 and are representative of four experiments with similar results. Bar = 50 µm. (B) The proportion of cells that showed vimentin collapse was quantified by examination by two independent observers of at least 200 cells from randomly acquired fields per experimental point. Results shown are average values of three independent experiments ± SEM. *P < 0.05 by t test versus GFP-vimentin-wt–transfected cells that were treated with 15d-PGJ2.
CyPG have been reported to exert anti-inflammatory, antiproliferative,and antiviral effects in several experimental systems. The abilityof these compounds to covalently modify cellular proteins isan important mechanism for these effects. Biotinylated analogsof cyPG have been used as probes to explore these interactions.Here we have shown that biotinylated 15d-PGJ2 mimics the effectsof 15d-PGJ2 on the heat-shock and inflammatory responses ofMC in culture and forms stable adducts with proteins that displayresistance to treatment with reducing agents but are disruptedunder alkaline conditions, as expected of Michael adducts betweenproteins and cyPG.
CyPG can bind to a broad but defined set of cellular proteins.Work from several laboratories, including ours, has led to theidentification of several targets for prostanylation. Amongthese targets are the transcription factors NF-B and AP-1 (12–14);IKK (11,23); proteins involved in cellular redox status regulation,such as thioredoxin and thioredoxin reductase (24,25); the proteinKeap-1, a sensor of electrophilic stress and regulator of thetranscription factor Nrf-2 (26,27); and H-Ras proteins (28).In this work, we used a proteomic approach to identify proteintargets for cyPG addition. Using biotinylated 15d-PGJ2, we detectedat least 50 biotin-positive spots in extracts from MC. The totalnumber of potential targets for cyPG action may be higher becausesome minor proteins may not be detected by this assay. Also,the biotin moiety may preclude access of the biotinylated cyPGto cysteine residues in some proteins or interactions for whichthe presence of the carboxyl group of the cyPG is important,as it has been proposed for PPAR- (29). In this study, we identifiedseveral important regulatory proteins. Hsp90 regulates proteinsthat are implicated in apoptotic, survival, and growth pathways(30). Hsp90 has been shown to undergo reversible cysteine-targetedoxidation during renal oxidative stress (31). Several reactivecysteine residues at Hsp90 C-terminus seem to be important forfunction (32,33). Therefore, it would be interesting to explorethe implications of cyPG addition to Hsp90 for chaperone functionor heat-shock response.
Other proteins detected include nucleoside diphosphate kinase,a multifunctional enzyme involved in the maintenance of thecellular pools of nucleoside triphosphate and in transcriptionalregulation (34). Nucleoside diphosphate kinase has been shownto undergo S-thiolation or disulfide cross-linking under conditionsof oxidative stress, which could have implications in functionswitching (34–36). The enzyme methylthioadenosine phosphorylaseis involved in the synthesis of methionine and in the regulationof polyamine synthesis (37). On the basis of previous evidence,modification of methylthioadenosine phosphorylase by cyPG couldhave implications for cell proliferation or apoptosis (38).
The major targets of biotinylated 15d-PGJ2 in MC seem to bevimentin, tubulin, and actin. These results shed light on thecellular fate of cyPG by showing that an important proportionof the cellular PG-protein adducts is constituted by cytoskeletalproteins. In addition, we found the contractile protein tropomyosin.The results presented here show extensive reorganization ofthe intermediate filament network, consisting in a perinuclearcollapse of vimentin filaments. It is interesting that a similarcollapse of the vimentin network has been reported in severalcell types that underwent a heat-shock response (39,40); however,this is the first report of this kind of reorganization in MC.These changes could constitute part of the defense mechanismstriggered by 15d-PGJ2 in MC. We also observed a fading of themicrotubule network in MC at early times of treatment with 15d-PGJ2.In contrast, the distribution of the actin cytoskeleton didnot show significant changes within the time frame explored.
The effects of 15d-PGJ2 on MC cytoskeletal distribution couldbe mediated by the direct modification of cytoskeletal proteins.All proteins that were identified in this study possess cysteineresidues that are susceptible to oxidative modifications, andsome of them have been reported recently to undergo thiolationunder oxidative stress (35,36,41). The modification of exposedsulfhydryl groups in cytoskeletal proteins may play a regulatoryrole, thus transducing oxidative stress signals into cytoskeletalchanges. Tubulin is a cysteine-rich redox-sensitive proteinthat plays a crucial function in cell division. Modificationof tubulin redox state or alkylation of functional sulfhydrylgroups may lead to impairment of microtubule polymerizationand inhibition of cellular proliferation (42). This featurehas been exploited for the development of anticancer agents(43,44). Thus, it could be hypothesized that modification oftubulin by cyPG may be involved in the antiproliferative effectsof these compounds. Vimentin contains a single cysteine residuethat is highly conserved among vertebrates. Vimentin glutathionylationhas been detected in oxidatively stressed T lymphocytes (41).However, the consequences of this modification for cytoskeletalorganization have not been explored. Using a C328S vimentinmutant, we observed that the presence of this cysteine residueis important for the full effect of 15d-PGJ2 on vimentin reorganization.Our results suggest that modification of this residue couldhave important consequences for the organization of intermediatefilaments.
The possibility should also be considered that 15d-PGJ2 couldalter cytoskeletal organization by indirect mechanisms, includingmodification of proteins that are involved in the regulationof redox status, chaperones, G-proteins, or microtubule-interactingproteins. It has been reported that 15d-PGJ2 can bind to theCRTH2 chemoattractant G-protein–coupled receptor at nanomolarconcentrations (45). However, involvement of this pathway inthe effects herein reported is unlikely because nanomolar concentrationsof 15d-PGJ2 or of the CRTH2 agonist indomethacin did not elicitHsp70 induction, iNOS inhibition, or cytoskeletal remodeling(unpublished observations). At micromolar concentrations, 15d-PGJ2can also activate PPAR. Nevertheless, the compound 9,10-dihydro-15d-PGJ2,a potent PPAR- agonist that lacks the cyclopentenone moiety,failed to induce cytoskeletal reorganization in MC. This observationalso supports the hypothesis that covalent modification of cellularthiols plays an important role in the effects of 15d-PGJ2 onintermediate filament and microtubule networks. The preciseconcentrations of 15d-PGJ2 in biologic systems are still a matterof debate, as it has been discussed previously (14,25). However,this issue does not preclude the interest of the protectiveeffects of cyPG and of their use as model compounds to explorethe biologic effects and targets of endogenous cyclopentenoneeicosanoids with similar chemical reactivity that have beendetected in various biologic systems (46,47).
In summary, we have identified physiologically relevant targetsfor modification by cyPG. These findings may open new avenuesfor the understanding of the pleiotropic effects of cyPG andmay help to define their potential use as therapeutic agents.
Acknowledgments
This work was supported by grants SAF2003-03713 from MEyC and04/179-01 from Fundación La Caixa. K.S. and F.S.-G. arerecipients of fellowships from C.S.I.C. and MEyC, respectively.
We thank Dr. Isabel Barasoaín from Centro de InvestigacionesBiológicas (Madrid, Spain) for the generous gift of anti-tubulinantibody and Dr. F.J. Cañada (Centro de InvestigacionesBiológicas) for the gift of biotinylated 15d-PGJ2. Weare indebted to Dolores Gutiérrez (Unidad de Proteómica,Parque Científico de Madrid) for expert assistance withprotein identification by mass spectrometry and to M. TeresaSeisdedos (Centro de Investigaciones Biológicas) forvaluable help with confocal microscopy. The technical assistanceof M. Jesús Carrasco is gratefully acknowledged.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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12,14-Prostaglandin J2 in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton
Top
Abstract
Introduction
12,14-PGJ2 (15d-PGJ2) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ2 inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ2 may be dependent on or independent from its role as a peroxisome proliferator–activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator–activated receptor–independent effects of 15d-PGJ2 is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ2 recapitulates the effects of 15d-PGJ2 on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ2 was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ2–treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ2 induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG. 
B and activator protein 1 (AP-1). Anti-inflammatory effects of 15d-PGJ2 have also been evidenced in cultured cells. In mesangial cells (MC), 15d-PGJ2 inhibited cytokine-elicited induction of cyclo-oxygenase-2 (8) and monocyte chemoattractant protein-1 (9). By using an analog of 15d-PGJ2 that retains full PPAR agonist activity but lacks the cyclopentenone structure, we showed recently that the capacity of 15d-PGJ2 to modify covalently cellular thiols plays a key role in the inhibition of proinflammatory genes such as inducible nitric oxide (iNOS), cyclo-oxygenase-2, and ICAM-1 in MC (10). Several proteins that are involved in the activation of NF-
(5 x 107 U/mg) was from Roche Diagnostics S.L. (Barcelona, Spain). TNF-
was from Serotec (Oxford, UK). Polyclonal anti-iNOS (sc-651) and anti-RhoGDI (sc-360) were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit Ig were from Dako (Glostrup, Denmark). Anti-vimentin, HRP-conjugated streptavidin, and enhanced chemiluminescence reagents were from Amersham Biosciences (Barcelona, Spain). Anti–heat-shock protein 90 (anti-Hsp90) was from Stressgen (Victoria, BC, Canada). The monoclonal anti-tubulin antibody was the gift of Dr. I. Barasoaín (Centro de Investigaciones Biológicas, Madrid, Spain). Secondary antibodies for immunofluorescence anti-rabbit–Texas Red, anti-mouse–Alexa488, and Phalloidin-Alexa546 were from Molecular Probes (Invitrogen Life Technologies S.A., Barcelona, Spain). All other reagents were of the highest purity available from Sigma Chemical Co. (St. Louis, MO). 
(29). In this study, we identified several important regulatory proteins. Hsp90 regulates proteins that are implicated in apoptotic, survival, and growth pathways (30). Hsp90 has been shown to undergo reversible cysteine-targeted oxidation during renal oxidative stress (31). Several reactive cysteine residues at Hsp90 C-terminus seem to be important for function (32,33). Therefore, it would be interesting to explore the implications of cyPG addition to Hsp90 for chaperone function or heat-shock response. 
