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J. Biol. Chem., Vol. 280, Issue 42, 35562-35570, October 21, 2005
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¶¶¶¶¶1
From the
Departments of Pharmacology, Cell and Developmental Biology, and ¶Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the ||Department of Organic Chemistry, University of Pavia, Viale Taramelli, 10-27100 Pavia, Italy
Received for publication, May 2, 2005 , and in revised form, July 27, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B
degradation and subsequent NF-B nuclear translocation and transcriptional activity. Expression of inducible nitric-oxide synthase and cyclooxygenase-2 were also inhibited by cyclopentenone IsoPs as was nitrite and prostaglandin production (IC50 
360 and 210 nM, respectively). 15-J2-IsoPs potently activated peroxisome proliferator-activated receptor 
(PPAR) nuclear receptors, whereas 15-A2-IsoP did not, although the anti-inflammatory effects of both molecules were PPAR-independent. Interestingly 15-A2-IsoPs induced oxidative stress in RAW cells that was blocked by the antioxidant 4-hydroxy-TEMPO (TEMPOL) or the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. TEMPOL also abrogated the inhibitory effect of 15-A2-IsoPs on lipopolysaccharide-induced NF-B activation, inducible nitricoxide synthase expression, and nitrite production, suggesting that 15-A2-IsoPs inhibit the NF-B pathway at least partially via a redox-dependent mechanism. 15-J2-IsoP, but not 15-A2-IsoP, also potently induced RAW cell apoptosis again via a PPAR -independent mechanism. These findings suggest that cyclopentenone IsoPs may serve as negative feedback regulators of inflammation and have important implications for defining the role of oxidative stress in the inflammatory response. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In macrophages, the inflammatory response is largely controlled through regulation of the transcription factor nuclear factor -B (NF-B). NF-B exists primarily in the cytosol as a p50/p65 heterodimer complexed with its inhibitor protein IB. Activation of the IB kinase (IK) complex by numerous stimuli, including exposure to the bacterial endotoxin lipopolysaccharide (LPS), leads to the phosphorylation of IB, causing its dissociation from the NF-B heterodimer and subsequent degradation by the proteasome. Loss of IB reveals a nuclear localization sequence on the NF-B heterodimer that allows its rapid translocation to the nucleus and facilitates transcription of proinflammatory proteins (7, 8). NF-B is a redox-sensitive transcription factor that can be either activated or inhibited by oxidative stress depending on cell type and circumstance (9, 10).
Excessive production of ROS leads to oxidation of membrane poly-unsaturated fatty acids, yielding a multitude of lipid peroxidation products. One such family of products is the isoprostanes (IsoPs), prostaglandin-like molecules produced by free radical-mediated peroxidation of arachidonic acid (11, 12). The measurement of IsoPs has emerged as arguably the most accurate and reliable index of oxidative stress in vivo (13). IsoP levels are elevated in many tissues exposed to chronic inflammation, including postmortem brain samples from patients with Alzheimer disease (14), atherosclerotic plaques from individuals with peripheral atherosclerosis (15), and synovial fluid from rheumatic joints (16). Furthermore injection of LPS into the mouse brain causes significant increases in IsoP levels, which can be abrogated by genetic deletion of the NF-B p50 subunit or the NF-B-responsive gene inducible nitric-oxide synthase (iNOS) (17, 18), demonstrating a link between NF-B activation and IsoP formation.
Several classes of IsoPs containing distinct prostane ring configurations arise from a common endoperoxide radical intermediate following arachidonate oxidation (Fig. 1). Our group has previously described the formation of a class of IsoPs containing an electrophilic ,
-unsaturated cyclopentenone ring known as cyclopentenone or A2/J2 IsoPs (19). These compounds react rapidly with thiol-containing molecules such as GSH and cellular proteins by Michael addition. We have also shown definitively that these compounds are formed in vivo and that their levels are elevated 22-fold under conditions of oxidative injury in rat liver (19). The biological effects of cyclopentenone IsoPs, however, are essentially unexplored.
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12,14-PGJ2, have demonstrated a wide variety of potent biological actions, including anti-inflammatory, antiviral, and both cytotoxic and cytoprotective effects (21). The anti-inflammatory effects of cyclopentenone PGs are likely due to inhibition of NF-B signaling through impairment of IK activity (22, 23), perturbation of NF-B DNA binding activity (24), activation of PPAR nuclear receptors (25), or induction of the anti-inflammatory proteins heat shock protein 70 (26, 27) or heme oxygenase-1 (28). PGA2 has also been shown previously to inhibit iNOS transcription and NF-B activation in a PPAR-independent manner in LPS-stimulated microglia (29). Given the abundant formation of IsoPs during inflammation and the potent immunomodulatory effects of the structurally similar cyclopentenone PGs, we sought to evaluate the biological effects of cyclopentenone IsoPs in a model of inflammation. Importantly the stereochemical differences between IsoPs and PGs often leads to divergent biological effects (30); thus the bioactivity of A2-IsoPs cannot necessarily be inferred from that of PGA2. The recent synthesis of both 15-A2-IsoPs and 15-J2-IsoPs (31, 32) allowed us to examined the effects of cyclopentenone IsoPs on the inflammatory response in LPS-stimulated RAW 264.7 murine macrophage cells and primary murine macrophages for the first time. The biological action of cyclopentenone IsoPs on macrophages represents a novel interaction between oxidative stress and inflammation, two processes central to many disease states.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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antagonists GW9662 and T0070907 were from Cayman Chemical (Ann Arbor, MI). Boc-aspartyl (OMe)-fluoromethyl ketone (BAF) was from Enzyme Systems Products (Dublin, CA). The NF-B inhibitor BAY 11-7082 was from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, PA). Primary antibodies to IB, iNOS, cyclooxygenase-2 (COX-2), p65, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA), and Hsc70 and ERK1/2 antibodies were from Cell Signaling Technologies (Beverly, MA). Cell Culture—RAW 267.4 murine macrophage cells were obtained from ATCC (Manassas, VA), and BV-2 murine microglial cells were a generous gift of Dr. Thomas Montine (University of Washington). Cells were grown in DMEM containing 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 mg/ml streptomycin and plated on 12-well plates or 100-mm tissue culture dishes 24 h before experimentation. No cells older than passage 10 were used as RAW cells showed highly variable responses at higher passage numbers. PC3 prostate carcinoma and LT234 colon carcinoma cells were from ATCC.
Primary Bone Marrow Macrophages and Macrophage Cell Lines—Primary macrophages and macrophage cell lines were generated using bone marrow from mice expressing a luciferase construct driven by the NF-B-dependent portion of the human immunodeficiency virus, type 1, long terminal repeat (33). Bone marrow cavities from femurs were flushed with phosphate-buffered saline (PBS) using a 27-gauge needle. The cells were then pelleted at 1000 rpm for 5 min, and red blood cells were lysed in 1 ml of NH4Cl lysis solution (Sigma). After 5 min, 4 ml of PBS was added, and cells were pelleted as above and resuspended in DMEM supplemented with 10% fetal bovine serum. Macrophages were cultured in DMEM, 10% fetal bovine containing 10% L-medium for 4-5 days to obtain adherent, differentiated macrophages.
Cell Treatment—Cells were rinsed twice with serum-free DMEM before treatment, and all treatments were carried out in serum-free DMEM containing penicillin and streptomycin. Cells were preincubated with IsoPs for 30 min prior to stimulation with LPS, and both IsoPs and LPS were present throughout the stimulation period in all experiments. Subsequently medium was removed and immediately stored at -80 °C. For experiments using inhibitors or antioxidants, cells were preincubated for 30 min with inhibitor and then for 30 min with inhibitor plus IsoPs prior to LPS addition. Appropriate vehicle controls (ethanol, 0.5%) were carried out in all cases.
Measurement of Nitrite—Nitrite, a stable breakdown product of nitric oxide, was measured in cell medium using the Griess reaction. Briefly 100 µl of conditioned medium was mixed with 50 µl each of Griess reagent R1 and R2 (Cayman Chemical) in a 96-well plate, and absorbance was measured at 540 nm. Quantitation was achieved by comparison with sodium nitrite standards in DMEM.
Tumor Necrosis Factor- (TNF) Quantification—TNF levels were quantified in conditioned cell medium using a Quantikine mouse TNF enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.
MTT Assay—Cell viability was assessed by measuring MTT reduction (34). Following IsoP treatment, cells were rinsed and incubated in 0.5 mg/ml MTT reagent dissolved in DMEM for 2 h. Cells were then permeabilized by addition of acidified (0.1 N HCl) isopropanol, and absorbance was measured at 595 nm.
Western Blotting—Total protein extract was prepared as described previously (35). Equal protein concentrations were separated using Criterion Tris-HCl gels (Bio-Rad). Proteins were then transferred to polyvinylidene difluoride membranes (Amersham Biosciences) and blocked for 1 h at room temperature with Odyssey blocking buffer (LiCor Biosciences, Lincoln, NE). Membranes were then incubated overnight in primary antibodies diluted in blocking solution. Membranes were washed in PBS containing 0.1% Tween 20 and then incubated in Alexa 800-conjugated fluorescent secondary antibodies (Molecular Probes, Eugene, OR) for 1 h at room temperature. Protein bands were visualized using an Odyssey infrared imaging system and software (version 1.2, LiCor Biosciences). Densitometry was performed using Scion Image software (version 4.0.2, Scion Corp., Frederick, MD).
F2-Isoprostane Assay—Total F2-IsoPs were measured in combined medium and cell pellet as described previously (12). Briefly cells were scraped directly into their conditioned medium and then collected by centrifugation. The cell pellet was resuspended in 0.5 ml of methanol containing 0.005% butylated hydroxytoluene, sonicated, and then subjected to chemical saponification using 15% KOH to hydrolyze bound F2-IsoPs. The conditioned medium was then added back to the cell lysate and adjusted to pH 3 followed by addition of 1 ng of 4H2-labeled 15-F2t-IsoP internal standard. Free F2-IsoPs were then purified by C18 and silica Sep-Pak extraction and thin layer chromatography and analyzed as pentafluorobenzyl ester, trimethylsilyl ether derivatives via gas chromatography, negative ion chemical ionization mass spectrometry.
PPAR Reporter Assay—PC3 cells were transiently transfected with 150 ng/ml of full-length PPAR, 150 ng/ml of (PPRE)3-TK-luciferase, and 2 ng/ml pRL-SV40 plasmids using FuGENE 6 at a lipid:DNA ratio of 4:1. Transfections were performed in RMPI 1640 cell medium supplemented with 10% charcoal-stripped fetal bovine serum. After 4 h, cells were treated either 0.1% vehicle (Me2SO) or the indicated concentration of 15-J2-IsoP and 15-A2-IsoP. The PPAR antagonist GW9662 (5 µM) was applied 2 h prior to IsoP exposure. After 24 h, cells were harvested in 1x luciferase lysis buffer. Relative light units from firefly luciferase activity were determined using a luminometer and normalized to the relative light units from Renilla luciferase using the Dual Luciferase kit (Promega).
In separate experiments, LT234 cells were transiently co-transfected with 0.3 µg of USA-thymidine kinase-luciferase reporter and 0.4 µg of PPAR (ligand binding domain)-GAL4 chimera and then treated with 15-A2-IsoPs for 16 h at which time luminescence was quantified as above.
Immunofluorescence Microscopy—For immunofluorescence staining, RAW cells were grown on glass coverslips. Following exposure to 15-A2-IsoPs or vehicle, cultures were fixed in 10% formaldehyde for 20 min, rinsed with PBS, permeabilized with 0.1% Triton X-100, and blocked for 1 h with 8% bovine serum albumin diluted in PBS. Coverslips were then incubated overnight at 4 °C in rabbit anti-p65 (1:100) primary antibody in 1% bovine serum albumin. Cells were then washed in PBS for a total of 20 min and incubated in Cy-2-labeled secondary antibodies for 1 h. Cells were then washed again and stained with 1.4 µM 4',6-diamidino-2-phenylindole (DAPI) for 10 min followed by further washes. Coverslips were mounted on microscope slides, and fluorescence was visualized with a Zeiss Axioplan microscope.
NF-B Reporter Assay—NF-B transcriptional activity was assessed using a reporter plasmid containing the human immunodeficiency virus long terminal repeat 36-base pair enhancer (containing a total of eight NF-B binding sites) upstream of the herpes simplex virus minimal thymidine kinase promoter driving expression of a green fluorescent protein-luciferase fusion protein (fusion protein from Clontech). DNA was mixed with Superfect liposome reagent (Qiagen, Valencia, CA) in serum-free medium, incubated at room temperature for 15 min, supplemented with serum-containing medium, and then added to cells and incubated for 4 h. The DNA/liposome solution was removed, and the cells were washed once with PBS. Serum-containing medium was then added to the cells and placed at 37 °C overnight. Experiments were performed the next day. Primary macrophages were obtained from transgenic mice expressing this construct and were not transfected. Following treatment, cells were scraped at select time points in Lysis Buffer (Promega). The cell debris was pelleted by centrifugation at 13,000 rpm for 5 min, and 20 µl of the sample was used to perform a standard luciferase assay using a luminometer.
Northern Blotting—Total cellular RNA was isolated from cells by TRI reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's protocol. Five micrograms of total RNA was fractionated with a MOPS-formaldehyde-agarose gel and transferred to Hybond N1 membrane (Amersham Biosciences). Following UV cross-linking, the blots were prehybridized for 30 min at 42 °C in Hybrisol I (Intergen Co., Purchase, NY), hybridized using 32P-labeled cDNA in the same buffer at 42 °C, washed, and subjected to autoradiography. Briefly an 860-bp Northern blot probe for COX-2 (generously provided by Dr. Sanjoy Das, Vanderbilt University) was generated by restriction endonuclease digestion of an 1156-bp COX-2 cDNA fragment with EcoRI and PstI.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B Degradation and NF-B Translocation and Transcriptional Activity Are Inhibited by 15-A2-IsoPs—To explore the effect of cyclopentenone IsoPs on the inflammatory response, we examined the effect of 15-A2-IsoPs on NF-B activation in LPS-stimulated RAW 264.7 macrophages. Following LPS exposure, translocation of NF-B p50/p65 heterodimers to the nucleus is required for NF-B-mediated transcription and can be inhibited by cyclopentenone PGs (36). As shown in Fig. 2A, treatment of RAW cells with LPS induced a marked translocation of NF-B p65 subunit into the nucleus. Application of 15-A2-IsoPs blocked this translocation. We quantified NF-B-dependent transcriptional activity using RAW cells transiently transfected with an NF-B reporter plasmid. LPS stimulation of these cells induced a 3-fold increase in luciferase production after 6 h that was inhibited dose-dependently by co-application of 15-A2-IsoPs (Fig. 2B).
The effects of cyclopentenone IsoPs on NF-B-dependent transcriptional activity were next examined in primary bone marrow-derived macrophages obtained from transgenic mice expressing an NF-B-responsive luciferase reporter construct. 15-A2-IsoPs inhibited NF-B reporter activity dose-dependently in LPS-stimulated in these primary macrophages to a degree similar to that seen in RAW cells, demonstrating that these effects are not unique to RAW cells (Fig. 2B).
Phosphorylation and subsequent degradation of IB is required for NF-B translocation to the nucleus and can be blocked by cyclopentenone PGs (22, 37). Thus, we examined by Western blot the degradation of IB in LPS-stimulated RAW cells treated with 15-A2-IsoPs. Stimulation of RAW cells with LPS led to a marked degradation of IB within 30 min, whereas co-treatment with 15-A2-IsoPs inhibited LPS-induced IB degradation in a dose-dependent manner (Fig. 2C). Densitometry revealed that treatment with 15-A2-IsoPs actually stabilized IB to greater-than-control levels (Fig. 2C), suggesting that these molecules inhibit basal IB degradation. Thus, our results demonstrate that 15-A2-IsoPs inhibit IB degradation and subsequent NF-B translocation and transcriptional activity.
15-A2-IsoPs Inhibit Nitrite Production and iNOS Induction in LPS-stimulated RAW 264.7 and Primary Macrophages—Activation of NF-B drives transcription of a diverse array of NF-B-responsive genes. In LPS-stimulated macrophages, iNOS and COX-2 are two proinflammatory gene products that are NF-B-responsive (38, 39). To determine the downstream effects of NF-B inhibition by 15-A2-IsoPs, we assessed iNOS protein expression and activity (as determined by production of nitrite, a stable metabolite of nitric oxide) in LPS-stimulated RAW 264.7 macrophages. In unstimulated cells, neither iNOS protein nor nitrite was detectable (data not shown). Stimulation of RAW cells with LPS alone led to marked expression of iNOS protein, an effect that was blocked in a dose-dependent manner by 15-A2-IsoPs (Fig. 3A). 15-A2-IsoP treatment also caused a dose-dependent inhibition of LPS-induced nitrite accumulation in cell medium with an IC50 of 360 nM (Fig. 3B). LPS-induced nitrite production was similarly inhibited by BAY 11-7082 (3 µM), a specific NF-B inhibitor, suggesting that blockade of nitrite production is due to NF-B inhibition (data not shown). 15-A2-IsoPs also inhibited LPS-induced nitrite production and expression of the proinflammatory cytokine TNF in primary bone marrow-derived macrophages (supplementary data).
To determine whether the effects of 15-A2-IsoPs are shared by all A2/J2-IsoPs, we examined the effects of 15-J2-IsoPs on LPS-stimulated RAW cells. 15-J2-IsoPs inhibited nitrite production with potency similar to that of 15-A2-IsoPs (Fig. 3F), suggesting that these two compounds share similar biology. We also observed that 4-hydroxynonenal (HNE), an abundant lipid peroxidation product also containing an ,-unsaturated carbonyl capable of forming Michael adducts, inhibited nitrite production in LPS-stimulated RAW cells with slightly less potency then either 15-A2- or 15-J2-IsoPs (Fig. 3F). This observation is in keeping with previous reports that HNE can inhibit the NF-B pathway (40, 41) and suggests that this is a common action of lipid peroxidation products containing reactive ,-unsaturated carbonyls.
LPS-induced COX-2 Induction and Prostaglandin Production Are Blocked by 15-A2-IsoPs—We next assessed the ability of 15-A2-IsoPs to modulate COX-2 induction and PG production in LPS-stimulated RAW264.7 macrophages. LPS-induced COX-2 mRNA and protein levels were also decreased in cells treated with 15-A2-IsoPs (Fig. 3, C and D). 15-A2-IsoPs blocked production of PGD2 in a dose-dependent manner with an IC50 of 210 nM (Fig. 3E).
Metabolism by Glutathione Adduction Renders 15-A2-IsoPs Biologically Inactive—Cyclopentenone IsoPs are rapidly metabolized in cells via enzymatic conjugation to GSH (42). We hypothesized that the bioactivity of 15-A2-IsoPs is dependent on the reactivity of the cyclopentenone ring and should be abrogated by conjugation to GSH. To test this hypothesis, GSH-15-A2-IsoP conjugates were prepared by incubation of 15-A2-IsoPs with excess GSH and bovine liver glutathione transferase followed by purification by high pressure liquid chromatography (42). The GSH-15-A2-IsoP conjugates, which can no longer adduct cellular thiols, failed to inhibit nitrite production in stimulated RAW cells even at concentrations as high as 25 µM (Fig. 4), suggesting that chemical reactivity is crucial to the bioactivity of 15-A2-IsoPs. Furthermore 15-F2t-IsoP, which lacks the reactive cyclopentenone ring but is otherwise structurally identical to 15-A2-IsoP, failed to inhibit LPS-induced nitrite production at concentrations up to 25 µM (data not shown), again implicating the cyclopentenone moiety in the bioactivity of 15-A2-IsoPs.
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Nuclear Receptors—The cyclopentenone prostaglandin metabolite 15-deoxy-12,14-prostaglandin J2 is a ligand for the PPAR nuclear receptor (43, 44), which is thought to mediate some of the anti-inflammatory effects of this molecule (45). To determine whether 15-A2- and 15-J2-IsoPs also act as PPAR agonists, we examined the ability of these molecules to activate a PPAR luciferase reporter system in PC3 cells. As shown in Fig. 5, 15-J2-IsoPs potently induced luciferase production in this system with an EC50 of 4 µM, and this activation of PPAR could be blocked by the specific PPAR antagonist GW9662 (5 µM). 15-A2-IsoPs, however, did not significantly activate PPAR at concentrations as high as 10 µM, whereas GW7845, a specific PPAR agonist, caused marked induction of luciferase production (Fig. 5A). 15-A2-IsoPs also failed to activate a PPAR ligand binding domain reporter system at concentrations up to 10 µM (data not shown). Thus, 15-J2-IsoPs, but not 15-A2-IsoPs, are novel ligands for the PPAR nuclear receptor and activate this receptor at concentrations consistent with their anti-inflammatory activity.
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-dependent as ligation of this receptor has been shown to lead to NF-B inhibition (45). However, we observed that preincubation of RAW cells with GW9662 (5 µM), which blocks 15-J2-IsoP-induced PPAR activation, or with T0070907 (0.5 µM), a second PPAR antagonist, had no effect on the ability of these compounds to inhibit LPS-induced nitrite production (Fig. 5B), demonstrating that like 15-A2-IsoPs, 15-J2-IsoPs inhibit the inflammatory response in a PPAR-independent manner.
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B activation and transcriptional activity are regulated by oxidative stress in a variety of cell types (9, 49). Thus, we sought to elucidate the potential relationship between 15-A2-IsoP-induced ROS production and NF-B inhibition. We thus examined the effect of TEMPOL co-administration on 15-A2-IsoP-induced impairment of IB degradation. Co-application of 1 mM TEMPOL partially rescued LPS-induced IB degradation in 15-A2-IsoP-treated cells (Fig. 7A), suggesting that 15-A2-IsoPs impair IB degradation in part via a ROS-dependent mechanism and that scavenging ROS with TEMPOL block this effect of 15-A2-IsoPs. TEMPOL alone did not alter LPS-induced IB degradation (supplemental data). TEMPOL also prevented the inhibition of NF-B transcriptional activity by 2.5 µM 15-A2-IsoPs in LPS-stimulated RAW cells, returning LPS-induced NF-B activation to near control levels (Fig. 7B). This result further supports the notion that 15-A2-IsoP-induced ROS production is important in mediating the anti-inflammatory effects of the molecule. Accordingly TEMPOL co-treatment also prevented 15-A2-IsoP-induced inhibition of LPS-stimulated iNOS production without increasing iNOS protein expression in cells treated with LPS but without IsoPs (Fig. 7C). Similarly we found that although treatment of LPS-stimulated RAW cells with 2.5 µM 15-A2-IsoPs alone caused a significant reduction in nitrite production, co-treatment with 1 mM TEMPOL blocked this effect, rescuing nitrite production to levels similar to those measured in cells treated with LPS alone (Fig. 7D). TEMPOL co-treatment caused a slight decrease in nitrite production in LPS-stimulated cells in the absence of IsoPs but rescued nitrite production in 15-A2-IsoP-treated cells, showing that this compound does not simply potentiate nitrite production. These results suggest that 15-A2-IsoPs can inhibit IB degradation and subsequent NF-B activation at least partially via a redox-dependent mechanism. Furthermore we found that the anti-inflammatory effects of 15-A2-IsoPs could also be partially abrogated by co-application of N-acetylcysteine (1 mM, data not shown), a thiol antioxidant, demonstrating that this effect is not unique to TEMPOL. Although antioxidants could mitigate the anti-inflammatory effects of 15-A2-IsoPs, this effect was not complete. Furthermore antioxidants were ineffective in preventing the anti-inflammatory effects of higher concentrations (>10 µM) of 15-A2-IsoPs (data not shown), suggesting that 15-A2-IsoPs can inhibit LPS-induced inflammation via two mechanisms, one of which is ROS-dependent and one of which is likely due to direct covalent modification of proteins.
15-J2-IsoPs, but Not 15-A2-IsoPs, Induce RAW Cell Apoptosis—The induction of macrophage apoptosis is a crucial anti-inflammatory mechanism and could underlie the observed effects of cyclopentenone IsoPs. However, we observed that 15-A2-IsoPs did not induce RAW cells death at concentrations as high as 25 µM (Fig. 8A), demonstrating that their effects are independent of cell toxicity. Conversely 15-J2-IsoPs were potent inducers of RAW cell death, killing these cells with an LD50 of 3.5 µM (Fig. 8A). 15-J2-IsoPs were equally toxic in the presence or absence of LPS (data not shown). As shown in Fig. 8A, 15-J2-IsoPs were also considerably more potent inducers of RAW cell death than HNE (LD50 14 µM). DAPI nuclear staining of RAW cells treated for 24 h with 5 µM 15-J2-IsoPs demonstrated pronounced nuclear condensation and abundant asymmetric chromatin formations, morphological indications of apoptotic cell death (Fig. 8D). Nuclei of cells treated with 10 µM 15-A2-IsoPs, however, appeared similar to control nuclei (Fig. 8, B and C). Furthermore co-application of the broad spectrum caspase inhibitor BAF (50 µM) provided significant, but not complete, protection from 15-J2-IsoP-induced RAW cell death (Fig. 8G) and prevented nuclear fragmentation (Fig. 8E), suggesting that this form of cell death is at least in part apoptotic.
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and induce macrophage apoptosis, we investigated the possibility that 15-J2-IsoP-induced RAW cell apoptosis is mediated by PPAR. Pre- and co-incubation of RAW cells with the PPAR antagonist GW9662 (5 µM) failed to protect cells from death induced by 5 µM 15-J2-IsoPs (Fig. 8F), suggesting that 15-J2-IsoP toxicity in RAW cells is PPAR-independent.
Taken together, these results demonstrate that 15-J2-IsoPs, but not 15-A2-IsoPs, are potent inducers of RAW cell death via a mechanism that has both caspase-dependent and -independent components. These experiments also suggest that the mechanisms of nitrite inhibition and cell death induction are independent as both 15-A2- and 15-J2-IsoPs inhibit nitrite production in RAW cells with similar potency, whereas only 15-J2-IsoPs induce RAW cell apoptosis, and that both processes are PPAR-independent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B activation via impairment of IB degradation. 15-A2-IsoPs attenuate LPS-induced expression of the NF-B-responsive proteins TNF, iNOS, and COX-2 and block nitric oxide and prostaglandin production in a dose-dependent manner at submicromolar concentrations. 15-J2-IsoPs exert similar effects, suggesting that all cyclopentenone IsoPs might be anti-inflammatory. 15-J2-IsoPs are also potent ligands for the PPAR nuclear receptor, whereas 15-A2-IsoPs are not, although the anti-inflammatory effects of both compounds are PPAR-independent. 15-A2-IsoPs promote lipid peroxidation in RAW cells, and the ability of these IsoPs to inhibit NF-B transcription and iNOS production appears to have a ROS-dependent component as the antioxidant TEMPOL ameliorates these effects to some extent. Furthermore 15-J2-IsoPs, but not 15-A2-IsoPs, induce RAW cell apoptosis again by a PPAR-independent mechanism. These findings show that cyclopentenone products of the IsoP pathway can modulate the inflammatory response in macrophages by several mechanisms.
The transcription factor NF-B plays a crucial role in the inflammatory response (8) as NF-B activation can lead to transcription of proinflammatory proteins including iNOS and COX-2 (38, 39). The modulation of NF-B activity by cyclopentenone PGs has been described thoroughly (21). Several groups have shown that IK activity is inhibited by cyclopentenone PGs presumably through direct adduction of vulnerable cysteine residues in IK (22, 36). Our data show that 15-A2-IsoPs inhibit IB degradation likely through inhibition of some upstream signaling event. It should be noted that although 15-A2-IsoPs completely suppress iNOS expression and nitrite production, they totally inhibit NF-B reporter activity or COX-2 expression, suggesting that these molecules likely also effect pathways other than NF-B that contribute to iNOS induction. As IsoPs are produced under different circumstances and often exert much different biological effects than their corresponding PGs (13), these findings have important implications for understanding the biology of lipid oxidation.
Previous work using cyclopentenone PGs (such as 15-deoxy-12,14-prostaglandin J2) has suggested that these molecules act via direct covalent adduction to cysteine residues in IK, particularly Cys-179 (22). However, modification of specific cysteine residues in IK, either through direct oxidation (10), nitrosylation (50), or covalent adduction (22), renders the complex inactive. A recent study shows that 15-deoxy-12,14-prostaglandin J2 can induce thiol oxidation of numerous proteins through induction of intracellular ROS production rather than through direct adduction (51). 15-A2-IsoPs induce ROS production in RAW cells, an effect that can be abrogated by either TEMPOL application or uncoupling of the mitochondrial electron transport chain with FCCP. In support of our findings, several studies have demonstrated increased radical production in cells treated with cyclopentenone PGs (46, 47). 15-A2-IsoPs are thus byproducts of oxidant injury that in turn promote further oxidative damage by stimulating ROS production, potentially setting in motion a feed-forward oxidation cycle that could rapidly alter the intracellular redox status of macrophages and thereby effect cellular function. Others have suggested that a similar cyclopentenone PG-induced shift in redox state underlies some of the biological actions of these molecules (51-53). TEMPOL or N-acetylcysteine scavenge 15-A2-IsoP-induced ROS, potentially preventing cysteine oxidation and preserving IK function, thereby facilitating IB degradation (see Fig. 7A, N-acetylcysteine data not shown). However, future experiments are required to directly examine IK cysteine oxidation following IsoP exposure. Our data demonstrate a novel indirect oxidative mechanism of NF-B inhibition that could potentially work in concert with direct covalent adduction to impair NF-B activation. Indeed cyclopentenone IsoPs can still partially inhibit the inflammatory response in the presence of TEMPOL, particularly at higher concentrations, suggesting that indirect oxidation and covalent adduction mechanisms both contribute to the effect of these compounds.
As products of the IsoP pathway, A2- and J2-IsoPs are expected to be formed in equal proportion during oxidative stress and are thus simultaneously present in oxidatively damaged tissue (19). We have shown that like 15-A2-IsoPs, 15-J2-IsoPs, cyclopentenone IsoPs that are isomeric to PGJ2, also inhibit nitrite production in LPS-stimulated RAW cells with similar potency, suggesting that anti-inflammatory properties are shared by all cyclopentenone IsoP isomers. Interestingly 15-J2-IsoPs also potently induce RAW cell apoptosis, whereas 15-A2-IsoPs do not. This finding suggests that cyclopentenone IsoP formation could inhibit macrophage function not only via NF-B inhibition but also through induction of apoptosis, a well described mechanism of inflammatory regulation (54). These results also show that J2-IsoPs can exert effects distinct from those of A2-IsoPs and likely modulate unique intracellular targets. Furthermore we have shown that cyclopentenone IsoPs are more biologically potent that HNE, a compound widely considered to be the major toxic product of lipid peroxidation.
Although many of the biological effects of cyclopentenone PGs (particularly J-ring PGs) were initially attributed to the ability of these compounds to activate PPAR nuclear receptors (43, 44), attention has more recently focused on the PPAR-independent actions of these molecules, which are thought to be mediated by thiol modification (24, 55, 56). We have demonstrated that 15-J2-IsoPs are novel and potent activators of PPAR, whereas 15-A2-IsoPs are not. However, our observations that the anti-inflammatory effects of 15-J2-IsoPs cannot be blocked by PPAR antagonists and that 15-A2-IsoP bioactivity can be abrogated by conjugation to GSH suggest that the PPAR-independent effects of these compounds are of primary importance. Nevertheless the finding that 15-J2-IsoPs can activate this receptor may have important implications for the role of lipid peroxidation in many other cellular processes.
In our studies, cyclopentenone IsoPs exert effects at high nanomolar and low micromolar concentrations. We have detected levels of esterified cyclopentenone IsoPs of 122 ng/g of tissue in CCl4-treated rat liver (19) and 179 ng/g in oxidized rat brain3, which roughly convert to 364 and 534 nM, respectively. These measurements are likely significant underestimates of total cyclopentenone levels as they do not account for non-esterified pools of IsoPs or for cyclopentenone molecules derived from eicosapentaenoic acid or docosahexaenoic acid (57). We believe that, taking these considerations into account, the concentrations used in this study have physiological relevance particularly under conditions of severe oxidant injury.
Reactive oxygen species can promote inflammation by increasing NF-B activation (58, 59), and their generation may be required for normal LPS signaling through Toll-like receptor 4 (60, 61). However, our results and the results of others (40, 41) suggest that products of ROS-mediated lipid peroxidation, such as cyclopentenone IsoPs, can also inhibit the inflammatory response. High levels of oxidative stress have also been shown to impair NF-B activation by several mechanisms. Oxidation of key cysteine residues in the NF-B DNA binding site can impair NF-B transcriptional activity (62). Further upstream, excessive ROS production can inhibit IB degradation via impairment of IK activity (10). Several studies have demonstrated that treatment of cells with the oxidant H2O2 can inhibit LPS- or TNF-induced IK activity, IB degradation, and cytokine production (63-65), suggesting that oxidative stress may exert complex or biphasic effects on the inflammatory response. As inflammation often leads to local oxidative damage (17), these findings would suggest that lipid peroxidation and subsequent formation of cyclopentenone IsoPs could serve as a brake to prevent excessive tissue damage due to inflammation. A similar role has been proposed for cyclopentenone PGs in the enzymatic modulation of inflammation (66). Thus, the degree of oxidant injury and the products generated during an oxidative insult are likely important factors in dictating the ultimate effect of oxidative stress on the inflammatory response. Furthermore as NF-B, PPAR, COX-2, and iNOS play important roles not only in inflammation but also in the pathogenesis of many disease states, the biological action of cyclopentenone IsoPs has broad implications for our understanding of the role of oxidative stress in human pathophysiology.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental data.
1 To whom correspondence should be addressed: Division of Clinical Pharmacology, Depts. of Medicine and Pharmacology, Vanderbilt University School of Medicine, 526 RRB, 23rd and Pierce Aves., Nashville, TN 37232-6602. E-mail: jason.morrow{at}vanderbilt.edu.
2 The abbreviations used are: ROS, reactive oxygen species; IsoP, isoprostane; LPS, lipopolysaccharide; NF-B, nuclear factor -B; IB, inhibitor -B ; IK, IB kinase; COX-2, cyclooxygenase-2; iNOS, inducible nitric-oxide synthase; PPAR, peroxisome proliferator-activated receptor ; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; DAPI, 4',6-diamidino-2-phenylindole; PG, prostaglandin; BAF, Boc-aspartyl (OMe)-fluoromethyl ketone; Boc, t-butoxycarbonyl; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TNF, tumor necrosis factor-; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MOPS, 4-morpholinepropanesulfonic acid; HNE, 4-hydroxynonenal; ANOVA, analysis of variance.
3 E. Musiek, G. Milne, and J. Morrow, unpublished data.
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ACKNOWLEDGMENTS |
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