HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giri, S. Articles by Singh, I. Search for Related Content PubMed PubMed Citation Articles by Giri, S. Articles by Singh, I.

The 15-Deoxy-12,14-Prostaglandin J₂ Inhibits the Inflammatory Response in Primary Rat Astrocytes via Down-Regulating Multiple Steps in Phosphatidylinositol 3-Kinase-Akt-NF-B-p300 Pathway Independent of Peroxisome Proliferator-Activated Receptor ¹

Shailendra Giri^*, Ramandeep Rattan^*, Avtar K. Singh^, and Inderjit Singh^2,*

Departments of ^* Pediatrics and Pathology and Laboratory Medicine, Medical University of South Carolina, and Department of Pathology and Laboratory Medicine, Ralph Johnson Veterans Affairs Medical Center, Charleston, SC 29425

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ligands for peroxisome proliferator-activated receptor (PPAR), such as 15-deoxy-12,14-PGJ₂ (15d-PGJ₂), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ₂ was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-, IL-1, IL-6, and iNOS gene expression) of 15d-PGJ₂ are observed to be independent of PPAR. Overexpression (wild-type and dominant-negative forms) of PPAR and its antagonist (GW9662) did not alter the 15d-PGJ₂-induced inhibition of LPS/IFN--mediated iNOS and NF-B activation. The 15d-PGJ₂ inhibited the inflammatory response by inhibiting IB kinase activity, which leads to the inhibition of degradation of IB and nuclear translocation of p65, thereby regulating the NF-B pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN--induced PI3K-Akt pathway. The 15d-PGJ₂ inhibited the recruitment of p300 by NF-B (p65) and down-regulated the p300-mediated induction of iNOS and NF-B luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ₂-mediated inhibition of p300-induced iNOS and NF-B luciferase activity. This study demonstrates that 15d-PGJ₂ suppresses inflammatory response by inhibiting NF-B signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPAR in primary astrocytes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the brain, inflammatory responses of astroglial cells occur during disease, infection, trauma, and ischemia (1, 2, 3). These responses include release of proinflammatory cytokines such as TNF-, IL-1, and IL-6 as well as synthesis and release of NO (4). In astrocytes, NO is primarily biosynthesized by the calcium-independent isoform of NO synthase (inducible NO synthase (iNOS)),³ which is not normally expressed, but induced in response to a variety of inflammatory stimuli. Increasing evidence points to a role for iNOS-derived NO in the pathogenesis of a variety of human neurological diseases such as Alzheimer’s disease, multiple sclerosis, X-adrenoleukodystrophy, globoid cell leukodystrophy (5, 6, 7, 8), cerebral ischemia (9), and traumatic brain injury (10). In experimental autoimmune encephalomyelitis and cerebral ischemia, it has been demonstrated that suppression of astroglial iNOS can be of therapeutic value in the prevention of neurological damage (11, 12).

Recently, studies with activated monocytes, microglia, and macrophages have shown that cyclopentenone PGs can inhibit certain proinflammatory responses possibly through the inhibition of various transcription factors such as AP-1, STAT1, and NF-B (13, 14, 15). The inhibition of the transcription factors was reported to occur through the activation of peroxisome proliferator-activated receptors (PPARs), particularly PPAR, whose natural ligand is 15-deoxy-12,14-PGJ₂ (15d-PGJ₂). The anti-inflammatory effect of 15d-PGJ₂ was found to be PPAR independent, but its effect on the modulation of macrophage lipid metabolism was PPAR dependent in PPAR-deficient cells (16). More recently, several studies have shown that 15d-PGJ₂ exhibits a potent anti-inflammatory effect by attenuating the expression of proinflammatory mediators in activated monocytes/macrophages mainly through the inhibition of transcription of NF-B-dependent inflammatory genes (15, 17, 18, 19). In addition to antagonizing the NF-B activity through a PPAR-dependent mechanism, 15d-PGJ₂ can directly inhibit the signaling steps leading to NF-B activation (15, 19). It has also been shown that the carbonyl group of 15d-PGJ₂ can act as an electrophile to react covalently with specific cysteine residues located in the activation loop of IB kinase- and the DNA binding domains of NF-B subunits in combination, leading to the suppression of NF-B-mediated trans activation (15, 19, 20). NF-B complexes are composed mainly of p50 and p65 subunits that translocate to the nucleus in response to cell stimulation with LPS and proinflammatory cytokines (21). This activation of NF-B requires phosphorylation by IB kinase (IKK) of IB proteins at specific serine residues, which target these proteins for ubiquitin conjugation and degradation by the 26S proteasome (21). The IKK complex contains two catalytic subunits, IKK and IKK, and a regulatory subunit, IKK (22, 23, 24). Activation of IKK is mediated by phosphorylation through various upstream kinases such as NF-B-inducing kinase, NF-B-activating kinase, Akt, and protein kinase C, which are involved in cellular signaling in response to proinflammatory stimuli (25, 26). IKK is rapidly activated after cell is challenged with LPS, IL-1, or TNF- and progressively undergoes phosphorylation at multiple serine residues, which induces the kinase activity and therefore contributes to the transient activation of this enzyme (25).

We investigated the mechanism of anti-inflammatory effect of 15d-PGJ₂ and report that 15d-PGJ₂ suppresses the production of iNOS and proinflammatory cytokines by down-regulating PI3K-Akt-NF-B-p300 pathway independent of PPAR in primary astrocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

DMEM (4.5 g of glucose/L), FBS, and HBSS were from Invitrogen Life Technologies (Carlsbad, CA). LPS (Escherichia coli, serotype 055:B5) and protease inhibitor mixture were from Sigma-Aldrich (St. Louis, MO). Abs against phosphospecific 3-phosphoinositide-dependent kinase (PDK) and Akt were purchased from Cell Signaling Technology (Beverly, MA). Abs against PPAR and iNOS were obtained from Upstate Biotechnology (Waltham, MA). [-³²P]ATP (3000 Ci/mmol) and [-³²P]dCTP (3000 Ci/mmol) were from PerkinElmer (Boston, MA). The 15d-PGJ₂ was purchased from BIOMOL (Plymouth Meeting, PA). The 9,10-dihydro-15d-PGJ₂ (CAY10410) was obtained from Cayman Chemical (Ann Arbor, MI). Abs for p65, p50, p300, IB, IB, IKK, and IKK; GST-IB fusion protein; and oligonucleotides for NF-B and NF-B-conjugated agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). PPAR EMSA kit was purchased from Geneka Biotechnology (Montreal, Canada). rIFN- and ELISA kits for TNF-, IL-1, and IL-6 were from R&D Systems (Minneapolis, MN). TRIzol, -galactosidase (-gal) kit, and Lipofectamine 2000 were from Invitrogen Life Technologies. MTT and lactose dehydrogenase (LDH) kits were obtained from Roche (Nutley, NJ). The ECL-detecting reagents and nitrocellulose membrane were purchased from Amersham Biosciences (Arlington Heights, IL). Luciferase assay system was from Promega (Madison, WI).

Plasmids

The peroxisome proliferator-response element (PPRE)-containing reporter plasmid (J6-thymidine kinase (TK)-Luc) was provided by B. Staels (Institut Pasteur de Lille, Lille, France) (27). NF-B luciferase was kindly provided by G. Rawadi (Institut Pasteur, Laboratoire des Mycoplasmes, Paris, France) (28). The construction of a –3.2-kb rat iNOS promoter luciferase was generously provided by H. Zhang (Medical College of Georgia, Augusta, GA) (29). The expression vector for hemagglutinin-IKK was a gift from Z.-G. Liu (National Institutes of Health, Bethesda, MD) (30). The iNOS (–234/+31) luciferase and iNOS (–331/+31 NF-B-mutated) luciferase were kind gifts from M. Perrella (Harvard University Medical School, Boston, MA) (31). FLAG-tagged wild-type (wt) PPAR and FLAG-tagged L468A/E471A PPAR were provided by V. Chatterjee (University of Cambridge, Cambridge, U.K.) (32). Constitutive active Akt was purchased from Upstate Biotechnology. Constitutive active PI3K p110 (p110*) was kindly provided by J. Raymond (Medical University of South Carolina, Charleston, SC) (33). The expression vectors of p50 and p65 were provided by R. Pope (Northwestern University Medical School, Chicago, IL) (34). The mutated p65 (S276A) was a kind gift from S. Ghosh (Yale University School of Medicine, New Haven, CT) (35). The expression vectors containing wt p300 (pCI.p300) was a gift of J. Boyes (Medical Research Council Clinical Sciences Center, London, U.K.) (36). The cDNA for TNF-, IL-1, and IL-6 were graciously provided by L. Van Eldik (Northwestern University, Chicago, IL) (37).

Cell culture

Astrocytes were prepared from rat cerebral tissue from 1- to 3-day-old postnatal Sprague-Dawley rat pups and maintained in DMEM (4.5 g of glucose/L) with 10% FBS and antibiotics, as described before (8, 38, 39). After 10 days of culture, astrocytes were separated from microglia and oligodendrocytes by shaking for 24 h in an orbital shaker at 240 rpm. To ensure complete removal of the oligodendrocytes and microglia, the shaking was repeated twice after a gap of 1 or 2 days. For induction of NO, cytokine production, and transfection, cells were plated onto polylysine-coated 24-well and 12-well plates, and then stimulated with LPS/IFN- in serum-free conditions. Based on glial fibrillary acidic protein staining, astrocytes were >95% pure.

Nitrite concentration

Synthesis of NO was determined by assay of culture supernatants for nitrite, a stable reaction product of NO with molecular oxygen, as mentioned before. Briefly, supernatants were mixed with an equal volume of the Griess reagent in 96-well plate, gently shaken, and read in a microplate reader at 570 nm. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO₂ in the assay.

Immunoblot analysis

Cells were harvested in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitor mixture), and protein was estimated using Bradford reagent (Bio-Rad, Hercules, CA). Fifty micrograms of total protein per lane was separated by SDS-PAGE and transferred to nitrocellulose (Amersham Biosciences). Blots were blocked for 1 h in 5% nonfat dry milk-TBS-0.1% Tween 20 and incubated overnight with primary Ab (1/1000) in 5% milk-TBS-0.1% Tween 20 at 4°C (in case of phospho-Abs, 3% BSA was used instead of milk). This was followed by 1-h incubation with appropriate secondary peroxidase-conjugated Ab (1/10,000; Sigma-Aldrich). Immunoreactivity was detected using the ECL detection method, according to the manufacturer’s instructions (Amersham Biosciences), and subsequent exposure of the membrane to x-ray film.

RNA isolation and Northern blot analysis

Cells were harvested from the culture dish directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories, Houston, TX), and total RNA was isolated, according to the manufacturer’s protocol. For Northern blot analyses, 20 µg of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, transferred to hybond-nylon membrane (Amersham Biosciences), and hybridized at 68°C with ³²P-labeled cDNA probe using Express Hyb hybridization solution (BD Clontech, Palo Alto, CA). The cDNA probe for iNOS was made by PCR amplification using forward (5'-CTC CTT CAA AGA GGC AAA AAT A-3') and reverse primers (5'-CAC TTC CTC CAG GAT GTT GT-3'). The cDNA for TNF-, IL-1, and IL-6 were graciously provided by L. Van Eldik (37). After hybridization, the filters were washed three times in solution I (2x SSC, 0.05% SDS) for 1 h at room temperature, followed by solution II (0.1x SSC, 0.1% SDS) at 50°C for another hour. Membranes were then dried and exposed to x-ray film (Kodak, Rochester, NY). The same membrane was probed for -actin to normalize the RNA loading. The relative mRNA content for iNOS (iNOS/-actin) was measured by scanning the bands with a Bio-Rad (model GS-670) imaging densitometer.

IKK assays

For IKK or IKK assays, primary astrocytes were pretreated with 15d-PGJ₂ (10 µM) and then stimulated with LPS/IFN- (0.5 µg/50 U/ml) for 15 min. Cells were washed with cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitor mixture). A total of 200 µg of cell extracts was incubated with anti-IKK or IKK Ab (Santa Cruz Biotechnology) for 2 h at 4°C, then 30 µl of protein A/G PLUS agarose for an additional 1 h. The immune complexes were washed twice in lysis buffer and twice in kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂) and incubated at 30°C in 30 µl of kinase buffer containing 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM DTT, 50 µM Na₃V0₄, 20 µM ATP, and 5 µCi of [-³²P]ATP. A total of 2 µg of GST-IB fusion protein (Santa Cruz Biotechnology) was used as substrate in each reaction. Reactions were stopped after 30 min by denaturation in SDS loading buffer. Proteins were resolved by SDS-PAGE, and substrate phosphorylation was visualized by autoradiography.

PI3K assay

After stimulation in serum-free medium, cells were lysed with ice-cold lysis buffer containing 1% v/v Nonidet P-40, 100 mM NaCl, 20 mM Tris (pH 7.4), 10 mM iodoacetamide, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors (Sigma-Aldrich). Lysates were incubated at 4°C for 15 min, followed by centrifugation at 13,000 x g for 15 min. The supernatant was precleared with protein A/G-Sepharose beads (Amersham Biosciences) for 1 h at 4°C, followed by the addition of 1 µg/ml p85 mAb. After 2-h incubation at 4°C, protein G-Sepharose beads were added, and the resulting mixture was further incubated for 1 h at 4°C. The immunoprecipitates were washed twice with lysis buffer, once with PBS, once with 0.5 M LiCl and 100 mM Tris (pH 7.6), once in water, and once in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, and 0.25 mM EDTA). PI3K activity was determined using a lipid mixture of 100 µl of 0.1 mg/ml phosphatidylinositol and 0.1 mg/ml phosphatidylserine dispersed by sonication in 20 mM HEPES (pH 7.0) and 1 mM EDTA. The reaction was initiated by the addition of 20 µCi of [-³²P]ATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA) and 100 µM ATP, and terminated after 15 min by the addition of 80 µl of 1 N HCl and 200 µl of chloroform:methanol (1:1). Phospholipids were separated by TLC and visualized by exposure to iodine vapor and autoradiography (40).

The p300 and p65 association

To determine the association between p300 and p65, nuclear extracts were prepared from the treated cells and were incubated with NF-B gel shift oligonucleotide agarose conjugate (25 µl) for 30 min at 4°C, as described before (41). Agarose beads were washed three times with buffer (20 mM HEPES, pH 7.9, 10% (v/v) glycerol, 0.2 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, and protease mixture) before addition of SDS loading buffer. Samples were resolved on SDS-PAGE, and p65 and p300 were detected by immunoblot analysis using Abs against p300 and p65 (Santa Cruz Biotechnology).

EMSA

Nuclear extracts from stimulated or unstimulated astrocytes were prepared, and EMSA was performed, as described previously (8, 39), with NF-B consensus sequence, which was end labeled with [-³²P]ATP. Nuclear extracts were normalized based on protein concentration, and equal amount of protein (5 µg) was loaded. DNA-protein complexes were resolved on 5% nondenaturing PAGE in 45 mM Tris (pH 7.8), 45 mM boric acid, and 1 mM EDTA (0.5x Tris-boric-EDTA), and run at 11 V/cm. The gels were dried and then autoradiographed at –70°C using x-ray film.

Cytokine assay

The levels of TNF-, IL-1, and IL-6 were measured in culture supernatant with ELISA using protocols supplied by the manufacturer (R&D Systems).

Transcriptional assays

Primary astrocytes were transiently transfected with NF-B or iNOS luciferase reporter gene (1.5 µg/well) with -gal (0.1 µg/well) in the presence or absence of cDNAs (as indicated in figures, 0.5 µg/well) by lipofectamine-2000 (Invitrogen Life Technologies), as described before (39). pcDNA3 was used to normalize all groups to equal amounts of DNA. Luciferase activity was determined using a luciferase assay kit (Promega).

Cell viability

Cytotoxic effects of treatments were determined by measuring the metabolic activity of cells with MTT and LDH release assay (Roche).

Statistical analysis

Results shown represent means ± SD. Statistical analysis was performed by ANOVA by the Student-Neuman-Keuls test using GraphPad InStat software (San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 15d-PGJ₂ down-regulates LPS/IFN--induced expression of proinflammatory cytokines in primary astrocytes

Rat primary astrocytes were pretreated for 30 min with different concentrations of 15d-PGJ₂ and then exposed to LPS/IFN- (0.5 µg/50 U/ml). LPS/IFN- markedly induced the production of proinflammatory cytokines (TNF-, IL-1, and IL-6) in astrocytes, as determined by ELISA (Fig. 1a). The 15d-PGJ₂ strongly inhibited the LPS/IFN--induced production of TNF-, IL-1, and IL-6 in the supernatants of primary astrocytes in a dose-dependent manner (Fig. 1a), whereas 15d-PGJ₂ alone had no effect on the production of cytokines. The 15d-PGJ₂-mediated inhibition of cytokine production in the supernatant corresponded with a decrease in their mRNA expression in cells (Fig. 1b). The concentration of 15d-PGJ₂ (1–20 µM) or LPS/IFN- had no effect on cell viability, as tested by LDH release and MTT assays (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. The 15d-PGJ2 inhibits LPS/IFN--induced cytokine synthesis in a dose-dependent manner. Primary rat astrocytes were incubated for 30 min with different concentrations of 15d-PGJ2, as indicated, followed by LPS/IFN- (0.5 µg/50 U/ml) treatment for 24 h. We measured the concentration of TNF-, IL-1, and IL-6 released in the medium using ELISA. For TNF- levels, medium was taken out at 6 h of LPS treatment, while for IL-1 and IL-6 at 24 h (a). Results are the mean ± SD of four determinations. ***, p < 0.001 as compared with untreated cells; #, p < 0.05; and @, p < 0.001 as compared with LPS treatment. For detection of cytokine message, RNA was isolated from astrocytes 6 h after treatment with LPS/IFN- and processed for Northern blot analysis, as mentioned in Materials and Methods (b). Blots are representatives of two different experiments. One blot was stripped and probed against -actin for equal loading.

The 15d-PGJ₂ inhibits LPS/IFN--induced NO production and iNOS gene expression

Along with the production of proinflammatory cytokines, NO production in response to cytokines has been shown to be a critical mediator in the pathophysiology of inflammatory diseases (4). Rat primary astrocytes were pretreated with different concentrations of 15d-PGJ₂ and then exposed to LPS/IFN-. LPS/IFN- treatment induced the NO production (measured as nitrite) several fold as compared with untreated cells. The 15d-PGJ₂ treatment inhibited the LPS/IFN--induced NO production in a dose-dependent manner (Fig. 2a). In contrast, 9,10-dihydro-15d-PGJ₂ (CAY10410) was not as potent as 15d-PGJ₂, suggesting that the ,-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ₂ is a prerequisite for its strong anti-inflammatory effect. To understand the inhibitory mechanism of 15d-PGJ₂ on LPS/IFN--mediated nitrite production, the effect of 15d-PGJ₂ on iNOS protein and mRNA level in primary rat astrocytes was examined. Consistent with the inhibition of nitrite, 15d-PGJ₂ also inhibited the LPS/IFN--induced expression of iNOS at the mRNA and the protein levels (Fig. 2, b and c). Similar to the NO production, 9,10-dihydro-15d-PGJ₂ did not inhibit iNOS protein expression at the concentration of 5 µM, while it significantly inhibited at higher concentration (10 µM). Furthermore, we examined the effect of 15d-PGJ₂ on activation of iNOS promoter in response to LPS/IFN-. A plasmid containing a 3.2-kb portion of the rat iNOS promoter cloned with the luciferase gene (iNOS-luc) was transiently transfected into subconfluent cultures of primary astrocytes. After 24 h, the cultures were pretreated with different concentration of 15d-PGJ₂, followed by LPS/IFN- treatment for 6 h (Fig. 2d). The iNOS promoter activity was substantially (2-fold) stimulated upon incubation with LPS/IFN-, which was significantly inhibited by 15d-PGJ₂ in a dose-response manner (Fig. 2d). Furthermore, to examine the specificity of 15d-PGJ₂, we examined the effect of other PGs (PGA₁, PGB₂, PGD₂, PGE₁, PGE₂, PGF₁, PGJ₂, 15d-PGJ₂, and 9,10-dihydro-15d-PGJ₂) on LPS/IFN--induced iNOS protein expression. Among them, 15d-PGJ₂ was the most potent, while PGJ₂ was found to be only slightly effective. In contrast, other PGs were ineffective in terms of inhibition of iNOS protein expression in primary astrocytes (Fig. 2e).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. The 15d-PGJ2 inhibits the expression of LPS/IFN--induced iNOS in primary astrocytes. Nitrite was measured in supernatant of primary astrocytes (a) after 24 h of LPS/IFN- and 15d-PGJ2/9,10-dihydro-15d-PGJ2 treatment. Data are mean ± SD of six different experiments. #, p < 0.001 as compared with control; ***, p < 0.001 as compared with LPS/IFN- treatment; *, p < 0.05 as compared with LPS/IFN- treatment; NS, not significant as compared with LPS/IFN- treatment. For detection of iNOS protein expression by immunoblot in response to 15d-PGJ2/9,10-dihydro-15d-PGJ2 treatment, cell lysate from astrocytes was prepared 24 h after LPS/IFN- treatment (b). Blots are the representative of three different experiments. For detection of iNOS message, RNA was isolated from astrocytes 6 h after treatment with LPS/IFN- and processed for Northern blot analysis, as mentioned in Materials and Methods (c). Blots are representative of two different experiments. Primary astrocytes were transiently transfected using Lipofectamine 2000 reagent with 1.5 µg/well iNOS luciferase reporter vector along with 0.1 µg/well pCMV--gal. After 24 h of transfection, cells were pretreated with the indicated concentrations of 15d-PGJ2 and 9,10-dihydro-15d-PGJ2 for 30 min, followed by LPS/IFN- (0.5 µg/50 U/ml) for 6 h. Cells were lysed and processed for luciferase activity (Promega) and -gal (Invitrogen Life Technologies). Luciferase activity was normalized with respect to -gal activity and expressed relative to the activity of the control (d). PGL3-Basic was transfected as a control to detect the basal levels of luciferase activity. Data are mean ± SD of three different values. #, p < 0.001 as compared with control; *, p < 0.05; **, p < 0.01; and NS, not significant as compared with LPS/IFN- treatment. e, Primary astrocytes were treated with various PGs (5 µM) for 30 min before the addition of LPS/IFN-. After 24 h, cell lysate was processed for iNOS and -actin Western blot analysis.

The 15d-PGJ₂ inhibits induction of iNOS in primary astrocytes independent of PPAR

To determine whether 15d-PGJ₂ mediates its effect through PPAR, we transfected primary astrocytes with PPRE luciferase reporter construct. Treatment with 15d-PGJ₂ was not able to induce PPRE luciferase activity; however, it induced a 4-fold increase in luciferase activity when cotransfected with an expression vector of PPAR gene (Fig. 3a). Similarly, addition of 9,10-dihydro-15d-PGJ₂ did not affect PPRE luciferase activity, but induced significantly in PPAR-transfected cells. In contrast, a dominant-negative construct of PPAR had no effect on PPRE luciferase activity. This suggests that primary astrocytes may not contain functional PPAR, because primary astrocytes do express PPAR protein (Fig. 3a), but are unable to induce PPRE luciferase activity in the absence of exogenously transfected PPAR expression vector. We also used a chimeric receptor system in which the putative ligand-binding domain of the PPARs is fused to the DNA binding domain of the yeast transcription factor galactose-responsive gene 4 (GAL4). The 15d-PGJ₂ and 9,10-dihydro-15d-PGJ₂ potently activated the PPAR-dependent as well as PPAR-dependent chloramphenicol acetyltransferase (CAT) reporter activity, whereas they had no effect on PPAR (Fig. 3b). To further investigate the effect of 15d-PGJ₂ on PPAR activation, EMSA was performed. The binding specificity of PPAR on its consensus sequence was confirmed by the addition of cold competitor (100x), which completely abolished PPAR binding; in contrast, mutant oligonucleotide had no effect (Fig. 3c). Untreated astrocytes showed a high basal level of PPAR, which was not modulated by LPS/IFN- in the presence or absence of 15d-PGJ₂ over the time period (Fig. 3c). These data are consistent with results from reporter assays, suggesting that the effect of 15d-PGJ₂ is mediated independent of PPAR. To further strengthen these conclusions, we overexpressed PPAR in primary astrocytes to examine its effect on the regulation of iNOS promoter. In the absence of PPAR expression vector, treatment of primary astrocytes with 15d-PGJ₂ significantly inhibited the iNOS-luciferase activity induced by LPS/IFN- (Fig. 3d). However, transfection with PPAR wt or dominant-negative expression vector did not affect the 15d-PGJ₂-mediated inhibition of iNOS promoter (Fig. 3d). Furthermore, we used GW9662, an irreversible PPAR antagonist, to investigate whether PPAR has any effect on iNOS gene expression. Treatment with GW9662 had no effect on LPS/IFN--induced iNOS protein expression even at higher concentrations (20 µM) (Fig. 3e). Although GW9662 treatment inhibited the 15d-PGJ₂/PPAR-induced PPRE luciferase activity (Fig. 3f), it could not reverse the 15d-PGJ₂-induced inhibition of iNOS protein expression (Fig. 3g). Taken together, these results demonstrate that 15d-PGJ₂-mediated anti-inflammatory effects (inhibition of iNOS gene expression) are independent of PPAR in primary astrocytes.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. The 15d-PGJ2 inhibits NO production and iNOS gene expression in primary astrocytes independent of PPAR. Primary astrocytes were transiently transfected with PPRE-luc vector (1.5 µg/well) and pCMV--gal (0.1 µg/well) along wt or dominant negative (DN) of PPAR. After 48 h, astrocytes were stimulated with 5 µM 15d-PGJ2/9,10-dihydro-15d-PGJ2, and luciferase activity was normalized with respect to -gal activity (a). Inset, The expression of PPAR in primary astrocytes when 50 µg of protein was used for its detection by Western blot analysis. PGL3-Basic was transfected as a control to detect the basal levels of luciferase activity. **, p < 0.01; ***, p < 0.001; and NS, not significant as compared with untreated control. $, p < 0.001 and @, not significant as compared with 15d-PGJ2-treated PPAR wt transfected cell. Data are mean ± SD of four different values. To examine the effect of 15d-PGJ2 on the trans activation of PPARs, primary astrocytes were cotransfected with PPARs-GAL4 chimeras and the reporter plasmid (upstream activating sequences)5-TK-CAT. After 48 h, cells were treated with 5 µM 15d-PGJ2/9,10-dihydro-15d-PGJ2 for 24 h. Cell extracts were subsequently assayed for CAT by ELISA (Roche) (b). pCMV-GAL4-binding domain (without insert) and (upstream activating sequences)5-TK-CAT were transfected as a control to detect the basal levels of CAT activity (first lane). Data are mean of four values ± SD. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 as compared with untreated cell. Nuclear extract was prepared, as mentioned before, from LPS/IFN--treated primary astrocytes in the presence or absence of 15d-PGJ2 at various times, as indicated. EMSA was performed for PPAR, per manufacturer’s instructions (Geneka Biotechnology) (c). EMSA data are representative of two different experiments. Primary astrocytes were transiently transfected with iNOS-luc vector (1.5 µg/well) and pCMV--gal (0.5 µg/well) along wt or dominant negative (dn) of PPAR. Luciferase activity was normalized with respect to -gal activity (d). Data are mean ± SD of six different values. ***, p < 0.001 as compared with untreated cells; @, p < 0.001 as compared with LPS/IFN- treatment; %, not significant as compared with LPS/IFN-/15d-PGJ2 treatment. To examine the effect of GW9662 on LPS/IFN--induced iNOS protein expression, primary astrocytes were pretreated with different concentration of GW9662 (1–20 µM) for 30 min, followed by LPS/IFN- treatment. After 24 h, cells were processed for iNOS/-actin protein detection by immunoblot, as mentioned in Materials and Methods (e). To examine the effect of GW9662 on PPRE luciferase activity, primary astrocytes were cotransfected with PPRE-luc vector (1.5 µg/well) and pCMV--gal (0.5 µg/well). After 24 h, cells were treated with GW9662 for 30 min before the treatment with 15d-PGJ2. After 6-h incubation, luciferase activity was examined, as described before (f). Data are mean ± SD of three different values. ***, p < 0.001 as compared with untreated cells; @, p < 0.001 as compared with 15d-PGJ2-treated PPAR wt transfected cells. Primary astrocytes were treated with the indicated concentration of GW9662 prior 30 min to 15d-PGJ2. After 30-min incubation with 15d-PGJ2, cells were stimulated with LPS/IFN- for 24 h and then processed for iNOS/-actin protein detection, as mentioned earlier (g). Blots are representative of two different results.

The 15d-PGJ₂ inhibits the activation of NF-B

Activation of NF-B is necessary for induction of iNOS, TNF-, and IL-6 (29, 34, 42). Therefore, to understand the basis of iNOS inhibition by 15d-PGJ₂, we examined the effect of 15d-PGJ₂ on the activation of NF-B. A dose-dependent effect of 15d-PGJ₂ on NF-B activity in LPS/IFN--stimulated cells is shown in Fig. 4a. Incubation of primary astrocytes with 15d-PGJ₂ (10 µM) significantly inhibited the activation of NF-B elicited by LPS/IFN- challenge, as measured by EMSA (Fig. 4a). However, 9,10-dihydro-15d-PGJ₂ had no effect on LPS/IFN--induced NF-B nuclear translocation (Fig. 4a). The inhibitory effect of 15d-PGJ₂ on NF-B activity was further analyzed in cells transfected with NF-B luciferase reporter by monitoring the reporter activity in response to LPS/IFN- challenge. Similar to the EMSA findings, 15d-PGJ₂ also decreased the LPS/IFN--dependent activation of NF-B luciferase activity in a dose-dependent manner (Fig. 4b). In contrast, 9,10-dihydro-15d-PGJ₂ did not have any effect on LPS/IFN--induced NF-B reporter activity (Fig. 4a). Modulation of NF-B by 15d-PGJ₂ and 9,10-dihydro-15d-PGJ₂ was further supported by immunoblot analysis of p65 and p50 in nuclear extracts of treated primary astrocytes (Fig. 4c). Moreover, these conclusions are further supported by the modulation of degradation of IB and IB by 15d-PGJ₂ and 9,10-dihydro-15d-PGJ₂ treatments (Fig. 4d). To examine the specificity of 15d-PGJ₂, we examined the effect of various PGs on NF-B luciferase activity. As shown in Fig. 4e, except 15d-PGJ₂, none of the other PGs had any effect on LPS/IFN--induced NF-B luciferase activity. In addition, 15d-PGJ₂ decreased 95% of LPS/IFN--mediated luciferase activity in cells transfected with the iNOS-luci (–234/+31) vector, a construct strictly dependent on NF-B activation (Fig. 4e). Interestingly, the use of a fragment of the iNOS promoter deleted in the B sites (iNOS (–331/+31)-luci) completely abolished the activity of the promoter, reflecting the necessity of this motif for expression of the gene in response to LPS/IFN- stimulation (Fig. 4e). Inhibition of NF-B by 15d-PGJ₂ was found to be completely independent of PPAR as cotransfection with wt and dominant-negative forms of PPAR expression vector did not affect the LPS/IFN--mediated NF-B luciferase activity. Moreover, the dominant-negative form of PPAR did not reverse the 15d-PGJ₂-induced inhibition in luciferase activity in primary astrocytes (Fig. 4f).

View larger version (48K): [in this window] [in a new window]   FIGURE 4. The 15d-PGJ2 inhibits LPS/IFN--induced NF-B activation in primary astrocytes. Nuclear extracts were prepared from LPS/IFN-/15d-PGJ2-treated primary astrocytes, as indicated, and analyzed by EMSA for NF-B (a). EMSA data are representative of three different experiments. Astrocytes were treated with 10 µM 15d-PGJ2/9,10-dihydro-15d-PGJ2 30 min before the addition of LPS/IFN-. After 1-h incubation, nuclear extract was prepared and processed for EMSA, as described before. Primary astrocytes were transiently cotransfected with 1.5 µg of p(NF-B)3LdLuc along with pCMV--gal (0.1 µg/well), followed by stimulation for 6 h with the indicated concentration of 15d-PGJ2/9,10-dihydro-15d-PGJ2 and LPS/IFN- (b). Luciferase activity was normalized with respect to -gal activity. PGL3-Basic was transfected as a control to examine the basal levels of luciferase activity. Data are mean ± SD of three different values. #, p < 0.001 as compared with control; **, p < 0.05; @, p < 0.001; and NS, not significant as compared with LPS/IFN- treatment. Immunoblot was performed for p65 and p50 in nuclear extracts from primary astrocytes stimulated with LPS/IFN- with or without 15d-PGJ2/9,10-dihydro-15d-PGJ2 (10 µM) (c). Blots are representative of two different experiments. Total cell lysate of primary astrocytes was processed after treatment with LPS/IFN- with or without 15d-PGJ2/9,10-dihydro-15d-PGJ2 (10 µM) for the detection of IB by immunoblot at indicated time period (d). Blots are representatives of two different experiments. Primary astrocytes were transiently cotransfected with 1.5 µg of p(NF-B)3LdLuc along with pCMV--gal (0.1 µg/well), followed by stimulation for 6 h with various PGs (5 µM) and LPS/IFN- (e). Data are mean ± SD of three different values. ***, p < 0.001 as compared with control; #, p < 0.001; and NS, not significant as compared with LPS/IFN- treatment. Primary astrocytes were transiently transfected with 1.5 µg of iNOS (–234/+31) luciferase or iNOS (–331/+31 NF-B-mutated) luciferase, followed by stimulation for 6 h with indicated treatment with 15d-PGJ2 (10 µM) and LPS/IFN- (f). Luciferase activity was normalized with respect to -gal activity. Data are mean ± SD of four different values. ***, p < 0.01 and #, not significant as compared with control; @, p < 0.01 as compared with LPS/IFN- treatment. NS, Not significant as compared with LPS/IFN- treatment in (iNOS (–331/+31 NF-B-mutated)-luciferase-transfected cells; %, p < 0.05 as compared with LPS/IFN- treatment in (iNOS (–234/+31)-luciferase-transfected cells. Primary astrocytes were transiently transfected with p(NF-B)3LdLuc vector (1.5 µg/well) and pCMV--gal (0.1 µg/well) along with wt or dominant negative (dn) of PPAR. After 48 h, astrocytes were stimulated with 15d-PGJ2 (10 µM), and luciferase activity was normalized with respect to -gal activity (g). Data are mean ± SD of three different values. ***, p < 0.001 as compared with untreated cells; NS, not significant and @, p < 0.001 as compared with LPS/IFN- treatment; %, not significant as compared with LPS/IFN-/15d-PGJ2 treatment.

The results from preceding studies indicate that inhibition of NF-B activity by 15d-PGJ₂ may be due to an impaired IB targeting in LPS/IFN--stimulated primary astrocytes. To investigate the possibility that 15d-PGJ₂ down-regulates the IKK activities, primary astrocytes were stimulated with LPS/IFN- in the presence or absence of 15d-PGJ₂, and whole cell extracts were tested for phosphorylation status of IKKs by using a phosphospecific Ab for IKK, which also recognizes IKK. The treatment with 15d-PGJ₂ inhibited the phosphorylation of IKK (Fig. 5a). To further evaluate the effect of 15d-PGJ₂ on the activity of IKKs, cells were treated with LPS/IFN- in the presence or absence of 15d-PGJ₂ (10 µM) for 15 min. The IKK complex was immunoprecipitated using either anti-IKK or IKK Abs, and the kinase activity was evaluated using GST-IB as the substrate. As shown in Fig. 5b, 15d-PGJ₂ inhibited the phosphorylation of GST-IB by IKK or IKK by 95 and 75%, respectively, as compared with corresponding LPS/IFN--treated cells. To further confirm these results, astrocytes were cotransfected with wt expression vector of IKK and NF-B luciferase. The 15d-PGJ₂ treatment significantly inhibited IKK-mediated NF-B luciferase activity as well as iNOS luciferase activity in primary astrocytes (Fig. 5, c and d). In contrast, 9,10-dihydro-15d-PGJ₂ did not affect IKK-mediated iNOS luciferase activity, but slightly inhibited NF-B activity (Fig. 5d). Taken together, these observations clearly demonstrate that 15d-PGJ₂ inhibits NF-B DNA binding as well as its transcriptional activity by inhibiting IKK activities.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. The 15d-PGJ2 inhibits LPS/IFN--induced IKK activity and IKK-mediated NF-B luciferase activity in primary astrocytes. Primary astrocytes were incubated with 15d-PGJ2 (10 µM) before LPS/IFN- (1 µg/50 U/ml). At various time periods, cell lysate was prepared and processed for the detection of phosphostatus of IKK by using phosphospecific Abs of IKK (Cell Signaling Technology), which also recognizes IKK (a). To determine the effect of 15d-PGJ2 on the activities of IKK and IKK, primary astrocytes were incubated with 15d-PGJ2 before LPS/IFN- (1 µg/50 U/ml). After 30 min, IKK or IKK was immunoprecipitated with its specific Abs, and their activity was measured using rGST-IB protein, as mentioned in Materials and Methods (b). Blots are representative of two different results. Primary astrocytes were transiently cotransfected with 1.5 µg of p(NF-B)3LdLuc or iNOS-luc along with 0.5 µg of hemagglutinin-IKK or pcDNA3 and 0.1 µg of pCMV--gal/well. After 24 h, cells were treated with the indicated concentration of 15d-PGJ2 and 9,10-dihydro-15d-PGJ2. Luciferase and -gal activities were performed, as mentioned earlier (c and d). Data are mean ± SD of three experiments. ***, p < 0.001 as compared with control; &, p < 0.001 as compared with LPS/IFN- treatment; #, p < 0.001; and NS, not significant as compared with LPS/IFN--treated (IKK-transfected) cells.

The 15d-PGJ₂ inhibits LPS/IFN--mediated PI3K-Akt pathway

It has previously been suggested that PI3K and Akt regulate the NF-B pathway via modulating IKK activity in response to proinflammatory cytokine (TNF-) and growth factor (platelet-derived growth factor) (43, 44). Inhibition of growth factor (insulin-like growth factor or epidermal growth factor) induced cell proliferation in primary astrocytes by 15d-PGJ₂, indicating that 15d-PGJ₂ might be affecting the PI3K and Akt proliferation pathway in these cells (data not shown). Therefore, we examined the effect of 15d-PGJ₂ on LPS/IFN--induced activation of Akt. As evident in Fig. 6a, 15d-PGJ₂ (10 µM) inhibited the LPS/IFN--mediated phosphorylation of Akt and PDK1 in a time-dependent manner. Treatment with 9,10-dihydro-15d-PGJ₂ (10 µM) had no effect on LPS/IFN--induced Akt phosphorylation (Fig. 6a). The upstream target of Akt/PDK1 is PI3K; therefore, we examined the effect of 15d-PGJ₂ on LPS/IFN--induced PI3K activity. Assays with phosphatidylinositol as a substrate revealed that the lipid kinase activity of PI3K was induced several fold upon LPS/IFN- treatment of primary astrocytes (Fig. 6b), which was inhibited by 15d-PGJ₂ (Fig. 6b). These observations indicate that the inhibitory mechanism of 15d-PGJ₂ on NF-B activation may be via the PI3K-Akt pathway. To further investigate the role of 15d-PGJ₂ on PI3K-Akt pathway, astrocytes were cotransfected with NF-B luciferase reporter and the constitutive active form of Akt (CA Akt) or PI3K catalytic subunit (P110*). Interestingly, cotransfection with CA Akt and P110* reversed not only 15d-PGJ₂-mediated inhibition of LPS/IFN--induced NF-B luciferase activity (Fig. 6, c and d), but also iNOS luciferase activity in primary astrocytes (Fig. 6, e and f). These studies clearly document that 15d-PGJ₂ down-regulates the LPS/IFN--mediated PI3K-Akt signal transduction pathway.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. The 15d-PGJ2 inhibits LPS/IFN--mediated PI3K-Akt pathway in primary astrocytes. Primary astrocytes were incubated for 30 min with 15d-PGJ2 (10 µM) before LPS/IFN- (1 µg/50 U/ml). At various time periods, cell lysates were prepared and processed for the detection of phosphostatus of PDK1 and Akt by using their phosphospecific Abs (Cell Signaling Technology). Astrocytes were treated with 15d-PGJ2 and 9,10-dihydro-15d-PGJ2, followed by LPS/IFN- treatment for 60 min. Cell lysate was processed for the detection of phosphor-Akt, as described above (a). For PI3K activity, primary astrocytes were preincubated with 15d-PGJ2 (1–10 µM), followed by LPS/IFN- treatment for 3 min. The p85 was immunoprecipitated with its Ab (Santa Cruz Biotechnology), and the lipid kinase activity of PI3K was determined in cell lysates, as described under Materials and Methods (b). Data are representative of two different experiments. Primary astrocytes were transiently cotransfected with 1.5 µg of p(NF-B)3LdLuc or iNOS-luc along with 0.5 µg of CA Akt or P110* or pcDNA3 and 0.1 µg of pCMV--gal/well. After 48 h, cells were treated with 15d-PGJ2 (10 µM) and LPS/IFN-, then luciferase and -gal activities were performed, as mentioned earlier (c–f). Data are mean ± SD of three experiments. **, p < 0.01 and ***, p < 0.001 as compared with control; #, p < 0.01 and %, p < 0.001 as compared with LPS/IFN- treatment; !, p < 0.05, @, p < 0.01, and &, p < 0.001 as compared with LPS/IFN- treated (Akt or P110*-transfected cells).

The 15d-PGJ₂ inhibits p65/p50-induced NF-B and iNOS luciferase activity in primary astrocytes

The predominant form of NF-B consists of p50 and p65 subunits that are sequestered in the cytoplasm of unstimulated cells by the inhibitory proteins IB and IB (21). Upon stimulation and degradation of IB complex, p50/p65 heterodimers translocate to the nucleus and bind to target genes. Earlier, p50 and p65 subunits have been demonstrated as a target for modification by 15d-PGJ₂ (15, 19). To further evaluate these findings, we cotransfected p65 and p50 with NF-B luciferase or iNOS luciferase reporter. As evident from Fig. 7, treatment with 15d-PGJ₂ inhibited the p65/p50-induced NF-B luciferase and iNOS luciferase activities in primary astrocytes. In contrast, 9,10-dihydro-15d-PGJ₂ did not affect iNOS luciferase, but inhibited p65/p50-mediated NF-B luciferase activity. All together, it is suggested that 15d-PGJ₂ directly modifies p65 and p50 subunits, resulting in the inhibition of the NF-B- and iNOS-reporter activities.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. The 15d-PGJ2 inhibits p65/p50-mediated iNOS and NF-B luciferase activities in primary astrocytes. Primary astrocytes were transiently cotransfected with 1.5 µg of iNOS-luc or p(NF-B)3LdLuc along with 0.5 µg of p65 and p50 or pcDNA3 (to normalize total DNA content) and 0.1 µg of pCMV--gal/well. After 48 h, cells were treated with 15d-PGJ2 and 9,10-dihydro-15d-PGJ2 (10–20 µM), and luciferase/-gal activities were performed, as mentioned earlier (a and b). Data are mean ± SD of six experiments. ***, p < 0.001 as compared with control; @, p < 0.001; $, p < 0.05; and NS, not significant as compared with LPS/IFN- treated (p65/p50-transfected astrocytes).

The 15d-PGJ₂ inhibits recruitment of p300 by NF-B (p65) in primary astrocytes

NF-B transcriptional competence requires interaction with the transcription coactivator p300 (45). To assess the physical interactions of p65 with p300, nuclear extracts from LPS/IFN--treated primary astrocytes in the presence or absence of 15d-PGJ₂ were incubated with NF-B gel shift oligonucleotides agarose conjugates. Interaction of NF-B complex with p300 was analyzed by Western blot analysis by using anti-p300 Ab/anti-p65 Ab. As shown in Fig. 8a, treatment with LPS/IFN- induced the binding of p65 on NF-B agarose as compared with untreated cells. In LPS/IFN--treated nuclear extract, p65 was able to recruit p300, suggesting a strong recruitment of coactivator p300 to the NF-B transcription factor. Pretreatment with 15d-PGJ₂ completely abolished the p65 nuclear translocation, which, in turn, inhibits the recruitment of p300 in primary astrocytes (Fig. 8a). This observation was further supported by TranSignal p300-TF (transcription factor) interaction array experiment (Panomics, Redwood City, CA). For this, we immunoprecipitated p300 protein from the nuclear extract of LPS/IFN--treated cells with or without the treatment of 15d-PGJ₂ (20 µM). Transcription factors that interacted with p300 were detected by the Panomics array. As depicted from Fig. 8b, treatment with 15d-PGJ₂ inhibits the interaction of p300 with NF-B. These results further support the conclusion that 15d-PGJ₂ inhibits the recruitment of p300 by NF-B in primary astrocytes (Fig. 8b). Serum response element (SRE) was used as an internal control because no change was observed in SRE intensity. Recently, Deng et al. (46) have shown that up-regulation of p300 binding leads to acetylation of p50. Therefore, we examined the effect of 15d-PGJ₂ on p300-mediated acetylation of p50. Nuclear extracts were prepared from cells treated with 15d-PGJ₂ for 30 min, followed by treatment with LPS/IFN- for 4 h. To detect the acetylation of p50, we immunoprecipitated p50 and detected by Western blot using an Ab specific for acetylated lysine. A low level of acetylated p50 was detected at basal state that was increased by LPS/IFN- stimulation (Fig. 8c). Acetylation of p50 was blocked by 15d-PGJ₂ treatment (Fig. 8c). The recruitment of p300 depends upon the phosphorylation status of p65 at Ser²⁷⁶ (35). To examine the effect of Ser²⁷⁶ of p65 on p300-mediated iNOS and NF-B luciferase activity, primary astrocytes were cotransfected with p300 in the presence or absence of p65 or p65 (S276A) expression vector (Fig. 8, d and e). Transfection with p300 or p65 significantly induced the iNOS and NF-B luciferase activities, and a synergistic effect was observed when p300 was cotransfected with p65. Interestingly, mutation in p65 at Ser²⁷⁶ completely abolished the synergistic effect, suggesting the crucial role of phosphorylation of p65 at S276 for recruitment of p300 in the regulation of NF-B and iNOS gene expression in primary astrocytes (Fig. 8, d and e).

View larger version (38K): [in this window] [in a new window]   FIGURE 8. The 15d-PGJ2 inhibits recruitment of p300 by NF-B (p65) and p300-mediated iNOS and NF-B luciferase activities in primary astrocytes. Nuclear extracts were prepared after 1 h and incubated with NF-B oligonucleotide agarose (Santa Cruz Biotechnology). The bound protein was resolved by SDS-PAGE gel. The membrane blot was probed with anti-p300 Ab. The same gel was stripped and reprobed with anti-p65 Ab (a). The gels are representative of three experiments. To detect the interaction between p300 and NF-B, a TranSignal p300-TF Interaction Array kit (Panomics) was used, according to the manufacturer’s instructions (b). Data were represented as an arbitrary ratio between NF-B to SRE, and SRE was taken as an internal control. Data are the mean of two different values. Nuclear extracts from primary astrocytes treated with 15d-PGJ2 stimulated with or without LPS/IFN- for 4 h were immunoprecipitated with p50 Ab, and the acetylated trans activator in the precipitate was detected by Western blot analysis using acetylated lysine mAb (c). Primary astrocytes were cotransfected with iNOS and NF-B luciferase reporter (1.5 µg/well) with p300 or p65 or p65(S276A) (0.5 µg/well), as indicated. Total DNA was kept constant in all wells with pcDNA3. Luciferase and -gal activities were performed, as mentioned earlier (d and e). Data are mean ± SD of four values. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 as compared with control; @, p < 0.001 as compared with p300-transfected cells; $, p < 0.001 as compared with p65-transfected cells; %, p < 0.001 as compared with p300/p65-transfected astrocytes. Primary astrocytes were transiently cotransfected with 1.5 µg of iNOS-luc or p(NF-B)3LdLuc along with 0.5 µg of p300 and CA Akt or P110* or cDNA3 (to normalize total DNA content) and 0.1 µg of pCMV--gal/well. Luciferase and -gal activities were performed, as mentioned earlier (f and g). Data are mean ± SD of six values. ***, p < 0.001 as compared with control; @, p < 0.001 as compared with p300-transfected astrocytes; #, p < 0.001 as compared with 15d-PGJ2 (5 µM)-treated cells (p300-transfected cells); %, p < 0.001 as compared with 15d-PGJ2 (10 µM)-treated cells (p300-transfected cells); !, p < 0.001 as compared with 15d-PGJ2 (20 µM)-treated cells (p300-transfected cells).

The 15d-PGJ₂ inhibits p300-mediated iNOS and NF-B luciferase activity independent of PPAR

It was of interest to examine whether the inhibitory effect of 15d-PGJ₂ on p300-mediated iNOS and NF-B luciferase activity in primary astrocytes is mediated through PPAR. For this, we cotransfected astrocytes with wt or dominant-negative expression vector of PPAR and p300 along with iNOS or NF-B luciferase reporter and examined the luciferase activity. Interestingly, cotransfection with p300 and PPAR wt significantly induced iNOS and NF-B luciferase activities as compared with p300 alone; in contrast, dominant-negative construct of PPAR had no additive effect over that of p300 (Fig. 9, a and b). Treatment with 15d-PGJ₂ significantly inhibited the iNOS and NF-B luciferase activities in primary astrocytes cotransfected with p300 along with PPAR wt or dominant negative, indicating that the effect of 15d-PGJ₂ may be independent of PPAR. In contrast, treatment with 9,10-dihydro-15d-PGJ₂ had no significant effect on p300-mediated iNOS luciferase and NF-B luciferase activity (Fig. 9, a and b). We also examined the effect of GW9662, an antagonist of PPAR, on 15d-PGJ₂-induced inhibition of p300-mediated iNOS and NF-B luciferase activity. Treatment with GW9662 did not reverse the 15d-PGJ₂-induced inhibition of p300-mediated luciferase activities (iNOS and NF-B) in primary astrocytes (Fig. 9, c and d). The p300 interacts with PPAR and modulates PPAR-mediated gene expression (47). To evaluate the effect of 15d-PGJ₂ on p300-mediated PPRE luciferase activities, astrocytes were cotransfected with p300 and PPRE luciferase reporter in the presence or absence of PPAR wt expression vector. As shown in Fig. 9e, p300 significantly induced the activity of PPRE luciferase, and cotransfection with PPAR did not further potentiate this activity. As shown previously, 15d-PGJ₂ induced PPRE luciferase activity only when cells were transfected with PPAR. Moreover, it did not affect p300- or p300/PPAR-mediated PPRE luciferase activity. Taken together, this study documents that 15d-PGJ₂ mediates its anti-inflammatory action independent of PPAR.

View larger version (36K): [in this window] [in a new window]   FIGURE 9. The 15d-PGJ2 inhibits p300-mediated iNOS and NF-B luciferase activities in primary astrocytes independent of PPAR. Primary astrocytes were transiently transfected with iNOS-luc or p(NF-B)3LdLuc vector (1.5 µg/well) and p300 (0.5 µg/well) along with wt or dominant negative (dn) of PPAR (a and b). Cells were treated with 15d-PGJ2 and 9,10-dihydro-15d-PGJ2 (10 µM) for 6 h, followed by luciferase activity, as described before. Luciferase activity was normalized with respect to -gal activity. Data are mean ± SD of six different values. ***, p < 0.001 as compared with control; @, p < 0.001; NS, not significant as compared with p300-transfected astrocytes; &, p < 0.001; $, not significant as compared with p300/PPAR wt transfected cells; &, p < 0.001 as compared with PPAR dn transfected cells. Primary astrocytes were transiently cotransfected with p300 and iNOS-luc or p(NF-B)3LdLuc along with pCMV--gal, as mentioned earlier. Cells were stimulated for 6 h with the indicated treatment with GW9662 (10 µM) in the presence or absence of 15d-PGJ2 (5–10 µM) (c and d). Luciferase activity was normalized with respect to -gal activity. Data are mean ± SD of three different values. ***, p < 0.001 as compared with control; @, p < 0.001 as compared with p300-transfected astrocytes; and NS, %, and &, not significant as compared with 15d-PGJ2-treated cells (p300 transfected) with 5, 10, and 20 µM concentration, respectively. Primary astrocytes were cotransfected with PPRE luciferase reporter (1.5 µg/well) with p300 or PPAR wt or dn (0.5 µg/well), as indicated. Total DNA was kept constant in all wells with pcDNA3. Luciferase and -gal activities were performed at 6 h (e), as mentioned earlier. Data are mean ± SD of six values. ***, p < 0.001 and @, not significant as compared with control; NS, not significant as compared with p300-transfected astrocytes; %, not significant as compared with p300/PPAR-transfected astrocytes; $, p < 0.001 as compared with 15d-PGJ2-treated astrocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The anti-inflammatory effect of 15d-PGJ₂ is thought to be mediated through the activation of PPAR (13, 14). In contrast to previous reports, in this study, we report that 15d-PGJ₂ inhibits the inflammatory response independent of PPAR in primary astrocytes. This conclusion is based on the following observations. First, 15d-PGJ₂ did not induce nuclear translocation of PPAR and PPRE-dependent luciferase activity, although primary astrocytes do express PPAR protein. Second, cotransfection with wt and dominant-negative expression vector of PPAR did not affect the 15d-PGJ₂-mediated inhibition of iNOS and NF-B luciferase activity. Third, GW9866 did not reverse 15d-PGJ₂-mediated inhibition of LPS/IFN- or p300-mediated iNOS or NF-B reporter activities, although it inhibited the 15d-PGJ₂-induced PPAR trans activation activities. Fourth, 15d-PGJ₂ did not induce p300/PPAR-induced PPRE luciferase activity. Fifth, an analog of 15d-PGJ₂, 9,10-dihydro-15d-PGJ₂ was not as potent as 15d-PGJ₂ in term of inhibition of iNOS and NF-B activation, but equally potent as 15d-PGJ₂ in activating PPRE reporter activity (in transfected PPAR astrocytes) as well as PPER-GAL4 reporter, suggesting that the ,-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ₂ is a prerequisite for strong anti-inflammatory effect of 15d-PGJ₂. Taken together, these results suggest that 15d-PGJ₂ does not require PPAR to inhibit the induction of iNOS in primary rat astrocytes. It may be that PPAR expressed in primary astrocytes is functionally inactive or the levels of constitutive PPAR are too low to induce the activation of PPRE elements; therefore, 15d-PGJ₂ was not able to further induce/activate PPAR, unless PPAR was exogenously expressed by transient transfection. Moreover, other specific PPAR agonists (ciglitazone and troglitazone) also had no effect on the iNOS luciferase and PPRE luciferase activities, thus further supporting the notion of existence of nonfunctional PPAR (data not shown). Ricote et al. (14) reported the absence of PPAR in RAW 264.7 cells, and most of the effects seen in these cells were observed only after transient expression of PPAR.

Recently, several studies have reported that 15d-PGJ₂ exhibits strong anti-inflammatory effect by attenuating the expression of proinflammatory mediators in activated monocytes/macrophages mainly through the inhibition of NF-B-dependent transcription of inflammatory genes (15, 17, 18, 19). Our data in primary astrocytes are in agreement with these studies and clearly demonstrate that 15d-PGJ₂ inhibits NF-B nuclear translocation via inhibition of IB degradation (15, 17, 18, 19). Studies described in this work document that 15d-PGJ₂ inhibits the degradation of IB, in vivo phosphorylation of IKK, and in situ phosphorylation of GST-IB substrate when IKK and IKK were immunoprecipitated from treated cells. One of the upstream kinases for IKKs is Akt, an important component of the PI3K pathway (43, 44). We examined the effect of 15d-PGJ₂ on LPS/IFN--induced Akt activity, a potential candidate to regulate the activity of IKKs (43, 44). Inhibition of Akt and PI3K activities modulated by the treatment of 15d-PGJ₂ and reversal of this effect via cotransfection of the constitutively active form of Akt and PI3K (P110*) further point toward the possibility that IKK regulation by Akt is the mechanism of anti-inflammatory action of 15d-PGJ₂. In addition, it has been reported that in vitro 15d-PGJ₂ also directly inhibits IKK activity. These studies support the conclusion that the anti-inflammatory actions of 15d-PGJ₂ include regulation of the PI3K-Akt-NF-B pathway. We also observed that 15d-PGJ₂ inhibits growth factor-induced cell proliferation of primary astrocytes via down-regulating PI3K-Akt pathway (our unpublished data).

Maximal NF-B transcriptional activity requires interaction with other components of transcriptional machinery, such as p300/CREB-binding protein (CBP) (45). Phosphorylation of p65 at Ser²⁷⁶ is critical for its interaction with p300 (35), and mutation at Ser²⁷⁶ completely abolished the recruitment of p300. The interaction between p65 and p300 was found to be critical for the induction of iNOS gene expression (Fig. 8d). Therefore, 15d-PGJ₂ may extend its anti-inflammatory activity by interfering with this pathway, thus impairing NF-B-dependent transcriptional activity without affecting the NF-B nuclear translocation or DNA binding. Our data clearly show that 15d-PGJ₂ inhibits the recruitment of p300 by p65 as well as p300-mediated iNOS and NF-B luciferase activity in primary astrocytes, which supports a putative nuclear role of PGs. Cyclooxygenase-2, the enzyme responsible for the production of PGs, is known to be localized both in the nuclear envelope as well as endoplasmic reticulum (56). The p300-mediated acetylation of p50 induced the affinity of p50/p65 for NF-B-binding sequence of promoters of NF-B-dependent genes (46). It was proposed that p300 binding to DNA-bound NF-B is limited and proinflammatory cytokines are capable of augmenting p300 binding by a positive feedback loop driven by p50 acetylation. The acetylation of p50 by histone acetyltransferase of p300 bound to the complex leads to an increased p50/p65 binding, which in turn recruits additional p300 to the complex. This autoregulatory loop ensures up-regulation of NF-B-mediated gene expression (46). Inhibition of recruitment of p300 and acetylation of p50 ultimately affects the NF-B-dependent gene expression by 15d-PGJ₂, thereby completely supporting their notion. Additionally, the reversal of 15d-PGJ₂ inhibition of p300-mediated iNOS and NF-B luciferase activity by constitutive active Akt and P110 further indicates the role of the PI3K-Akt pathway in the regulation of p300-mediated NF-B and iNOS gene regulation in primary astrocytes.

PGJ₂ and its metabolites are characterized by the presence of a cyclopentenone ring system containing an electrophilic carbon. This ring system can react covalently by means of the Michael addition reaction with nucleophiles such as free sulfhydryls of glutathione and cysteine residues in cellular proteins (15, 19, 20). In p65 and p50 NF-B subunits, there is a conserved cysteine residue located in the DNA binding domain (Cys⁶² in p50, Cys³⁸ in p65) (57). It has been reported that PPAR-independent actions of 15d-PGJ₂ may be due to the reactive ring system (15), and that a mutation at Cys³⁸ to Ser of p65 abolished the inhibitory effect of 15d-PGJ₂ on DNA-binding ability of homodimer of p65 (15). Therefore, the anti-inflammatory effect of 15d-PGJ₂ in p65/p50-mediated NF-B and iNOS luciferase activities may possibly be mediated by reactive cyclopentenone ring in this compound, which acts in a receptor-independent manner (PPAR). Moreover, use of 9,10-dihydro-15d-PGJ₂ suggests that reduction of the double bond in the cyclopentenone ring of 15d-PGJ₂ significantly abolishes the anti-inflammatory effect of 15d-PGJ₂. This indicates that the biological activities of 15d-PGJ₂ can be attributed to its electrophilic center.

Based on the data reported in this work and elsewhere, a model of LPS/IFN- induction of iNOS gene expression in primary astrocytes is shown in Fig. 10. The current study demonstrates that LPS stimulation of astrocytes leads to activation of the PI3K-Akt pathway, which activates the NF-B pathway by phosphorylation of IKKs. Inhibition of these pathways by 15d-PGJ₂ limits the release and phosphorylation of p65 and recruitment of p300, which cooperatively regulate iNOS gene expression. Thus, the PI3K-Akt pathway imposes a regulatory mechanism of 15d-PGJ₂ to inhibit the expression of TNF-, IL-6, and iNOS in LPS-stimulated primary astrocytes. In contrast, IFN- induces rapid tyrosine phosphorylation of STAT1, which then forms homodimers, translocates to the nucleus, and binds to the -activating sequence. STAT1 can directly interact with the CBP/p300 family of transcriptional coactivators (58). In addition, 15d-PGJ₂ was previously reported to inhibit the phosphorylation of STAT1 as well as JAK1 and JAK2 activation in activated astrocytes and microglia (59).

View larger version (24K): [in this window] [in a new window]   FIGURE 10. Schematic model of the mechanism of action of 15d-PGJ2 in the regulation of the iNOS gene expression in primary astrocytes. Binding of LPS to the CD14 and TLR4/MD2 complex (60 ) activates the PI3K-Akt signaling pathway (a). The 15d-PGJ2 inhibits PI3K-Akt pathway, which results in the down-regulation of IKK activity (b), degradation of IB proteins, and release and phosphorylation of p65. The 15d-PGJ2 inhibits the recruitment of p300 coactivator by p65 (c), finally affecting the NF-B-dependent genes expression (d). In contrast, IFN- induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, STAT1, which then forms homodimers, translocates to the nucleus, and binds to the -activating sequence. STAT1 can directly interact with the CBP/p300 family of transcriptional coactivators (58 ). The 15d-PGJ2 reduces the phosphorylation of STAT1 as well as JAK1 and JAK2 in activated astrocytes and microglia (e) (59 ).

	Acknowledgments

 
We thank Drs. Georges Rawadi, Hanfang Zhang, Zheng-Gang Liu, Steven A. Kliewer, V. Krishna, K. Chatterjee, Shankar Ghosh, Linda J. Van Eldik, and Bruce C. Kone for their kind gift of constructs; Hope Terry for secretarial assistance; and Joyce Bryan for technical assistance. We are thankful to Drs. Miguel A. Contreras, Anne G. Gilg, and Ehtishamul H. Shah for proofreading this manuscript.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants NS-22576, NS-34741, NS-40810, NS-37766, and NS-40144.

² Address correspondence and reprint requests to Dr. Inderjit Singh, 316, Clinical Science Building, Medical University of South Carolina, Charleston, SC 29425. E-mail address: singhi{at}musc.edu

³ Abbreviations used in this paper: iNOS, inducible NO synthase; 15d-PGJ₂, 15-deoxy-12,14-PGJ₂; -gal, -galactosidase; CA Akt, constitutive active form of Akt; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; GAL4, galactose-responsive gene 4; IKK, IB kinase; LDH, lactose dehydrogenase; PDK, 3-phosphoinositide-dependent kinase-1; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; SRE, serum response element; TK, thymidine kinase; wt, wild type.

Received for publication January 20, 2004. Accepted for publication August 6, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Minagar, A., P. Shapshak, R. Fujimura, R. Ownby, M. Heyes, C. Eisdorfer. 2002. The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associated dementia, Alzheimer disease, and multiple sclerosis. J. Neurol. Sci. 202:13.[Medline]
2. Little, A. R., J. P. O’Callagha. 2001. Astrogliosis in the adult and developing CNS: is there a role for proinflammatory cytokines?. Neurotoxicology 22:607.[Medline]
3. Dong, Y., E. N. Benveniste. 2001. Immune function of astrocytes. Glia 36:180.[Medline]
4. Smith, K. J., R. Kapoor, P. A. Felts. 1999. Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol. 9:69.[Medline]
5. Gebicke-Haerter, P. J.. 2001. Microglia in neurodegeneration: molecular aspects. Microsc. Res. Tech. 54:47.[Medline]
6. Smith, K. J., H. Lassmann. 2002. The role of nitric oxide in multiple sclerosis. Lancet Neurol. 1:232.[Medline]
7. Gilg, A. G., A. K. Singh, I. Singh. 2000. Inducible nitric oxide synthase in the central nervous system of patients with X-adrenoleukodystrophy. J. Neuropathol. Exp. Neurol. 59:1063.[Medline]
8. Giri, S., M. Jatana, R. Rattan, J. S. Won, I. Singh, A. K. Singh. 2002. Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease. FASEB J. 16:661.[Abstract/Free Full Text]
9. Bolanos, J. P., A. Almeida. 1999. Roles of nitric oxide in brain hypoxia-ischemia. Biochim. Biophys. Acta 1411:415.[Medline]
10. Horton, J. W.. 2003. Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy. Toxicology 189:75.[Medline]
11. Cross, A. H., T. P. Misko, R. F. Lin, W. F. Hickey, J. L. Trotter, R. G. Tilton. 1994. Aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in SJL mice. J. Clin. Invest. 93:2684.
12. Iadecola, C., F. Zhang, X. Xu. 1995. Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. Am. J. Physiol. 268:R286.
13. Jiang, C., A. T. Ting, B. Seed. 1998. PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82.[Medline]
14. Ricote, M., A. C. Li, T. M. Willson, C. J. Kelly, C. K. Glass. 1998. The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391:79.[Medline]
15. Straus, D. S., G. Pascual, M. Li, J. S. Welch, M. Ricote, C. H. Hsiang, L. L. Sengchanthalangsy, G. Ghosh, C. K. Glass. 2000. 15-deoxy- 12,14-prostaglandin J2 inhibits multiple steps in the NF-B signaling pathway. Proc. Natl. Acad. Sci. USA 97:4844.[Abstract/Free Full Text]
16. Chawla, A., Y. Barak, L. Nagy, D. Liao, P. Tontonoz, R. M. Evans. 2001. PPAR- dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat. Med. 7:48.[Medline]
17. Petrova, T. V., K. T. Akama, L. J. Van Eldik. 1999. Cyclopentenone prostaglandins suppress activation of microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-12,14-prostaglandin J₂. Proc. Natl. Acad. Sci. USA 96:4668.[Abstract/Free Full Text]
18. Castrillo, A., M. J. Diaz-Guerra, S. Hortelano, P. Martin-Sanz, L. Bosca. 2000. Inhibition of IB kinase and IB phosphorylation by 15-deoxy-(12,14)-prostaglandin J₂ in activated murine macrophages. Mol. Cell. Biol. 20:1692.[Abstract/Free Full Text]
19. Cernuda-Morollon, E., E. Pineda-Molina, F. J. Canada, D. Perez-Sala. 2001. 15-Deoxy- 12,14-prostaglandin J₂ inhibition of NF-B-DNA binding through covalent modification of the p50 subunit. J. Biol. Chem. 276:35530.[Abstract/Free Full Text]
20. Rossi, A., P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, M. G. Santoro. 2000. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IB kinase. Nature 403:103.[Medline]
21. Ghosh, S., M. Karin. 2002. Missing pieces in the NF-B puzzle. Cell 109:S81.
22. Zandi, E., M. Karin. 1999. Bridging the gap: composition, regulation, and physiological function of the IB kinase complex. Mol. Cell. Biol. 19:4547.[Free Full Text]
23. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, M. Karin. 1997. The IB kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B activation. Cell 91:243.[Medline]
24. DiDonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, M. Karin. 1997. A cytokine-responsive IB kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
25. Delhase, M., M. Hayakawa, Y. Chen, M. Karin. 1999. Positive and negative regulation of IB kinase activity through IKK subunit phosphorylation. Science 284:309.[Abstract/Free Full Text]
26. Nakano, H., M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura. 1998. Differential regulation of IB kinase and by two upstream kinases, NF-B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95:3537.[Abstract/Free Full Text]
27. Delerive, P., K. De Bosscher, S. Besnard, W. Vanden Berghe, J. M. Peters, F. J. Gonzalez, J. C. Fruchart, A. Tedgui, G. Haegeman, B. Staels. 1999. Peroxisome proliferator-activated receptor negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-B and AP-1. J. Biol. Chem. 274:32048.[Abstract/Free Full Text]
28. Rawadi, G., J. Garcia, B. Lemercier, S. Roman-Roman. 1999. Signal transduction pathways involved in the activation of NF-B, AP-1, and c-fos by Mycoplasma fermentans membrane lipoproteins in macrophages. J. Immunol. 162:2193.[Abstract/Free Full Text]
29. Zhang, H., X. Chen, X. Teng, C. Snead, J. D. Catravas. 1998. Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells. Biochem. Pharmacol. 55:1873.[Medline]
30. Devin, A., Y. Lin, S. Yamaoka, Z. Li, M. Karin, Z. Liu. 2001. The and subunits of IB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF. Mol. Cell. Biol. 21:3986.[Abstract/Free Full Text]
31. Perrella, M. A., A. Pellacani, P. Wiesel, M. T. Chin, L. C. Foster, M. Ibanez, C. M. Hsieh, R. Reeves, S. F. Yet, M. E. Lee. 1999. High mobility group-I(Y) protein facilitates nuclear factor-B binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer. J. Biol. Chem. 274:9045.[Abstract/Free Full Text]
32. Gurnell, M., J. M. Wentworth, M. Agostini, M. Adams, T. N. Collingwood, C. Provenzano, P. O. Browne, O. Rajanayagam, T. P. Burris, J. W. Schwabe, et al 2000. A dominant-negative peroxisome proliferator-activated receptor (PPAR) mutant is a constitutive repressor and inhibits PPAR-mediated adipogenesis. J. Biol. Chem. 275:5754.[Abstract/Free Full Text]
33. Tanti, J. F., T. Gremeaux, S. Grillo, V. Calleja, A. Klippel, L. T. Williams, E. Van Obberghen, Y. Le Marchand-Brustel. 1996. Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J. Biol. Chem. 271:25227.[Abstract/Free Full Text]
34. Zagariya, A., S. Mungre, R. Lovis, M. Birrer, S. Ness, B. Thimmapaya, R. Pope. 1998. Tumor necrosis factor gene regulation: enhancement of C/EBP-induced activation by c-Jun. Mol. Cell. Biol. 18:2815.[Abstract/Free Full Text]
35. Zhong, H., M. J. May, E. Jimi, S. Ghosh. 2002. The phosphorylation status of nuclear NF-B determines its association with CBP/p300 or HDAC-1. Mol. Cell 9:625.[Medline]
36. Boyes, J., P. Byfield, Y. Nakatani, V. Ogryzko. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396:594.[Medline]
37. Petrova, T. V., K. T. Akama, L. J. Van Eldik. 1999. Selective modulation of BV-2 microglial activation by prostaglandin E₂: differential effects on endotoxin-stimulated cytokine induction. J. Biol. Chem. 274:28823.[Abstract/Free Full Text]
38. McCarthy, K. D., J. de Vellis. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890.[Abstract/Free Full Text]
39. Giri, S., N. Nath, B. Smith, B. Viollet, A. K. Singh, I. Singh. 2004. 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J. Neurosci. 24:479.[Abstract/Free Full Text]
40. Pahan, K., J. R. Raymond, I. Singh. 1999. Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells. J. Biol. Chem. 274:7528.[Abstract/Free Full Text]
41. Rattan, R., S. Giri, A. K. Singh, I. Singh. 2003. Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKK. Free Radical Biol. Med. 35:1037.[Medline]
42. Hu, H. M., Q. Tian, M. Baer, C. J. Spooner, S. C. Williams, P. F. Johnson, R. C. Schwartz. 2000. The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1. J. Biol. Chem. 275:16373.[Abstract/Free Full Text]
43. Ozes, O. N., L. D. Mayo, J. A. Gustin, S. R. Pfeffer, L. M. Pfeffer, D. B. Donner. 1999. NF-B activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401:82.[Medline]
44. Romashkova, J. A., S. S. Makarov. 1999. NF-B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:86.[Medline]
45. Gerritsen, M. E., A. J. Williams, A. S. Neish, S. Moore, Y. Shi, T. Collins. 1997. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc. Natl. Acad. Sci. USA 94:2927.[Abstract/Free Full Text]
46. Deng, W. G., Y. Zhu, K. K. Wu. 2003. Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor--induced cyclooxygenase-2 promoter activation. J. Biol. Chem. 278:4770.[Abstract/Free Full Text]
47. Gelman, L., G. Zhou, L. Fajas, E. Raspe, J. C. Fruchart, J. Auwerx. 1999. p300 interacts with the N- and C-terminal part of PPAR2 in a ligand-independent and -dependent manner, respectively. J. Biol. Chem. 274:7681.[Abstract/Free Full Text]
48. Goetzl, E. J., S. An, W. L. Smith. 1995. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J. 9:1051.[Abstract]
49. Phipps, R. P., S. H. Stein, R. L. Roper. 1991. A new view of prostaglandin E regulation of the immune response. Immunol. Today 12:349.[Medline]
50. Harris, S. G., J. Padilla, L. Koumas, D. Ray, R. P. Phipps. 2002. Prostaglandins as modulators of immunity. Trends Immunol. 23:144.[Medline]
51. Kliewer, S. A., J. M. Lenhard, T. M. Willson, I. Patel, D. C. Morris, J. M. Lehmann. 1995. A prostaglandin J₂ metabolite binds peroxisome proliferator-activated receptor and promotes adipocyte differentiation. Cell 83:813.[Medline]
52. Xin, X., S. Yang, J. Kowalski, M. E. Gerritsen. 1999. Peroxisome proliferator-activated receptor ligands are potent inhibitors of angiogenesis in vitro and in vivo. J. Biol. Chem. 274:9116.[Abstract/Free Full Text]
53. Sarraf, P., E. Mueller, W. M. Smith, H. M. Wright, J. B. Kum, L. A. Aaltonen, A. de la Chapelle, B. M. Spiegelman, C. Eng. 1999. Loss-of-function mutations in PPAR associated with human colon cancer. Mol. Cell 3:799.[Medline]
54. Sawano, H., M. Haneda, T. Sugimoto, K. Inoki, D. Koya, R. Kikkawa. 2002. 15-Deoxy-12,14-prostaglandin J₂ inhibits IL-1-induced cyclooxygenase-2 expression in mesangial cells. Kidney Int. 61:1957.[Medline]
55. Marx, N., G. Sukhova, C. Murphy, P. Libby, J. Plutzky. 1998. Macrophages in human atheroma contain PPAR: differentiation-dependent peroxisomal proliferator-activated receptor (PPAR) expression and reduction of MMP-9 activity through PPAR activation in mononuclear phagocytes in vitro. Am. J. Pathol. 153:17.[Abstract/Free Full Text]
56. Morita, I., M. Schindler, M. K. Regier, J. C. Otto, T. Hori, D. L. DeWitt, W. L. Smith. 1995. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J. Biol. Chem. 270:10902.[Abstract/Free Full Text]
57. Chen, F. E., D. B. Huang, Y. Q. Chen, G. Ghosh. 1998. Crystal structure of p50/p65 heterodimer of transcription factor NF-B bound to DNA. Nature 391:410.[Medline]
58. Zhang, J. J., U. Vinkemeier, W. Gu, D. Chakravarti, C. M. Horvath, J. E. Darnell, Jr. 1996. Two contact regions between Stat1 and CBP/p300 in interferon signaling. Proc. Natl. Acad. Sci. USA 93:15092.[Abstract/Free Full Text]
59. Park, E. J., S. Y. Park, E. H. Joe, I. Jou. 2003. 15d-PGJ2 and rosiglitazone suppress Janus kinase-STAT inflammatory signaling through induction of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 in glia. J. Biol. Chem. 278:14747.[Abstract/Free Full Text]
60. Beutler, B.. 2000. Tlr4: central component of the sole mammalian LPS sensor. Curr. Opin. Immunol. 12:20.[Medline]

This article has been cited by other articles:

				J. H. Lee, J. H. Woo, S. U. Woo, K. S. Kim, S. M. Park, E.-h. Joe, and I. Jou The 15-Deoxy-{Delta}12,14-Prostaglandin J2 Suppresses Monocyte Chemoattractant Protein-1 Expression in IFN-{gamma}-Stimulated Astrocytes through Induction of MAPK Phosphatase-1 J. Immunol., December 15, 2008; 181(12): 8642 - 8649. [Abstract] [Full Text] [PDF]

				U. Panzer, G. Zahner, U. Wienberg, O. M. Steinmetz, A. Peters, J.-E. Turner, H.-J. Paust, G. Wolf, R. A. K. Stahl, and A. Schneider 15-Deoxy-{Delta}12,14-prostaglandin J2 inhibits INF-{gamma}-induced JAK/STAT1 signalling pathway activation and IP-10/CXCL10 expression in mesangial cells Nephrol. Dial. Transplant., December 1, 2008; 23(12): 3776 - 3785. [Abstract] [Full Text] [PDF]

				G. Alba, R. El Bekay, P. Chacon, M. E. Reyes, E. Ramos, J. Olivan, J. Jimenez, J. M. Lopez, J. Martin-Nieto, E. Pintado, et al. Heme oxygenase-1 expression is down-regulated by angiotensin II and under hypertension in human neutrophils J. Leukoc. Biol., August 1, 2008; 84(2): 397 - 405. [Abstract] [Full Text] [PDF]

				P T Gutierrez, E Folch-Puy, O Bulbena, and D Closa Oxidised lipids present in ascitic fluid interfere with the regulation of the macrophages during acute pancreatitis, promoting an exacerbation of the inflammatory response Gut, May 1, 2008; 57(5): 642 - 648. [Abstract] [Full Text] [PDF]

				N. Mizutani, T. Sakurai, T. Shibata, K. Uchida, J. Fujita, R. Kawashima, Y. I. Kawamura, N. Toyama-Sorimachi, T. Imai, and T. Dohi Dose-Dependent Differential Regulation of Cytokine Secretion from Macrophages by Fractalkine J. Immunol., December 1, 2007; 179(11): 7478 - 7487. [Abstract] [Full Text] [PDF]

				R. N. Saha, M. Jana, and K. Pahan MAPK p38 Regulates Transcriptional Activity of NF-{kappa}B in Primary Human Astrocytes via Acetylation of p65 J. Immunol., November 15, 2007; 179(10): 7101 - 7109. [Abstract] [Full Text] [PDF]

				L. Yang, C.-C. Chan, O.-S. Kwon, S. Liu, J. McGhee, S. A. Stimpson, L. Z. Chen, W. W. Harrington, W. T. Symonds, and D. C. Rockey Regulation of peroxisome proliferator-activated receptor-{gamma} in liver fibrosis Am J Physiol Gastrointest Liver Physiol, November 1, 2006; 291(5): G902 - G911. [Abstract] [Full Text] [PDF]

				Z. Wang, V. M. Aris, K. D. Ogburn, P. Soteropoulos, and M. E. Figueiredo-Pereira Prostaglandin J2 Alters Pro-survival and Pro-death Gene Expression Patterns and 26 S Proteasome Assembly in Human Neuroblastoma Cells J. Biol. Chem., July 28, 2006; 281(30): 21377 - 21386. [Abstract] [Full Text] [PDF]

				R. K. Selvaraj and K. C. Klasing Lutein and Eicosapentaenoic Acid Interact to Modify iNOS mRNA Levels through the PPAR{gamma}/RXR Pathway in Chickens and HD11 Cell Lines J. Nutr., June 1, 2006; 136(6): 1610 - 1616. [Abstract] [Full Text] [PDF]

				A. S. Paintlia, M. K. Paintlia, I. Singh, and A. K. Singh IL-4-Induced Peroxisome Proliferator-Activated Receptor {gamma} Activation Inhibits NF-{kappa}B Trans Activation in Central Nervous System (CNS) Glial Cells and Protects Oligodendrocyte Progenitors under Neuroinflammatory Disease Conditions: Implication for CNS-Demyelinating Diseases J. Immunol., April 1, 2006; 176(7): 4385 - 4398. [Abstract] [Full Text] [PDF]

				M. Hughes-Fulford, C.-F. Li, J. Boonyaratanakornkit, and S. Sayyah Arachidonic Acid Activates Phosphatidylinositol 3-Kinase Signaling and Induces Gene Expression in Prostate Cancer Cancer Res., February 1, 2006; 66(3): 1427 - 1433. [Abstract] [Full Text] [PDF]

				J.-H. Lee, H. S. Jung, P. M. Giang, X. Jin, S. Lee, P. T. Son, D. Lee, Y.-S. Hong, K. Lee, and J. J. Lee Blockade of Nuclear Factor-{kappa}B Signaling Pathway and Anti-Inflammatory Activity of Cardamomin, a Chalcone Analog from Alpinia conchigera J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 271 - 278. [Abstract] [Full Text] [PDF]

				R. Rattan, S. Giri, A. K. Singh, and I. Singh 5-Aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase J. Biol. Chem., November 25, 2005; 280(47): 39582 - 39593. [Abstract] [Full Text] [PDF]

				Y. Kobayashi, S. Ueki, G. Mahemuti, T. Chiba, H. Oyamada, N. Saito, A. Kanda, H. Kayaba, and J. Chihara Physiological Levels of 15-Deoxy-{Delta}12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemotaxis on Human Eosinophils through Peroxisome Proliferator-Activated Receptor-{gamma} Ligation J. Immunol., November 1, 2005; 175(9): 5744 - 5750. [Abstract] [Full Text] [PDF]

				R. Luna-Medina, M. Cortes-Canteli, M. Alonso, A. Santos, A. Martinez, and A. Perez-Castillo Regulation of Inflammatory Response in Neural Cells in Vitro by Thiadiazolidinones Derivatives through Peroxisome Proliferator-activated Receptor {gamma} Activation J. Biol. Chem., June 3, 2005; 280(22): 21453 - 21462. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giri, S. Articles by Singh, I. Search for Related Content PubMed PubMed Citation Articles by Giri, S. Articles by Singh, I.