Cell Biology

Identification of Novel Protein Targets for Modification by 15-Deoxy-Δ12,14-Prostaglandin J2 in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton

Konstantinos Stamatakis, Francisco J. Sánchez-Gómez and Dolores Pérez-Sala
JASN January 2006, 17 (1) 89-98; DOI: https://doi.org/10.1681/ASN.2005030329

Abstract

The cyclopentenone prostaglandin 15-deoxy-Δ12,14-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.

Prostaglandins with cyclopentenone structure are endogenous eicosanoids that are generated by nonenzymatic dehydration of arachidonic acid metabolites. Cyclopentenone prostaglandins (cyPG) exert varied biologic actions, including inhibition of cell proliferation in several cancer cell lines and anti-inflammatory and antiviral activities. The molecular basis for these varied effects is multiple. The common feature of these prostaglandins is the presence of an unsaturated carbonyl group in the cyclopentane ring (cyclopentenone; see Figure 1). This structure was found early to be an important requirement for the antitumoral and antiviral effects of cyPG (1). This moiety confers cyPG the capacity to form covalent adducts with thiol groups in glutathione or in proteins by Michael addition (Figure 1). Modification of critical cysteine residues in signaling proteins can modulate cell function. Moreover, some cyPG, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), may act as ligands for the transcription factors of the nuclear receptor superfamily known as peroxisome proliferator–activated receptors (PPAR), which have been reported to play important roles in the regulation of lipid metabolism (2) and in cardiovascular and renal pathophysiology (3,4). For these reasons, the potential use of cyPG as therapeutic agents has been explored in numerous studies using cellular and animal models of inflammation or injury. Protective effects of micromolar concentrations of 15d-PGJ2 against ischemia-reperfusion injury and multiple-organ failure caused by endotoxic shock have been reported (5–7). These beneficial effects were associated with an inhibition of the inflammatory response and of the activation of transcription factors NF-κB 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-κB and AP-1, as well as components of the transcription factors themselves, have been identified as targets for modification by 15d-PGJ2. Modification of IKK reduces NF-κB activation (11,12), whereas 15d-PGJ2 addition to critical cysteines in the DNA binding domains of NF-κB and AP-1 proteins results in inhibition of DNA binding (13,14). CyPG also modulate cell proliferation. In MC, a biphasic effect of 15d-PGJ2 has been reported, with low concentrations promoting cell proliferation and higher concentrations inducing cell death (15). On this basis, a potential for the use of cyPG in the restoration of glomerular architecture in progressive glomerular disease has been postulated (15). Another important feature of the effect of 15d-PGJ2 is the induction of a heat-shock response in many cell types, including MC (16,17). This stress response, which requires the cyclopentenone moiety, could contribute to the beneficial effects of 15d-PGJ2 in renal cell injury during inflammation or ischemia (17). Therefore, the modification of protein thiols by 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-PGJ2 should also be considered. The identification of proteins that are susceptible to be modified by cyPG addition could help define novel targets for therapeutic intervention and identify potential adverse effects. In a previous study, we observed the presence of multiple targets for modification by biotinylated 15d-PGJ2 in MC (10). Here we address the identification of the modified proteins by proteomic approaches (Figure 1) and report the binding of biotinylated 15d-PGJ2 to several previously unknown targets. In addition, we provide evidence for the potential involvement of protein prostanylation in the regulation of MC cytoskeletal organization.

Figure 1.
Figure 1.

Strategy for the detection and identification of proteins that were modified by biotinylated 15-deoxy-Δ12,14-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 and Methods

Materials

15d-PGJ2 was from Calbiochem-Novabiochem (San Diego, CA) or from Cayman Chemical (Ann Arbor, MI). 15d-PGJ2 biotinylated at the carboxyl group was provided by Dr. F.J. Cañada (Centro de Investigaciones Bilógicas, Madrid, Spain). Recombinant human IL-1β (5 × 107 U/mg) was from Roche Diagnostics S.L. (Barcelona, Spain). TNF-α 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).

Cell Culture

Cell culture media and supplements were from Invitrogen. Rat MC were obtained as reported earlier (18). Cells were grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Throughout this study, passages 7 to 18 were used. Cell treatments were performed in serum-free medium. CyPG were added in DMSO. Final DMSO concentration was 0.1% (vol/vol). Control cells received an equivalent amount of DMSO. For cytokine stimulation, confluent MC were incubated in serum-free medium for 24 h before the addition of a combination of 3 ng/ml IL-1β plus 37 ng/ml TNF-α.

Incorporation of Biotinylated 15d-PGJ2 into MC Proteins

MC were incubated with biotinylated 15d-PGJ2 for 2 h in serum-free medium. These conditions were found to yield maximal protein labeling. Lysates were obtained by disrupting cells in 50 mM Tris (pH 7.5); 0.1 mM EDTA; 0.1 mM EGTA; 0.1 mM β-mercaptoethanol; 0.5% SDS that contained 2 μg/ml of each of the protease inhibitors leupeptin, pepstatin A, and aprotinin; and 1.3 mM Pefablock (Roche). Biotin incorporation was assessed by Western blot.

Protein Electrophoresis and Identification

For two-dimensional electrophoresis, cells were lysed in 20 mM Hepes (pH 7.2), 50 mM NaCl, 1% NP-40, 0.3% sodium deoxycholate, and 0.1% SDS plus protease inhibitors. Aliquots of cell lysates that contained 600 μg of protein were precipitated with 10% 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 ReadyStrip IPG Strips (pH 3 to 10; Bio-Rad, Hercules, CA) for isoelectric focusing on a Protean IEF cell (Bio-Rad), following the instructions of the manufacturer. For the second dimension, strips were loaded on duplicate 15% polyacrylamide SDS gels. Gels were stained with GelCode Blue (Pierce, Rockford, IL). One of the gels was subsequently transferred to Immobilon P membrane (Millipore, Bedford, MA) and used for localization of biotinylated 15d-PGJ2–labeled spots by Western blot (10). The Coomassie-stained spots that co-migrated with the biotin-positive proteins were excised from the duplicate gel. The accuracy of the procedure was confirmed by the disappearance of the biotin signal in the Western blot of the gel used for picking. The confirmed spots were subjected to in-gel digestion with trypsin (19) and analysis by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using α-cyano-4-hydroxy-cinnamic acid as the matrix. Mass spectra were calibrated internally using the peptide mix resulting from trypsin autolysis. Proteins were identified with the MASCOT (Matrix Science, London, UK) searching algorithms using the monoisotopic peptide masses and a peptide mass tolerance of ±50 ppm. When indicated, protein identity was confirmed by MALDI-TOF MS-MS analysis of selected peptides using the MALDI-tandem TOF mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA).

Avidin Pull-Down Assays

MC were incubated in the presence of 5 μM 15d-PGJ2 or biotinylated 15d-PGJ2. Cells were lysed, and biotinylated proteins were purified by adsorption onto Neutravidin beads (Pierce) following the manufacturer’s instructions. Proteins of interest were detected in the eluate by Western blot.

Fluorescence Microscopy

Cells that were grown on glass coverslips were treated with various agents for 2 h. For immunofluorescence, cells were fixed with 3.5% formaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 1% BSA, they were incubated with primary antibodies at 1:200 dilution. Subsequently, coverslips were washed with PBS and incubated with secondary antibodies at 1:200 dilution and/or with DAPI for 1 h. Coverslips were mounted with Fluorsafe (Calbiochem) and images were obtained with a Leica TCS-SP2-AOBS-UV confocal inverted microscope, using a ×63/1.4 objective.

Plasmids and Transfections

Full-length human cDNA vimentin (Origene, Rockville, MD) was cloned into the EcoRI, SmaI sites of the pEGFP-C1 vector (Clontech, Palo Alto, CA) to obtain GFP-vimentin-wt. Cysteine 328 was mutated to serine using the Quickchange XL site-directed mutagenesis kit from Stratagene (La Jolla, CA) and primers forward 5′-GGTGCAGTCCCTCACCTCTGAAGTGGATGCCC-3′ and reverse 5′-GGGCATCCACTTCAGAGGTGAGGGACTGCACC-3′ to obtain GFP-vimentin-C328S. Cells that were grown on glass coverslips were transfected with constructs that were purified with Endofree plasmid kit (Qiagen, Valencia, CA) using Lipofectamine 2000 (Invitrogen). After 24 h, cells were treated as described above.

Results

Effects of Biotinylated 15d-PGJ2 on the Stress and Inflammatory Responses of MC

The protective effects of cyPG have been attributed to their ability to induce a cell stress response and attenuate the inflammatory response. Covalent protein modification is important for these effects (10,20). To substantiate the use of biotinylated 15d-PGJ2 as a tool to identify potential targets for cyPG action, we assessed its ability to mimic the effects of 15d-PGJ2. The induction of Hsp70 is a hallmark of the heat-shock response. Therefore, we assessed Hsp70 protein levels in cyPG-treated MC. As previously reported (17), micromolar concentrations of 15d-PGJ2 potently elicited Hsp70 expression. This effect was mimicked by the biotinylated analog (Figure 2A). We have previously shown that micromolar concentrations of 15d-PGJ2 abolish cytokine-elicited iNOS induction in MC (10). Treatment of MC with biotinylated 15d-PGJ2 also provoked a marked inhibition of iNOS levels (95% reduction with 5 μM and undetectable levels with 10 μM biotinylated 15d-PGJ2, respectively; Figure 2B). These observations show that the biotinylated analog recapitulates the anti-inflammatory and stress-inducing effects of 15d-PGJ2 in MC.

Figure 2.
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 associated with the modification of cellular proteins (Figure 3A). Binding of cyPG to cellular proteins occurs through the formation of adducts with free cysteine residues by Michael addition (Figure 1). Using radioactively labeled cyPG, this binding has been shown to be stable under reducing conditions; however, it can be hydrolyzed by alkali (21). As shown in Figure 3B, binding of biotinylated 15d-PGJ2 to proteins in MC lysates was resistant to treatment with 10 mM dithiothreitol, but it was clearly reduced after treatment with 0.1 N NaOH, thus showing the same susceptibility as the binding of nonmodified cyPG. Taken together, these results support the use of biotinylated 15d-PGJ2 as a tool to identify protein targets for covalent modification potentially involved in the biologic effects of cyPG.

Figure 3.
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–treated cells by two-dimensional electrophoresis. The patterns that were given by Coomassie staining and detection of biotin with HRP-streptavidin are shown in Figure 4. After superimposition of both patterns, the Coomassie-stained proteins that coincided with the biotin-positive spots were excised and analyzed by tryptic digestion and MALDI-TOF MS. Figure 5 shows a representative MALDI-TOF mass spectrum and peptide mass fingerprinting analysis that corresponds to spot 3 from Figure 4, identified as vimentin. Table 1 displays a list of the proteins identified along with a summary of the identification data. As control for the selectivity of cyPG addition, we analyzed several spots that were clearly detected with Coomassie but gave no signal with HRP-streptavidin. However, several biotin-positive spots were detected by Western blot and could not be matched to any of the Coomassie-stained spots and may represent less abundant proteins that get modified in a high proportion.

Figure 4.
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.
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).

View this table:
Table 1.

Proteins identified by trypsin digestion and mass spectrometrya

The binding of biotinylated 15d-PGJ2 to some of the proteins identified was confirmed by pull-down assays using Neutravidin-agarose. Figure 6 shows that Hsp90, vimentin, tubulin, and actin, present in lysates from MC that were treated with biotinylated 15d-PGJ2, were selectively retained on avidin beads. In contrast, the abundant cytosolic protein RhoGDI was not detected in the avidin-binding fraction, thus suggesting that this protein is not a target for modification by cyPG. The various proteins that were found in the avidin-binding fraction were present in different proportions with respect to their levels in total lysates. Vimentin and tubulin were the proteins retained in a higher proportion, whereas only a minor fraction of total Hsp90 was retained on avidin beads. This may reflect the different degree of modification of these targets by biotinylated 15d-PGJ2.

Figure 6.
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 proteins identified are constituents of microfilaments, microtubules, or intermediate filaments and are involved in cell architecture and dynamics. Therefore, we explored the effect of 15d-PGJ2 on MC cytoskeletal organization by immunofluorescence (Figure 7). Incubation of MC with 15d-PGJ2 induced marked morphologic changes in the vimentin filament network. Control MC showed a typical vimentin pattern characterized by abundant fine filaments extending from the nuclear periphery toward the plasma membrane (Figure 7A). The position and the integrity of cell nuclei were shown by DAPI staining. Treatment with 15d-PGJ2 resulted in the disappearance of vimentin filaments from the cell periphery and accumulation in the perinuclear region. For better evidencing this effect, cells were stained with an antibody against the cytosolic protein RhoGDI. This clearly showed the loss of vimentin staining from extensive areas of the cytoplasm. The organization of 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 treatment under these conditions. The reorganization of vimentin and tubulin networks was not accompanied by changes in the levels of these proteins as assessed by Western blot (Figure 7B). It is interesting that 9,10-dihydro-15d-PGJ2, an analog of 15d-PGJ2 that lacks the cyclopentenone moiety and displays reduced ability to bind to proteins (10), did not induce vimentin or tubulin redistribution, thus suggesting that thiol modification is important for the effects of 15d-PGJ2 on MC cytoskeletal organization. Consistent with this, biotinylated 15d-PGJ2 induced a marked redistribution of intermediate vimentin filaments and microtubules that resembled the effects of nonmodified 15d-PGJ2 (Figure 7C).

Figure 7.
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 effects of cyPG, GFP-vimentin-wt and C328S mutant constructs were transfected in MC. GFP-vimentin was incorporated into the endogenous intermediate filament network, as it has been characterized previously (22). Cells that were transfected with GFP-vimentin-wt showed marked vimentin collapse in response to 15d-PGJ2 treatment (Figure 8). It is interesting that the proportion of cells that underwent vimentin reorganization was significantly lower in cells that were transfected with the GFP-vimentin-C328S mutant. These results indicate that mutation of this cysteine residue partially protects the vimentin network against 15d-PGJ2–elicited disruption.

Figure 8.
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.

Discussion

CyPG have been reported to exert anti-inflammatory, antiproliferative, and antiviral effects in several experimental systems. The ability of these compounds to covalently modify cellular proteins is an important mechanism for these effects. Biotinylated analogs of cyPG have been used as probes to explore these interactions. Here we have shown that biotinylated 15d-PGJ2 mimics the effects of 15d-PGJ2 on the heat-shock and inflammatory responses of MC in culture and forms stable adducts with proteins that display resistance to treatment with reducing agents but are disrupted under alkaline conditions, as expected of Michael adducts between proteins and cyPG.

CyPG can bind to a broad but defined set of cellular proteins. Work from several laboratories, including ours, has led to the identification of several targets for prostanylation. Among these targets are the transcription factors NF-κB and AP-1 (12–14); IKK (11,23); proteins involved in cellular redox status regulation, such as thioredoxin and thioredoxin reductase (24,25); the protein Keap-1, a sensor of electrophilic stress and regulator of the transcription factor Nrf-2 (26,27); and H-Ras proteins (28). In this work, we used a proteomic approach to identify protein targets for cyPG addition. Using biotinylated 15d-PGJ2, we detected at least 50 biotin-positive spots in extracts from MC. The total number of potential targets for cyPG action may be higher because some minor proteins may not be detected by this assay. Also, the biotin moiety may preclude access of the biotinylated cyPG to cysteine residues in some proteins or interactions for which the presence of the carboxyl group of the cyPG is important, as it has been proposed for PPAR-γ (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.

Other proteins detected include nucleoside diphosphate kinase, a multifunctional enzyme involved in the maintenance of the cellular pools of nucleoside triphosphate and in transcriptional regulation (34). Nucleoside diphosphate kinase has been shown to undergo S-thiolation or disulfide cross-linking under conditions of oxidative stress, which could have implications in function switching (34–36). The enzyme methylthioadenosine phosphorylase is involved in the synthesis of methionine and in the regulation of polyamine synthesis (37). On the basis of previous evidence, modification of methylthioadenosine phosphorylase by cyPG could have implications for cell proliferation or apoptosis (38).

The major targets of biotinylated 15d-PGJ2 in MC seem to be vimentin, tubulin, and actin. These results shed light on the cellular fate of cyPG by showing that an important proportion of the cellular PG-protein adducts is constituted by cytoskeletal proteins. In addition, we found the contractile protein tropomyosin. The results presented here show extensive reorganization of the intermediate filament network, consisting in a perinuclear collapse of vimentin filaments. It is interesting that a similar collapse of the vimentin network has been reported in several cell 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 mechanisms triggered by 15d-PGJ2 in MC. We also observed a fading of the microtubule network in MC at early times of treatment with 15d-PGJ2. In contrast, the distribution of the actin cytoskeleton did not show significant changes within the time frame explored.

The effects of 15d-PGJ2 on MC cytoskeletal distribution could be mediated by the direct modification of cytoskeletal proteins. All proteins that were identified in this study possess cysteine residues that are susceptible to oxidative modifications, and some of them have been reported recently to undergo thiolation under oxidative stress (35,36,41). The modification of exposed sulfhydryl groups in cytoskeletal proteins may play a regulatory role, thus transducing oxidative stress signals into cytoskeletal changes. Tubulin is a cysteine-rich redox-sensitive protein that plays a crucial function in cell division. Modification of tubulin redox state or alkylation of functional sulfhydryl groups may lead to impairment of microtubule polymerization and inhibition of cellular proliferation (42). This feature has been exploited for the development of anticancer agents (43,44). Thus, it could be hypothesized that modification of tubulin by cyPG may be involved in the antiproliferative effects of these compounds. Vimentin contains a single cysteine residue that is highly conserved among vertebrates. Vimentin glutathionylation has been detected in oxidatively stressed T lymphocytes (41). However, the consequences of this modification for cytoskeletal organization have not been explored. Using a C328S vimentin mutant, we observed that the presence of this cysteine residue is important for the full effect of 15d-PGJ2 on vimentin reorganization. Our results suggest that modification of this residue could have important consequences for the organization of intermediate filaments.

The possibility should also be considered that 15d-PGJ2 could alter cytoskeletal organization by indirect mechanisms, including modification of proteins that are involved in the regulation of redox status, chaperones, G-proteins, or microtubule-interacting proteins. It has been reported that 15d-PGJ2 can bind to the CRTH2 chemoattractant G-protein–coupled receptor at nanomolar concentrations (45). However, involvement of this pathway in the effects herein reported is unlikely because nanomolar concentrations of 15d-PGJ2 or of the CRTH2 agonist indomethacin did not elicit Hsp70 induction, iNOS inhibition, or cytoskeletal remodeling (unpublished observations). At micromolar concentrations, 15d-PGJ2 can also activate PPAR. Nevertheless, the compound 9,10-dihydro-15d-PGJ2, a potent PPAR-γ agonist that lacks the cyclopentenone moiety, failed to induce cytoskeletal reorganization in MC. This observation also supports the hypothesis that covalent modification of cellular thiols plays an important role in the effects of 15d-PGJ2 on intermediate filament and microtubule networks. The precise concentrations of 15d-PGJ2 in biologic systems are still a matter of debate, as it has been discussed previously (14,25). However, this issue does not preclude the interest of the protective effects of cyPG and of their use as model compounds to explore the biologic effects and targets of endogenous cyclopentenone eicosanoids with similar chemical reactivity that have been detected in various biologic systems (46,47).

In summary, we have identified physiologically relevant targets for modification by cyPG. These findings may open new avenues for the understanding of the pleiotropic effects of cyPG and may help to define their potential use as therapeutic agents.

Acknowledgments

This work was supported by grants SAF2003-03713 from MEyC and 04/179-01 from Fundación La Caixa. K.S. and F.S.-G. are recipients of fellowships from C.S.I.C. and MEyC, respectively.

We thank Dr. Isabel Barasoaín from Centro de Investigaciones Biológicas (Madrid, Spain) for the generous gift of anti-tubulin antibody and Dr. F.J. Cañada (Centro de Investigaciones Biológicas) for the gift of biotinylated 15d-PGJ2. We are indebted to Dolores Gutiérrez (Unidad de Proteómica, Parque Científico de Madrid) for expert assistance with protein identification by mass spectrometry and to M. Teresa Seisdedos (Centro de Investigaciones Biológicas) for valuable help with confocal microscopy. The technical assistance of M. Jesús Carrasco is gratefully acknowledged.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Identification of Novel Protein Targets for Modification by 15-Deoxy-Δ12,14-Prostaglandin J2 in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton
Konstantinos Stamatakis, Francisco J. Sánchez-Gómez, Dolores Pérez-Sala
JASN Jan 2006, 17 (1) 89-98; DOI: 10.1681/ASN.2005030329
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