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15-Deoxy-∆12,14-prostaglandin J2: An Electrophilic Trigger of Cellular Responses Koji Uchida* and Takahiro Shibata Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan ReceiVed May 22, 2007
Electrophilic molecules are endogenously generated and are causally involved in many pathophysiological effects. Prostaglandin D2 (PGD2), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J2-series such as 15-deoxy-∆12,14PGJ2 (15d-PGJ2). 15d-PGJ2 is an electrophile, which can covalently react via the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins that play an important role in the control of the redox cell-signaling pathways. Covalent binding of 15dPGJ2 to cellular proteins may be one of the mechanisms by which 15d-PGJ2 induces a cellular response involved in most of the pathophysiological effects associated with inflammation. In the present perspective, we provide a comprehensive summary of 15d-PGJ2 as an electrophilic mediator of cellular responses. Contents Introduction Formation of 15d-PGJ2 from PGD2 Detection of 15d-PGJ2 Quantification of 15d-PGJ2 15d-PGJ2 As an Electrophile Cellular Responses Induced by 15d-PGJ2 6.1. Antioxidant Response 6.2. Anti-Inflammation 6.3. Redox Alteration 6.4. Protein Turnover 6.5. Apoptosis 6.6. Cell Growth and Differentiation 6.7. Cytoskeletal Dysfunction 6.8. Neuronal Differentiation 7. Conclusions 1. 2. 3. 4. 5. 6.
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1. Introduction The prostaglandins (PGs1) are a family of structurally related molecules that are produced by cells in response to a variety of extrinsic stimuli and regulate cellular growth, differentiation, and homeostasis (1, 2). Among them, PGD2 is a major cyclooxygenase (COX) product in a variety of tissues and cells and has marked effects on a number of biological processes, including platelet aggregation, relaxation of vascular and nonvascular smooth muscles, and nerve cell functions (3). PGD synthase (PGDS) catalyzes the isomerization of the 9,10endoperoxide group of PGH2, a precursor of prostanoids, to produce PGD2. Two distinct types of PGDS have been identified: lipocalin enzyme (L-PGDS) and hematopoietic enzyme (H-PGDS). Although L-PGDS is expressed in the central nervous system and male genital organs of various mammals, H-PGDS is widely expressed in peripheral tissues as well as in * To whom correspondence should be addressed. Tel: 81-52-789-4127. Fax: 81-52-789-5741. E-mail:
[email protected]. 1 Abbreviations: COX, cyclooxygenase; HNE, 4-hydroxy-2-nonenal; LCMS/MS, liquid chromatography/tandem mass spectrometry; LDL, lowdensity lipoprotein; mAb, monoclonal antibody; MALDI-TOF MS, matrixassisted laser desorption and ionization time-of-flight mass spectrometry; PG, prostaglandin; 15d-PGJ2, 15-deoxy-∆12,14-prostaglandin J2; PPARγ, peroxisome proliferator-activated receptor γ; Trx, thioredoxin.
antigen-presenting cells, mast cells, and megakaryocytes (4). The PGs are physiologically present in body fluids in picomolarto-nanomolar concentrations (5); however, arachidonate metabolism is significantly increased under several pathological conditions, including hyperthermia, infection, and inflammation (6), and local PG concentrations in the micromolar range have been detected at sites of acute inflammation (7). The two major enzymatic pathways responsible for the catabolism of PGD2 are an 11-ketoreductase and NADP-linked 15-hydroxy-PGdehydrogenase (8, 9). The former pathway leads to the production of 11β-PGF2R. The second pathway leads to the formation of 13,14-dihydro-15-keto-PGD2, which does not appear to be biologically active. In the early 1980s, it was discovered that PGD2 readily undergoes dehydration in ViVo and in Vitro to yield biologically active PGs of the J2 series, such as PGJ2, ∆12-PGJ2, and 15deoxy-∆12,14-PGJ2 (15d-PGJ2) (10, 11). These cyclopentenonetype PGs are actively transported into cells and accumulated in the nuclei, where they act as potent inducers of cell growth inhibition and cell differentiation and exhibit their own unique spectrum of biological effects, including the inhibition of cell cycle progression, the suppression of viral replication, the induction of the heat shock protein expression, and the stimulation of osteogenesis (12). One of the most important findings about this type of PG is that 15d-PGJ2 as the endogenous lipid mediator is a ligand for the nuclear receptor, the peroxisome proliferator-activated receptor γ (PPARγ), which promotes adipocyte differentiation (13, 14). However, members of the J2 series of the PGs, unlike other classes of eicosanoids, characterized by the presence of an electrophilic R,β-unsaturated carbonyl group in the cyclopentenone ring, have their own unique spectrum of biological effects. The reactive center of the cyclopentenone PGs has been proposed to account for some of their receptor-independent biological actions (15, 16). They can covalently react via the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins that play an important role in the control of the redox cell signaling pathways (12, 15, 16). In the present perspective, we provide a comprehensive summary of 15d-PGJ2 as an endogenous electrophile and
10.1021/tx700177j CCC: $40.75 2008 American Chemical Society Published on Web 12/04/2007
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15d-PGJ2 accumulates in the cytoplasm of most of the foamy or spindle macrophages in human atherosclerotic plaques (17) and in the spinal cord of sporadic amyotrophic lateral sclerosis (ALS) patients (20). In addition, the 15d-PGJ2 immunoreactivity was also detected in the activated macrophages by lipopolysaccharides (LPS), carrageenan, and IL-13 (17, 21–23). These immunochemical studies revealed that intracellular and extracellular 15d-PGJ2 could function as both autocrine and paracrine factors, respectively. These findings also suggested that (i) because PGD2 is the major prostaglandin in most tissues, these cyclopentenone-type PGD2 metabolites are likely to be produced at a number of sites and may reach functionally significant levels in inflammatory processes and that (ii) COX-2 up-regulation, through its pivotal role in inflammation, followed by the enhanced intracellular production of 15d-PGJ2 might be ubiquitously involved in inflammation and its related disorders.
4. Quantification of 15d-PGJ2
Figure 1. Conversion of PGD2 to 15d-PGJ2.
illustrate some recent approaches to establish the involvement of this PGD2 metabolite as a possible electrophilic mediator of cellular response in the pathogenesis of diseases.
2. Formation of 15d-PGJ2 from PGD2 The in Vitro synthesis of 15d-PGJ2 was first reported by Fitzpatrick and Wynalda (10). These authors have shown that human serum albumin catalyzes the in Vitro transformation of PGD2 into three dehydration products, such as ∆12-PGJ2, 15dPGD2, and 15d-PGJ2. In a later study, Shibata et al. (17) investigated the mechanism of formation of 15d-PGJ2 using a chiral-phase HPLC method for separation of the PGD2 metabolites and established that (i) the ketone group of PGD2 at C11 facilitates the β-elimination of the hydroxyl group at C9 to form PGJ2, (ii) PGJ2 is directly converted into 15d-PGJ2 in an albumin-independent manner, (iii) PGJ2 is stoichiometrically converted into ∆12-PGJ2 in an albumin-dependent manner, and (iv) both 15d-PGD2 and ∆12-PGJ2 are not converted to 15dPGJ2 (Figure 1). It has been speculated that the formation of R,β-unsaturated ketone within the cyclopentenone ring of PGJ2 facilitates the ∆12 isomerization followed by dehydration of the C15 hydroxyl group to generate 15d-PGJ2.
3. Detection of 15d-PGJ2 It is not known whether the PGD2 pathway is utilized in an organism, but it is clear that the J2-type prostanoids are produced in ViVo. This is based on the observations that ∆12-PGJ2 is a natural component of human body fluids and that ∆12-PGJ2 synthesis is suppressed by treatment with COX inhibitors (18). The natural precursor of the PGJ2 derivatives appears to be PGD2 because its in ViVo administration leads to a significant increase in ∆12-PGJ2 (18). The in ViVo presence of 15d-PGJ2 has been demonstrated by immunochemical procedures. Girloy et al. (19) first determined the inflammatory exudate levels of 15d-PGJ2 by enzyme immunoassay throughout carrageenininduced pleurisy (19). Later, Shibata et al. (17) developed a murine monoclonal antibody against 15d-PGJ2 and found that
Immunologic detection is a powerful tool that can be used to evaluate the presence of a desired target and its subcellular localization. Major advantages of this technique over biochemical approaches are the evaluation of small numbers of cells or archival tissues that may otherwise not be subject to analysis. The anti-15d-PGJ2 mAb 11G2 has been reported to be able to measure the extracellular amount of 15d-PGJ2 in medium from RAW264.7 macrophage stimulated by LPS (17). However, the specificity and sensitivity of the antibody still cast a shadow of doubt. As an alternative approach, Bell-Parikh et al. (24) developed a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for quantification of 15d-PGJ2 and demonstrated that 3T3-L1 fibroblasts could produce a very small amount of 15d-PGJ2. The authors have also shown that the levels of 15d-PGJ2 excreted in urine are very low compared to the levels of other PGs and are not altered in diabetes or after the administration of LPS, leading to the conclusion that the amounts of 15dPGJ2 generated in ViVo are insufficient for it to play a physiological role in modulating most of the responses. However, this study also lacks positive controls showing that they could detect 15d-PGJ2 added directly or formed from exogenous PGD2. In addition, because of its hydrophobic properties, 15dPGJ2 may be localized and concentrated in membrane compartments, leading to the accumulation of greater local amounts of 15d-PGJ2 than the amounts determined using the whole cells. Furthermore, there are intrinsic difficulties associated with measuring something that is so reactive. Like other reactive species (e.g., hydrogen peroxide and reactive aldehydes), 15dPGJ2 reacts readily with sulfhydryl groups of proteins and glutathione (GSH) to form conjugates, although no study has addressed the concentration of these conjugates in Vitro and in ViVo. On the basis of the reactivity and instability of 15d-PGJ2, both chemical and immunochemical procedures are still of limited usefulness in defining the absolute concentrations of free 15d-PGJ2 and its related prostanoids inside the cells. Thus, it may be still too early to underestimate the physiological and pathophysiological roles of this molecule.
5. 15d-PGJ2 As an Electrophile It has been suggested that some of the effects of PGJ2 are mediated through its covalent binding to proteins (Figure 2). This is due to the reactive cyclopentenone ring of 15d-PGJ2, which readily reacts with substances containing nucleophilic groups such as the cysteinyl thiol groups of proteins. Such reactions are termed Michael addition reactions. The regioselectivity of the thiol addition has been established by Suzuki et
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Figure 2. Michael-type addition of 15d-PGJ2 to cysteine residues of protein.
al. (25), in which the reaction of the cyclopentenone PGs with thiol molecules occurs at the endocyclic β-position within the cyclopentenone ring and affords a single monoadduct. Moreover, using (E)-4-methyl-5-ethylidene-2-cycolopentenone, a model of cyclopentenone PGs, the preferential reactivity of this position has been demonstrated by the molecular orbital calculation (25). This reactive center has been proposed to account for some of the receptor-independent biological actions of 15d-PGJ2, on the basis of the observations that (i) 9,10-dihydro-15d-PGJ2, a 15dPGJ2 analogue lacking the reactive R,β-unsaturated carbonyl group, could not reproduce many effects of 15d-PGJ2 (26–28) and that (ii) site-directed mutation at cysteine residues of the target protein virtually abolishes the effect of 15d-PGJ2 (15, 29–31).
6. Cellular Responses Induced by 15d-PGJ2 6.1. Antioxidant Response. Elevation of the activities of the antioxidant and phase 2 detoxification enzymes of cells provides protection against neoplasia. It has been suggested that the induction of electrophile-processing enzymes, for example, glutathione S-transferases (GSTs), UDP-glucuronosyltransferases, and NAD(P)H: quinone oxidoreductase-1 (NQO1), is a major protective mechanism. The induction of these enzymes is evoked by an extraordinary variety of chemical agents, including Michael reaction acceptors (32). The transcriptional activation of the chemoprotective response genes by these compounds has been traced to a cis-acting transcriptional enhancer called the antioxidant response element (ARE) (33). In humans, detoxification enzymes involved with the insult of oxidative stress are up-regulated by the ARE, for example, NQO-1, aldo-keto reductase 1C1 (AKR1C1)-AKR1C3, and γ-glutamyl cysteine ligase (GCL). The Nrf2-Keap1 system coordinately regulates the cytoprotective gene expression via the ARE (34). Kawamoto et al. (35) developed a cell culture system that potently responds to the phenolic antioxidants resulting in the induction of the GST activity and first reported that 15d-PGJ2 is an excellent inducer of antioxidant response. Later, several studies have established that 15d-PGJ2 induces the response via the Keap1Nrf2 pathway (21, 22, 36, 37). It was first proposed that the inducers disrupt the Keap1–Nrf2 complex by modifying the cysteine residues of Keap1 (38), allowing Nrf2 to translocate to the nucleus where, in heterodimeric combinations with other basic leucine zipper proteins, it binds to the ARE of the detoxification enzyme genes and accelerates their transcription. More recently, however, several studies have shown that the electrophile modification of Keap1 inhibits ubiquitin conjugation to Nrf2 by the Keap1–Cullin3 complex, thereby provoking the opening of the Keap1 gate and resulting in the nuclear accumulation of Nrf2 (39, 40). The high cysteine content of Keap1 has suggested that it is an excellent candidate as the sensor for 15d-PGJ2. Among the several candidate cysteines for the actual targets for inducers, Cys-273 and Cys-288 were first proposed to be critical for exposure to the inducers. Meanwhile, in other studies, an alternative cysteine (Cys-151)
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has been suggested to be involved in the sensing mechanisms of electrophiles (41, 42). 6.2. Anti-Inflammation. 15d-PGJ2 is emerging as a likely regulator of acute and chronic inflammation. The anti-inflammatory effect of 15d-PGJ2 was first deduced from an animal model of carrageenin-induced inflammation in which a twophase PG release was described after the expression of COX-2; the PGE2 synthesis predominates during the early inflammatory step, whereas 15d-PGJ2 substitutes for PGE2 formation at the end of the process, co-incident with the accumulation of macrophages (19). The physiopathological relevance of the enhanced synthesis of 15d-PGJ2 in activated macrophages was speculated to represent a mechanism in which 15d-PGJ2 functions as a feedback regulator of the inflammatory responses (19). Cuzzocrea et al. (43) also demonstrated the effects of 15dPGJ2 in rodent models of chronic collagen-induced arthritis inflammation. Molecular mechanisms of anti-inflammatory functions of 15dPGJ2 have been well studied. Given the critical importance of the NF-κB activity for the expression of pro-inflammatory genes, a number of studies have focused on 15d-PGJ2 as the inhibitor of the NF-κB pathway. It has been reported that 15d-PGJ2 inhibits NF-κB activation by covalently binding to the IκB kinase (IKK) or the p65 or p50 subunit of NF-κB (15, 44, 45). Rossi et al. (15) demonstrated a mechanism of anti-inflammatory activity, which is based on the direct inhibition and modification of the IKKβ subunit of IKK. They found that the mutation at Cys-179 in the activation loop of IKKβ virtually abolishes the effect of 15d-PGJ2. In addition, Straus et al. (44) demonstrated that 15d-PGJ2 could inhibit the DNA binding of the p65 subunit of NF-κB in a manner that is dependent on the presence of Cys38, located in the DNA binding domain. Moreover, CernudaMorollon et al. (45) also demonstrated that 15d-PGJ2 could inhibit the DNA binding activity of the p50 subunit by interacting with Cys-62, which is located in the DNA binding domain. More recently, selenium supplementation was shown to increase the production of 15d-PGJ2 as an adaptive response to protect cells against the oxidative stress-induced proinflammatory gene expression via inhibition of IKK (46). However, 15d-PGJ2 has been reported to serve as a natural ligand of PPARγ (13, 14). Previous studies have demonstrated that the molecular mechanisms of PPAR-dependent anti-inflammatory responses are based on (i) the interaction of PPAR with various transcription factors stimulating inflammation, such as NF-κB, C/EBP, and NF-AT, (ii) the formation of complexes between PPARs and transcriptional coactivators and corepressors, and (iii) the ability of PPARs to modulate the activity of different kinases involved in various pro-inflammatory pathways (47–49). 15d-PGJ2 represses several genes in activated macrophages, including the inducible nitric oxide synthase and tumor necrosis factor R genes, and this repression is suggested to be at least partly dependent on PPARγ expression (13, 50, 51). In the meantime, Shiraki et al. (29) recently reported that 15d-PGJ2 covalently binds to a cysteine residue in the PPARγ ligand binding pocket through a Michael addition reaction by an R,βunsaturated ketone. Using rhodamine–maleimide as well as mass spectrometry, the authors have shown that the binding of 15dPGJ2 to the receptor is covalent and irreversible. Moreover, although the electrophilic carbon C9 (endocyclic β-position) within the cyclopentenone ring is considered to react with the cysteine residue in NF-κB and other proteins (26, 44), the carbon at the exocyclic β-position (C13) has been shown to react with the sulfur atom of the cysteine residue in PPARγ. More recently, Shiraki et al. (30) further investigated the functional significance
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Figure 3. Targets and biological functions of 15d-PGJ2.
of the covalent binding of the ligands and observed a two-step reaction that employed a dock and lock mechanism of ligand binding, in which 15d-PGJ2 first enters into the ligand-binding pocket (dock), and then the covalent binding of the ligand occurs at a relatively low rate (lock). It has also been suggested that the anti-inflammatory functions of 15d-PGJ2 may be associated with a pathway related to antioxidant response. Using the carrageenan-induced pleurisy model, Itoh et al. (21) provided evidence to suggest correlations between the accumulation of 15d-PGJ2 and the activation of Nrf2 during carrageenan-induced pleurisy and proposed a mechanism by which Nrf2 regulates the acute inflammatory response by orchestrating the recruitment of inflammatory cells and regulating the expression of the antioxidative stress genes downstream of 15d-PGJ2. 6.3. Redox Alteration. As part of an effort to identify the endogenous inducer of intracellular oxidative stress and to elucidate the molecular mechanism underlying oxidative stressmediated cell degeneration, Kondo et al. (52) examined the oxidized fatty acid metabolites for their ability to induce intracellular ROS production in SH-SY5Y neuroblastoma cells in Vitro and found that the J2 series of the PGs represent the most potent inducers. On the basis of the observations that (i) 15d-PGJ2 partially reduced the intracellular GSH levels, (ii) 15dPGJ2 treatment of the cells resulted in a significant decrease in the GSH-peroxidase activity, and (iii) the N-acetylcysteine pretreatment significantly inhibited both the ROS production and cytotoxicity by 15d-PGJ2, the intracellular redox status appeared to represent a critical parameter for the PG-induced ROS production and cytotoxicity. Furthermore, it was observed that intracellular ROS production was accompanied by the alteration of the cellular redox status and that the production of lipid peroxidation derived highly cytotoxic aldehydes, such as acrolein and 4-hydroxy-2-nonenal (52). On the basis of these findings, it has been hypothesized that intracellular oxidative stress constitutes a pivotal step in the pathway of cellular dysfunction induced by electrophilic molecules. The 15d-PGJ2-induced oxidative stress is associated with the accumulation of oxidized proteins. Ishii et al. (53) analyzed the protein carbonyl, the most widely studied marker of protein oxidation, generated in the 15d-PGJ2-treated cells and found that protein carbonylation specifically occurred on a 19 S proteasome regulatory subunit S6 ATPase, which was associated
with (i) a dramatic increase in protein carbonyls within S6 ATPase, (ii) a significant decrease in S6 ATPase activities, and (iii) a decreased ability of the 26 S proteasome to degrade substrates. Moreover, using biotin-cysteine as the probe, the occurrence of cysteine-targeted oxidation of protein in the 15dPGJ2-treated cells has been investigated. The proteomics approach revealed that 15d-PGJ2-promoted oxidation of a range of proteins involved in various processes, including cytoskeletal proteins, glycolytic enzymes, redox enzymes, and the chaperone protein (54). The observation that oxidation targets specific enzymes indicates that this protein modification may serve a regulatory role, rather than a simple function in the protection of protein SH groups against irreversible oxidation. 15d-PGJ2 may alter the cellular redox state by the functional impairment of proteins involved in the regulation of cellular antioxidant defenses by direct or indirect interactions. Thioredoxin (Trx) and thioredoxin reductase (TrxR) play a key role in cell defence against oxidative stress. Shibata et al. (28) examined the mechanism of the 15d-PGJ2 modification of Trx by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) and showed that the 15dPGJ2-treated Trx demonstrates the addition of one molecule of 15d-PGJ2 per protein molecule. Moreover, the authors identified two cysteine residues, Cys-35 and Cys-69, as the targets of 15dPGJ2 by LC-MS/MS analysis. Of interest, these data are in striking contrast to the observation that the reaction of Trx with an electrophilic aldehyde, HNE, results in the formation of at least five adducts (Sakurai, T. and Uchida, K., unpublished data). In addition, Moos et al. (55) reported that cyclopentenone PG modifies and inactivates TrxR. The modification of TrxR by cyclopentenone PG results in the inactivation of this enzyme, and this interaction has been shown by means of a biotinylated PGA1 analogue and is associated with impairment of the activity of TrxR. 6.4. Protein Turnover. Recent studies demonstrated that the treatment of human neuroblastoma SH-SY5Y cells with 15dPGJ2 induced the formation of the 15d-PGJ2/proteasome conjugates (56) and oxidation of the S6 ATPase subunit of the 26 S proteasome (53). These modifications could contribute to decreased proteasome activity. In addition, it has also been demonstrated that ∆12-PGJ2 inhibits the proteasome pathway via inhibition of the cellular ubiquitin isopeptidase activity (57).
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Wang et al. (58) assessed the proteasome activity by an in-gel assay in cells harvested with a buffer that preserves the assembly of the 26 S proteasome and revealed a PGJ2-dependent decline in proteasome activity. This decline paralleled a shift in the 26 S proteasome to the 20 S core particle, indicating that PGJ2 disrupted the assembly state of the 26 S proteasomes. Proteasome has a role to play in controlling cellular processes, such as metabolism and the cell cycle, through the signal-mediated proteolysis of key enzymes and regulatory proteins (59–61). It also operates in the stress response by removing abnormal proteins and in the immune response by generating antigenic peptides. The fact that 15d-PGJ2 inhibits the ubiquitin proteasome system provides a mechanism independent of Keap1 modification that could explain the increase in ARE signaling. This may lead to an indirect action of this PG as a trigger of anti-inflammatory and antioxidant responses. Moreover, substrates of proteasome include a number of cell regulatory molecules, such as cyclins, the Myc oncogene protein, and p53; the regulated degradation of these molecules has been linked to the control of cell proliferation and cell cycle progression (59–61). Thus, it is likely that the 15d-PGJ2-induced disruption of the proteasome pathway results in the buildup of not only antioxidant and detoxification enzymes but also pro-apoptotic and detrimental proteins, such as p53 and ubiquitinated proteins. 6.5. Apoptosis. It has been shown that 15d-PGJ2 plays a role in the resolution of inflammation by inducing apoptosis of activated macrophages (62). In addition, the observations (63) that the J2 series of the PGs represent potent inducers of intracellular oxidative stress and that the production of reactive oxygen species in the cells is closely associated with the excitotoxic effect of the PGs raises the possibility that the production of 15d-PGJ2 and related PGs during inflammation may be causally involved in the pathophysiological effects associated with degenerative cell loss. Kondo et al. (20) investigated the molecular mechanisms involved in neuronal cell death induced by 15d-PGJ2 and revealed that the 15d-PGJ2 stimulates the p53-dependent apoptotic pathway, leading to the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand. More recently, 15d-PGJ2 has been shown to activate ATM (ataxia-telangiectasia mutated), leading to p53 activation and apoptosis (64). Fitzpatrick and co-workers have reported that cyclopentenone prostaglandins such as PGA1 and PGA2 inactivate p53-dependent transcription and induce p53independent apoptotic cell death (55, 65, 66). It has also been shown that 15d-PGJ2 inhibits G2–M phase progression via the down-regulation of expression of antiapoptotic proteins including cyclin B1 (67). Moreover, 15d-PGJ2 potentiates tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL)induced apoptosis (68), in which 15d-PGJ2 up-regulates death receptor 5 (DR5) expression at both mRNA and protein levels in human malignant tumor cells. This up-regulation is not mediated through PPARγ activation by 15d-PGJ2 but through the increases in DR5 mRNA stability. DR5 up-regulation results in the synergistic sensitization of soluble recombinant human TRAIL-induced apoptosis. 6.6. Cell Growth and Differentiation. The transcription factor AP-1, an active factor composed of homo or heterodimers of the jun and fos family proteins, plays a central role in the regulation of cell proliferation in response to mitogens, in inflammation, and in the cellular response. Pérez-Sala et al. (69) revealed a molecular basis for the direct inhibition of AP-1 DNA binding by 15d-PGJ2 and showed that the 15d-PGJ2 modification of c-Jun occurs both in Vitro and in intact cells and found that the attachment of the cyclopentenone prostaglandin occurs at
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cysteine 269, which is located in the c-Jun DNA binding domain, thereby inhibiting DNA binding and hence AP-1 inactivation. More interestingly, they have also observed that 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15dPGJ2-bonded dimers. More recently, Kin et al. (27) demonstrated that 15d-PGJ2 can block estrogen receptor-R (ERR) function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERR DNA-binding domain, resulting in fundamental inhibition of both hormonedependent and hormone-independent ERR transcriptional activity, thereby inhibiting cell proliferation. However, Oliva et al. (31) showed that 15d-PGJ2-induced cell proliferation and activation of mitogen-activated protein (MAP) kinase are mediated by 15d-PGJ2-elicited H-Ras activation. They demonstrated that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras but not with N-Ras or K-Ras. Mutation of this cysteine residue blocks both the attachment of 15d-PGJ2 to H-Ras and the stimulation of its activity. These findings describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by the formation of a covalent adduct with 15d-PGJ2. 6.7. Cytoskeletal Dysfunction. Using proteomic analysis in combination with biotinylated 15d-PGJ2, potential targets of the electrophile-responsive proteome have been identified in cultured mesangial cells (70), isolated liver mitochondria (71), and NIH3T3 cells (72). Aldini et al. (73) identified β-actin as the major adducted protein in human neuroblastoma SH-SY5Y cells; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), a redox enzyme (biliverdin reductase), and an adaptor protein (14-3-3γ). In addition, they have characterized the 15d-PGJ2 modification of action and found that (i) 15d-PGJ2 induces marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization, (ii) using a mass spectrometric approach, 15d-PGJ2 reacts with the isolated G-actin in a 1:1 stoichiometric ratio and selectively binds for the Cys-374 site through a Michael adduction mechanism, and (iii) the covalent binding of 15d-PGJ2 induces a significant unfolding of the actin structure and in particular that 15d-PGJ2 distorts actin subdomains 2 and 4 that define the nucleotide binding sites impeding the nucleotide exchange. It has also been shown that 15d-PGJ2 can directly bind to vimentin and induce marked morphologic changes in the vimentin filament network in mesangial cells (74). These findings suggest that 15d-PGJ2 modifies cytoskeletal protein and may cause cytoskeletal derangement. 6.8. Neuronal Differentiation. 15d-PGJ2 and its analogues have been shown to promote neurite outgrowth from PC12 cells (75, 76). Recently, Tanaka et al. (77) conducted an efficient solution-phase synthesis of 15d-PGJ2 derivatives and determined their neurite-outgrowth-promoting activity. Biological assays that elucidated neurite-outgrowth-promoting activity in PC12 cells revealed that the side-chains influenced the outgrowthpromoting activity.
7. Conclusions On the basis of a number of reports concerning the detection of 15d-PGJ2 in human diseases, there is no doubt that the steadystate levels of 15d-PGJ2 increase under pathophysiological states associated with inflammation. Considerable progress has also been recently made toward understanding the mechanisms of
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action of 15d-PGJ2. As described in this perspective, 15d-PGJ2 and its related cyclopentenone-type compounds could trigger diverse aspects of in Vitro cellular responses, including detoxification and pro-inflammatory and anti-inflammatory responses, through redox-sensitive signaling mechanisms. However, there are a number of interesting questions remaining to be answered. Particularly, the physiological role of this compound in ViVo remains an intriguing issue. In addition, development of a highly sensitive and specific assay for the quantification of the intracellular 15d-PGJ2 would also be desirable. Finally, since 15d-PGJ2 exerts its biological effects at least in part through a reaction with cellular proteins, the identification of target molecules of 15d-PGJ2 may facilitate efforts to develop new aspects of electrophilic mediators.
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